CHIMERIC PROTEINS

Abstract

This invention relates to modular proteins that interact with one or more target molecules. The chimeric proteins comprise two or more repeat domains, such as tetratricopeptide repeat domains; inter-repeat loops linking the repeat domains; and one or more peptide ligands. Each peptide ligand is located in an inter-repeat loop or at the N or C terminus of the chimeric protein. The peptide ligands may include heterologous peptidyl binding motifs, such as short linear motifs (SLiMs). Chimeric proteins with various configurations and methods for their production and use are provided.

Description

FIELD

This invention relates to chimeric proteins and their production and uses.

BACKGROUND

A priority area in medicine, particularly cancer research, is the expansion of the ‘druggable’ proteome, which is currently limited to narrow classes of molecular targets. For example, protein-protein interactions (PPIs) are fundamental to all biological processes and represent a large proportion of potential drug targets, but they are not readily amenable to conventional small molecule inhibition. The architecture of tandem repeat proteins has tremendous scope for rational design (Kobe & Kajava 2000, Longo & Blaber, 2014, Rowling et al., 2015). The key features of tandem repeat proteins are relatively small size, modularity and extremely high stability (and therefore recombinant production) without the need of disulphide bonds. Individual consensus-designed repeats are self-compatible and can be put together in any order; function is therefore also modular, which means that multiple functions can be independently designed and incorporated in a combinatorial fashion within a single molecule (WO2017106728).

Novel repeat protein functions, e.g. DARPins (Tamaskovic et al., 2012), have been developed based on the natural type of PPI interface of these proteins i.e. spanning many repeat units to create an extended, high-affinity binding interface for the target. Mutations have been introduced into the surface residues in the tetratricopeptide (TPR) repeats of the cytosolic receptor peroxin 5 (Sampathkumar et al. (2008) J. Mol. Biol., 381, 867-880). Binding of peptide ligands to peroxin 5 is shown to be mediated by residues located in several different TPR repeats. The interactions of TPR containing protein kinesin-1 with different cargo proteins has also been reported (Zhu et al PLoS One 2012 7 3 e33943). The specificity and stability of ankyrin repeat proteins has been modified through the introduction of mutations into ankyrin repeat sequences (Li et al (2006) Biochemistry 45 15168-15178).

SUMMARY OF THE INVENTION

The present inventors have found that chimeric proteins which comprise peptidyl ligands, such as short linear motifs (SLiMs), on scaffolds. Such chimeric proteins (i.e., modular binding proteins), may be useful for example, as single- or multi-function protein therapeutics.

An aspect of the invention provides a chimeric protein comprising:

• • a scaffold comprising a first end and a second end, and two or more repeat domains linked by inter-repeat loops between the ends; and
  • one or more peptide ligands, wherein a single peptide ligand is located in the scaffold in (i), an inter-repeat loop, (ii) at the first end, or (iii) at the second end of the scaffold, thereby forming a chimeric protein (a grafted scaffold).

In a preferred embodiment, the scaffold is a continuous polypeptide strand such that the first end is the N terminus and the second end is the C terminus of the scaffold.

In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule. One of the first or second target molecules may be an E3 ubiquitin ligase. Where a chimeric protein comprises two or more peptide ligands, the ligands are different ligands (bind to different targets) and are not located in the same loop or at the same end of a scaffold.

Another aspect of the invention provides a method of producing a chimeric protein comprising:

• • inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a scaffold comprising two or more repeat domains linked by inter-repeat loops, to produce a chimeric nucleic acid encoding a chimeric protein as described herein; and
  • expressing the chimeric nucleic acid to produce the chimeric protein.

Another aspect of the invention provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising:

• • providing a nucleic acid encoding a scaffold comprising two or more repeat domains linked by inter-repeat loops, and
  • incorporating into the nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising the first and second peptide ligands, wherein the peptide ligands are independently located in an inter-repeat loop or at the N or C terminus of the chimeric protein; and
  • expressing the nucleic acid to produce the protein.

In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.

In another aspect, the invention provides a chimeric protein, comprising

(i) a tetratricopeptide (TPR) scaffold comprising first and second α-helices linked by an inter-repeat loop, and,

(ii) a first heterologous peptide that binds to a target protein, and

(iii) a second heterologous peptide that binds to an E3 ubiquitin ligase,

wherein the first and second heterologous peptides are, independently, located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.

In a preferred embodiment, each of the first and second α-helices comprises the amino acid sequence Y-X1X2X3X4; wherein Y is an amino acid sequence shown in Tables 4 to 6 and X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or optionally wherein X2 is P.

In another preferred embodiment, the first and second α-helices each comprise the amino acid sequence:

AEAWYNLGNAYYKQGDYQKAIEYYQKALEL-X1X2X3X4;
or
AEALNNLGNVYREQGDYQKAIEYYQKALEL-X1X2X3X4;
or
AEAWYNLGNAYYRQGDYQRAIEYYQRALEL-X1X2X3X4;
or
AEALNNLGNVYREQGDYQRAIEYYQRALEL-X1X2X3X4;
or
AEALRNLGRVYRRQGRYQRAIEYYRRALEL-X1X2X3X4,

wherein X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or optionally wherein X2 is P.

In another preferred embodiment, the chimeric protein comprising third, fourth and fifth TPR repeats.

The invention also provides a chimeric protein comprising

(i) a TPR scaffold comprising first and second α-helices linked by an inter-repeat loop, and,

(ii) a heterologous peptide ligand that binds an E3 ligase,

wherein the heterologous peptide is located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.

The invention also provides a chimeric protein comprising

(i) a TPR scaffold comprising first and second α-helices linked by an inter-repeat loop, and,

(ii) a heterologous peptide ligand that binds a target protein,

• • wherein the heterologous peptide ligand is located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.

Another aspect of the invention provides a library comprising chimeric proteins, each chimeric protein in the library comprising;

• • (i) two or more repeat domains,
  • (ii) inter-repeat loops linking the repeat domains; and
  • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
    • wherein at least one amino acid residue in the peptide ligands in the library is diverse.

Another aspect of the invention provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;

• • (i) two or more repeat domains,
  • (ii) inter-repeat loops linking the repeat domains; and
  • (iii) a peptide ligand comprising at least one diverse amino acid residue,

wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (i) an inter-repeat loop; (ii) the N terminus or (iii) the C terminus of the chimeric protein, and

• • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (i) an inter-repeat loop; (ii) the N terminus or (iii) the C terminus of the chimeric protein.

Another aspect of the invention provides a method of producing a library of chimeric proteins comprising;

• • (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
    • (i) two or more repeat domains,
    • (ii) inter-repeat loops linking the repeat domains; and
    • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
    • wherein the peptide ligands in the population are diverse, and
  • (b) expressing the population of nucleic acids to produce the diverse population,
    • thereby producing a library of chimeric proteins.

Another aspect of the invention provides a method of screening a library comprising;

• • (a) providing a library of chimeric proteins, each chimeric protein in the library comprising;
    • (i) two or more repeat domains,
    • (ii) inter-repeat loops linking the repeat domains; and
    • (iii) a peptide ligand located in the inter-repeat loop, at the N terminus or at the C terminus of the protein.
    • wherein at least one amino acid residue in the peptide ligands in the library is diverse,
  • (b) screening the library for chimeric proteins which display a binding activity, and
  • (c) identifying one or more chimeric proteins in the library which display the binding activity.

Other aspects and embodiments of the invention are described in more detail below.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the thermostability of consensus-designed tetratricopeptide (CTPR) proteins containing loop- or helix-grafted binding motifs: Thermal denaturation, monitored by circular dichroism, of 2-repeat RTPR (a CTPR in which lysine residues have been replaced with arginine residues) proteins: RTPR2 (in diamonds), RTPR2 containing a loop binding-module (circles) and RTPR2 containing a helix binding-module (squares). All samples are at 20 μM in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl.

FIG. 2 shows the thermostability of CTPR proteins of increasing length containing an increasing number of binding modules (alternating with blank modules): Thermal denaturation curves, monitored by circular dichroism, of TPR proteins containing 1, 2, 3 and 4 loops comprising a tankyrase-binding sequence: 1TBP-CTPR2, 2TBP-CTPR4, 3TBP-CTPR6, 4TBP-CTPR8. All samples are at 20 μM in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl.

FIG. 3 shows an example of helix grafting. FIG. 3A (i) shows the crystal structures of SOS1 (son-of-sevenless homologue 1) bound to KRAS (Kirsten rat sarcoma) (PDB 1NVU, Margarit et al. Cell (2003) 112(5):685-95), and (ii) shows the SOS1 helix grafted onto a helix at the N-terminus of a CTPR2 protein. The modelled structure of SOS-RTPR2 is shown, and the sequence of the helix is given with the key KRAS-binding residues in grey and the residues that form the interface with the CTPR helices in black. (iii) shows the modelled structure of SOS-TPR2 in complex with KRAS. FIG. 3B shows binding of SOS-TPR2 to KRAS measured by competitive fluorescence polarization (FP). The complex between mant-GTP and KRAS was pre-formed, and 0.1-300 μM SOS-RTPR2 was then titrated in to the complex, displacing the mant-GTP from KRAS resulting in a decrease in FP. EC50 is 3 μM.

FIG. 4 shows another example of helix grafting. FIG. 4A shows the modelled structure of the Mdm2 (Mouse double minute 2 homolog) N-terminal domain in complex with the p53-TPR2 comprising the Mdm2-binding helix of p53 grafted onto a helix at the C-terminus of a CTPR2 protein. FIG. 4B shows an ITC analysis of the interaction between p53-TPR2 and Mdm2 N-terminal domain. The N-terminal domain of Mdm2 was titrated into the cell containing 10 μM p53-TPR2.

FIG. 5 shows an example of single and multivalent loop-grafted CTPRs. FIG. 5A shows an ITC analysis of the interaction between a series of tankyrase-binding loop-grafted CTPR2 proteins (TBP-CTPR2) and the substrate-binding ARC4 (ankyrin-repeat cluster) domain of tankyrase. There is an enhancement of both binding affinity and dissociation constant with increasing number of binding modules. FIG. 5B shows native gel analysis (using a native gel in Tris-Glycine buffer pH 8.0, 40 μM protein concentration) of multivalent TBP-CTPR proteins expressed as fusion constructs with the foldon trimerisation domain (Boudko et al 2002; Meier et al. 2004). 1TBP-CTPR2, 2TBP-CTPR4 and 4TBP-CTPR8 (all lacking the foldon domain) were purified and run as monomeric controls. Constructs having the foldon domain run at much higher molecular weights than their monomeric counterparts.

FIG. 6 shows an example of loop-grafted CTPRs comprising the 10-residue Skp2-binding sequence derived from p27 grafted into a loop of a CTPR protein (CTPR-p27). FIG. 6A shows that HA-CTPR2-p27 is able to co-IP FLAG-Skp2 from HEK293T cells. FIG. 6B shows E. coli-expressed and purified TPR5-p27 inhibits p27 ubiquitination in vitro.

FIG. 7 shows another example of loop-grafted CTPRs. FIG. 7A shows (left) ITC analysis of the interaction between the Keap1 (Ketch-like ECH-associated protein I) KELCH domain and a CTPR2 protein containing a loop-grafted Keap1-binding sequence derived from the protein Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) (Nrf-CTPR2). No binding is observed for the blank CPTR2 protein (right). FIG. 7B shows that three variants of Nrf-CTPR2 (Nrf-CTPR2 (i), Nrf-CTPR2 (ii), Nrf-CTPR2 (iii) can co-IP Keap1 from HEK293T cells.

FIG. 8 shows live-cell imaging of intracellular delivery of an RTPR achieved by resurfacing (by introducing Arginine residues at surface sites). PC3 (left) and U2OS (right) cells incubated with 10 μM FITC-labelled resurfaced TBP-RTPR2 for 3 hours at 37° C., 5% CO₂. Overlay of DIC (differential interference contrast) and confocal image. Intracellular fluorescence was also observed at lower concentrations of protein.

FIG. 9 shows the induced degradation of the target protein beta-catenin by designed hetero-bifunctional RTPRs. FIG. 9A shows the beta-catenin levels in cells transfected with either HA-tagged beta-catenin plasmid alone or HA-tagged beta-catenin plasmid together with one of two different hetero-bifunctional RTPR plasmids (LRH1-TPR-p27 and axin-TPR-p27, designed to bind simultaneously to beta-catenin and to E3 ligase SCF^Skp2). FIG. 9B shows a quantitative analysis of the beta-catenin levels in the presence of different hetero-bifunctional RTPRs designed to bind simultaneously to beta-catenin and to either E3 ligase SCF^Skp2 or E3 ligase Mdm2. The analysis was performed using densitometry of the bands detected by Western blots corresponding to HA-tagged beta-catenin normalised to actin bands using ImageJ. Negative controls used were single-function TPRs or blank (non-functional) TPRs.

FIG. 10 shows examples of different chimeric protein formats. A chimeric protein may comprise: two repeat domains with a helical target-binding peptide and a helical E3-binding peptide at the N and C termini (FIG. 10A); three repeat domains with a helical E3-binding peptide at the C terminus and a target peptide ligand in the first inter-repeat loop from the N terminus (FIG. 10B); three repeat domains with a helical target-binding peptide at the N terminus and an E3 peptide ligand in the second inter-repeat loop from the N terminus (FIG. 10C), four repeat domains with a target-peptide ligand and an E3 peptide ligand in the first and third inter-repeat loop from the N terminus (FIG. 10D).

FIG. 11 shows a schematic of a chimeric protein with four peptide ligands located in alternate inter-repeat loops. The binding sites are arrayed at 90° to each other.

FIG. 12 shows a schematic of a chimeric protein engineered so that peptide ligands in alternate inter-repeat loops bind adjacent epitopes on the target.

FIG. 13 shows the modelled structure of a hetero-bifunctional chimeric protein comprising TPR repeat domains, an LRH1-derived peptide ligand designed to bind target beta-catenin, and a p53-derived N-terminal peptide ligand designed to bind to the E3 ubiquitin ligase mdm2.

FIG. 14 shows a schematic of the combinatorial assembly of a module comprising a repeat domain and a terminal helical peptide ligand and a module comprising repeat domains and an inter-repeat loop peptide ligand to generate a chimeric protein.

FIG. 15 shows examples of different chimeric protein formats. (i) shows the blank proteins; (ii) shows binding peptides inserted into one or more inter-repeat loops. (iii) shows helical binding peptides at one or both of the termini; (iv) is a combination of loop and helical binding peptides; (v) and (vi) show examples of how multivalency can be achieved.

FIG. 16 shows a schematic of the assembly of a chimeric protein by the progressive screening of chimeric proteins comprising modules with a diverse peptide ligand in addition to modules already identified in previous rounds of screening.

FIG. 17 shows the effect of designed multi-valent tankyrase-binding TPR proteins on Wnt signalling. HEK293T cells were transfected with TPR-encoding plasmids using Lipofectamine2000. The TPR proteins contained 1-4 copies of a tankyrase-binding peptide (TBP) grafted onto the inter-repeat loop(s). For example, 2TBP-CTPR4 is a protein comprising 4 TPR modules with one TBP grafted onto the loop between the first and second TPR and one between the third and fourth TPR. ‘Foldon’ indicates a trimeric TPR-foldon fusion protein.

FIG. 18 shows characterisation of the size and charge of liposome-encapsulated TPR proteins.

FIG. 19 shows the delivery of TPR proteins into cells by liposome encapsulation. FITC dye-labelled liposomes stain the cell membrane upon membrane fusion (red panel), and RITC-labelled TPR protein cargo is then delivered into the cytoplasm. The green panel and red-green merge show that the proteins have entered the cells and are spread diffusely in the cytoplasm.

FIG. 20 shows that liposome-encapsulated TPR proteins are not toxic to HEK293T cells at the concentrations used.

FIG. 21 shows the effect of designed hetero-bifunctional TPR proteins (delivered by liposome encapsulation) on Wnt signalling. The TPR proteins contained a tankyrase-binding peptide and a SCF^Skp2-binding peptide to direct tankyrase for ubiquitination and subsequent degradation. Cells were treated with liposomes for 2 hr.

FIG. 22 shows the effect of designed hetero-bifunctional TPR proteins (delivered by liposome encapsulation) on Wnt signalling. The TPR proteins contained a beta-catenin-binding peptide and a SCF^Skp2-binding peptide to direct beta-catenin for ubiquitination and subsequent degradation. Cells were treated with liposomes encapsulating 32 μg protein for variable times (2-8 h) indicated in the figure.

FIG. 23 shows the effect of designed hetero-bifunctional TPR proteins on KRAS levels in HEK 293T cells. The TPR proteins contained a binding sequence for KRAS (a non-helical peptide sequence, referred to as KBL, grafted onto an inter-repeat loop of the RTPR) and a degron derived from p27 grafted onto another inter-repeat loop. Cells were transiently transfected with 50 ng or 500 ng of TPR encoding plasmids, as indicated, and with KRAS plasmid or empty vector as control. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. In dark grey are cells treated transfected with single-function TPR plasmid (containing degron only).

FIG. 24 shows the effect of hetero-bifunctional TPR proteins targeting endogenous KRAS to the CMA (chaperone-mediated autophagy) pathway. The TPR proteins contained a binding sequence for KRAS (either a grafted helix derived from son-of-sevenless-homolog 1 (SOS) or a non-helical peptide sequence (referred to as ‘KBL’) displayed in a loop of the RTPR) and targeted for degradation using two different chaperone-mediated autophagy peptides (referred to as ‘CMA Q’ or ‘CMA K’) at the N- or C-terminus of the construct. Constructs or empty vector (light grey) were transiently transfected into either HEK293T or DLD1 (colorectal cancer cell line). 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. Those constructs that resulted in significant reduction in KRAS compared to the empty vector control are shown in white.

FIG. 25 shows examples of variations in the linker sequence connecting a peptide ligand to an inter-repeat loop in order to optimise the binding affinity for the target. The example shown is Nrf-TPR, a TPR protein designed to bind to Keap1 (see FIG. 7 of the original patent application). Glycine residues were introduced into the linker to provide flexibility and increased spatial sampling. The introduction of this more flexible linker sequence was found to increase the binding affinity of the Nrf-TPR protein (labelled ‘Flexible’) when compared with the consensus-like linker sequence. Altering the charge content of the linker sequence (‘labelled ‘Charged’) and altering the conformational properties (based on the predictions of the program CIDER (Holehouse et al. Biophys. J. 112, 16-21 (2017)) of the loop by changing the amino acid composition of the linker sequence (labelled ‘CIDER-optimised’) also affected the Keap1-binding affinity.

FIG. 26 shows the schematic representation of a matrix of degradation-inducing chimeric proteins. The matrix shown is for use in targeting β-catenin for degradation. These proteins comprise a scaffold (grey rectangles) onto which are grafted: (1) a target-binding peptide ligand and (2) a binding peptide for an E3 ubiquitin ligase or a component of another degradation pathway. Each of the target-binding peptides is derived from a different protein that interacts with β-catenin (see Table 2). Each of the degradation pathway-binding peptides (referred to as “degrons”) is derived from a substrate or binding partner of one of many different E3s or from a binding partner for one of a component of another cellular degradation pathway (including chaperone-mediated autophagy, selective autophagy and ESCRT (endosome-lysosome) pathways); ‘etc.’ denotes the fact that there are many such proteins that can be harnessed for degradation, as detailed further in Table 3. The schematic illustrates the combinatorial “plug-and-play” nature of these matrices, in terms of the ability to slot in any target-recruiting peptide and degradation-pathway-recruiting peptide. The other factor that can be varied in the matrix arises from the fact that the two peptides can also be grafted onto different positions in the scaffold so as to present the target in different configurations with respect to the E3 or other degradation machinery. Once the matrix is constructed, it can then be screened in cell-based assay in order to identify the best combination of two peptides and their positions within the scaffold that induces the greatest reduction in target protein levels. The same panel of diverse degradation pathway components can be used for screen for degradation of any target.

DETAILED DESCRIPTION

This invention relates to the chimeric proteins that comprise multiple repeat domains. These repeat domains are linked to each other in the polypeptide chain by inter-repeat loops. One or more peptide ligands (i.e., peptidyl binding motifs or binding domains), are located in one or more of the inter-repeat loops and/or in N or C terminal helices of the chimeric protein. The peptide ligands may be to the same or different target molecules and the chimeric protein may be multi-functional and/or multi-valent. The geometrical display of the grafted binding sites may be precisely and predictably tuned by adjusting the positions of the binding sites and the number and shape of the repeat domains. Chimeric proteins as described herein may be useful in a range of therapeutic and diagnostic applications.

A “repeat domain” is a repetitive structural element of 30 to 100 amino acids that forms a defined secondary structure. Multiple (two or more) repeat domains stack sequentially in a modular fashion to form a stable protein, which may for example have a solenoid or toroid structure. Repeat domains may be synthetic or may be naturally-occurring repeats from tandem repeat proteins, or variants thereof.

Due to the identical form of their building blocks, solenoid domains can only assume a limited number of shapes. Two main topologies are possible: linear (or open, generally with some degree of helical curvature) and circular (or closed). Patthy, László (2007). Protein Evolution. Wiley-Blackwell. ISBN 978-1-4051-5166-5.

If the two terminal repeats in a solenoid do not physically interact, it leads to an open or linear structure. Members of this group are frequently rod- or crescent-shaped. The number of individual repeats can range from 2 to over 50. A clear advantage of this topology is that both the N- and C-terminal ends are free to add new repeats and folds, or even remove existing ones during evolution without any gross impact on the structural stability of the entire domain. Kinch L N, Grishin N V (June 2002). Curr. Opin. Struct. Biol. 12 (3): 400-8. doi:10.1016/s0959-440x(02)00338-x. PMID 12127461. This type of domain is extremely common among extracellular segments of receptors or cell adhesion molecules. A non-exhaustive list of examples include: EGF repeats, cadherin repeats, leucine-rich repeats, HEAT repeats, ankyrin repeats, armadillo repeats, tetratricopeptide repeats, etc. Whenever a linear solenoid domain structure participates in protein-protein interactions, frequently at least 3 or more repetitive subunits form the ligand-binding sites. Thus—while individual repeats might have a (limited) ability to fold on their own—they usually cannot perform the functions of the entire domain alone.

In the case when the N- and C-terminal repeats lie in close physical contact in a solenoid domain, the result is a topologically compact, closed structure. Such domains typically display a high rotational symmetry (unlike open solenoids that only have translational symmetries), and assume a wheel-like shape. Because of the limitations of this structure, the number of individual repeats is not arbitrary. In the case of WD40 repeats (perhaps the largest family of closed solenoids) the number of repeats can range from 4 to 10 (more usually between 5 and 7). (Vogel C, Berzuini C, Bashton M, Gough J, Teichmann S A (February 2004). J. Mol. Biol. 336 (3): 809-23). Kelch repeats, beta-barrels and beta-trefoil repeats are further examples for this architecture.

A repeat domain may have the structure of a solenoid repeat. The structures of solenoid repeats are well known in the art (see for example Kobe & Kajava Trends in Biochemical Sciences 2000; 25(10):509-15). For example, a repeat domain may have an α/α or α/3₁₀ (helix-turn-helix or hth) structure, for example a tetratricopeptide repeat structure; α/α/α (helix-turn-helix-turn-helix or hthth) structure, for example an armadillo repeat structure; a β/β/α/α structure; a α/β or 3₁₀/β structure, for example a leucine rich repeat (LRR) structure; a β/β/β structure, for example, an IGF1RL, HPR or PelC repeat structure; or a β/β structure, for example a serralysin or EGF repeat structure.

A “scaffold” refers to two or more repeat domains, and a “grafted scaffold” refers to a continuous polypeptide comprising a scaffold and a heterologous binding site (e.g., a peptide ligand).

Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops. The loops between adjacent Ankyrin repeats are semi-structured and therefore are quite rigid. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics.

The armadillo (Arm) repeat is an approximately 40 amino acid long tandemly repeated sequence motif first identified in the Drosophila melanogaster segment polarity gene armadillo involved in signal transduction through wingless. Animal Arm-repeat proteins function in various processes, including intracellular signalling and cytoskeletal regulation, and include such proteins as beta-catenin, the junctional plaque protein plakoglobin, the adenomatous polyposis coli (APC) tumour suppressor protein, and the nuclear transport factor importin-alpha, amongst others [(PUBMED:9770300)].

Suitable repeat domains may include domains of the Ankyrin clan (Pfam: CL0465), such as ankyrin (PF00023), which may comprise a 30-34 amino-acid repeat composed of two beta strands and two alpha helices; domains of the leucine-rich repeat (LRR) clan (Pfam; CL0022), such as LRR1 (PF00560), which may comprise a 20-30 amino acid repeat composed of an α/β horseshoe fold; domains of the Pec Lyase-like (CL0268) clan, such as pec lyase C (PF00544), which may comprise a right handed beta helix; domains of the beta-Roll (CL0592) clan such as Haemolysin-type calcium-binding repeat (PF000353), which may comprise short repeat units (e.g. 9-mers) that form a beta-roll made up of a super-helix of beta-strand-turns of two short strands each, stabilised by Ca²⁺ ions; domains of the PSI clan (CL0630), such as trefoil (PF00088); and domains of the tetratricopeptide clan (CL0020), such as TPR-1 (PR00515), which may comprise a 24 to 30, or 24 to 40, or 24 to 90 amino acid repeat composed of a helix-turn-helix.

Consensus Sequences for ANK repeats (SMART database, see Table 10) include the following:

O04242/1-30   NGHTALHIAASK------------------GDEQCVKLLLEHGA------DPNA
CONSENSUS/80% .t.sslhhsh.t..................tp.phhphllp.t.......pht.
CONSENSUS/65% pstosLphAstp..................sphphlphLlptss......shsh
CONSENSUS/50% sGpTsLHhAsps..................sshcllchLlspus......slst

Consensus Sequence for ARM repeats (SMART database, see Table 11) include the following:

IMO2HUMANb    PND-KIQAVIDAG--VCRRLVELLM----------------------
              HNDYKVVSPALRA
CONSENSUS/80% pt...h..hhp.t..hl..lhphlt........................p.pl.t.shhs
CONSENSUS/65% ssp.ptphlhpts..slshLlpLLp......................pts.plhptsshs
CONSENSUS/50% ssc.sppsllcsG..slstLlpLLs......................sscsclppsAstA
IMO2HUMANb    VGNIVT
CONSENSUS/80% ltpls.
CONSENSUS/65% LpNlst
CONSENSUS/50% LsNlus

Suitable repeat domains may be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44:D279-D285).

In some preferred embodiments, the repeat domain may have the structure of an α/α-solenoid repeat domain, such as a helix-turn-helix. A helix-turn-helix domain comprises two antiparallel α-helices of 12-45 amino acids.

Suitable helix-turn-helix domains include tetratricopeptide-like repeat domains. Tetratricopeptide-like repeats may include domains of the TPR clan (CL0020), for example and Arm domains (see for example Armadillo; PF00514; Huber et al Cell 1997; 90: 871-882), HEAT domains (Huntingtin, EF3, PP2A, TOR1; PF02985; see for example Groves et al. Cell. 96 (1): 99-110), PPR domains (pentatricopeptide repeat PF01535; see for example Small (2000) Trends Biochem. Sci. 25 (2): 46-7), TALE domains (TAL (transcription activator-like) effector; PF03377; see for example Zhang et al Nature Biotechnology. 29 (2): 149-53) and TPR1 domains (tetratricopeptide repeat-1; PF00515; see for example Blatch et al BioEssays. 21 (11): 932-9).

Other suitable helix-turn-helix domain may be synthetic, for example DHR1 to DHR83 as disclosed in Brunette et al., Nature 2015 528 580-584.

In some preferred embodiments, the helix-turn-helix scaffold may be a tetratricopeptide repeat domain (TPR) (D'Andrea & Regan, 2003) or a variant thereof. TPR repeat domains may include naturally occurring or synthetic TPR domains. Suitable TPR repeat domains are well known in the art (see for example Parmeggiani et al., J. Mol. Biol. 427 563-575) and may have the amino acid sequence:

AEAWYNLGNAYYKQGDYQKAIEYYQKALEL-X1X2 X3X4,

• • wherein X_1-4 are independently any amino acid, preferably X₁ and X₂ being D and P respectively, or may be a variant of this sequence.
  • Additional TPR repeat consensus sequences (SMART database, see Table 9) include the following:

S75991        ALTLNNIGTI YYAREDYDQA LNYYEQALSL SRAV
CONSENSUS/80% XXhhXthuXh hXXXtphppA htXhppsltht XpX
CONSENSUS/65% spshhphGth hhphsphppA lphappAlpl pspX
CONSENSUS/50% spsatslGps atptucaccA lcsap+ALcl sPss

• • Other TPR repeat domain sequences are shown in Tables 4-6 and 9 below.

The grouping of amino acids to classes and class abbreviation (the key) used within consensus sequences are shown below.

Class	Key	Residues
alcohol	o	S, T
aliphatic	l	I, L, V
any	.	A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
		V, W, Y
aromatic	a	F, H, W, Y
charged	c	D, E, H, K, R
hydrophobic	h	A, C, F, G, H, I, K, L, M, R, T, V, W, Y
negative	−	D, E
polar	p	C, D, E, H, K, N, Q, R, S, T
positive	+	H, K, R
small	s	A, C, D, G, N, P, S, T, V
tiny	u	A, G, S
turnlike	t	A, C, D, E, G, H, K, N, Q, R, S, T

Preferred TPR domains may include CTPR, RTPRa, RTPRb and KTPRb domains, for example a domain having a sequence shown in Table 4 or Table 6 or a variant of a sequence shown in Table 4 or Table 6.

In some embodiments, a TPR repeat domain may be a human TPR repeat domain, preferably a TPR repeat domain from a human protein in blood. TPR repeat domains from human blood may have reduced immunogenicity in vivo. Suitable human blood TPR repeat domains may include repeat domains from IFIT1, IFIT2 or IFIT3. Other examples of human blood repeat domains identified in the plasma proteome database are shown in Table 5.

Suitable human blood repeat domains may be identified from the plasma proteome database (Nanjappa et al Nucl Acids Res 2014 January; 42 (Database issue):D959-65) for example by searching for sequences with high sequence identity to the TPR repeat domain using standard sequence analysis tools (e.g. Altschul et al Nucleic Acids Res. 25:3389-34021; Altschul et al FEBS J. 272:5101-5109).

A variant of a reference repeat domain or binding site sequence set out herein may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a repeat domain shown above by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids. Preferred variants of a TPR repeat domain may comprise one or more conserved residues, for example, 1, 2, 3, 4, 5, 6 or more preferably all of Leu at position 7, Gly or Ala at position 8, Tyr at position 11, Ala at position 20, Ala at position 27, Leu or Ile at positions 28 and 30 and Pro at position 32.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

Sequence comparison may be made over the full-length of the relevant sequence described herein.

For example, a repeat domain may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands. For example, aromatic residues in the repeat domain may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of repeat domain sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions. A suitable TPR repeat domain for grafting hydrophobic peptide ligands may have the amino acid sequence:

AEAWYNLGNAYYRQGDYQRAIEYYQRALEL-X1X2 X3X4,

• • wherein X_1-4 are independently any amino acid, preferably X₁ and X₂ being D and P respectively.

In some embodiments, lysine residues in the repeat domain may be replaced by arginine residues to prevent ubiquitination and subsequent degradation. This may be particularly useful when the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC). For example, a suitable TPR repeat domain may have the amino acid sequence:

AEALNNLGNVYREQGDYQRAIEYYQRALEL-X1X2 X3X4,

• • wherein X_1-4 are independently any amino acid, preferably X₁ and X₂ being D and P respectively.

In preferred embodiments, the chimeric protein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 repeat domains. Preferably, the chimeric protein comprises 2 to 5 repeat domains. Chimeric proteins with fewer repeat domains may display increased cell penetration. For example, a chimeric protein with 2-3 repeat domains may be useful in binding intracellular target molecule. Chimeric proteins with more repeat domains may display increased stability and functionality. For example, a chimeric protein with 4 or more repeat domains may be useful in binding extracellular target molecules. A chimeric protein with 6 or more repeat domains may be useful in producing long linear molecules for targeting or assembling extracellular complexes in bi- or multivalent formats.

In other embodiments, sufficient stability and functionality may be conferred by a single repeat domain with N and C terminal peptide ligands. For example, a chimeric protein may comprise:

• • (i) a repeat domain, and
  • (ii) peptide ligands at the N and C terminal of the repeat domain.

The repeat domains of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the repeat domains.

A “binding domain” (“peptide ligand”) is a contiguous amino acid sequence that specifically binds to a target molecule. Suitable peptide ligands that are capable of grafting onto a terminal helix or inter-repeat loop are well-known in the art and include peptide sequences selected from a library, antigen epitopes, natural protein-protein interactions (helical, extended or turn-like) and short linear motifs (SLiMs). Viral SLiMs (that hijack the host machinery) may be particularly useful because they may display high binding affinities (Davey et al (2011) Trends Biochem. Sci. 36, 159-169).

A suitable peptide ligand for a target molecule may be selected from a library, for example using phage or ribosome display, or identified or designed using rational approaches or computational design, for example using the crystal structure of a complex or an interaction. In some embodiments, peptide ligands may be identified in an amino acid sequence using standard sequence analysis tools (e.g. Davey et al Nucleic Acids Res. 2011 Jul. 1; 39 (Web Server issue): W56-W60).

Peptide ligands may be 5 to 25 amino acids in length, preferably 8 to 15 amino acids, although in some embodiments, longer peptide ligands may be employed.

Generally in chimeric proteins of the invention, the two or more peptide ligands are 40 angstroms apart from each other, they may be 35 angstroms, 30 angstroms, 25 angstroms, 20 angstroms, 15 angstroms but no less than 10 angstroms apart. A person of skill in art can use a 3D structural software such as Chimera or Pymol to determine the minimum distances between positions for ideal positioning in three dimensional orientation.

The peptide ligands and the repeat domains of the chimeric protein are heterologous i.e. the peptide ligand is not associated with the repeat domain in naturally occurring proteins and the binding and repeat domains are artificially associated in the chimeric protein by recombinant means.

A chimeric protein described herein may comprise 1 to n+1 peptide ligands, where n is the number of repeat domains in the chimeric protein. The number of peptide ligands is determined by the required functionality and valency of the chimeric protein. For example, one peptide ligand may be suitable for a mono-functional chimeric protein and two or more peptide ligands may be suitable for a bi-functional or multi-functional chimeric protein.

Chimeric proteins may be monovalent. A target molecule may be bound by a single peptide ligand in a monovalent chimeric protein. Chimeric proteins may be multivalent. A target molecule may be bound by two or more of the same or different peptide ligands in a multivalent chimeric protein.

Chimeric proteins may be monospecific. The peptide ligands in a monospecific chimeric protein may all bind to the same target molecule, more preferably the same site or epitope of the target molecule.

Chimeric proteins may be multi-specific. The peptide ligands in a multi-specific chimeric protein may bind to different target molecules. For example, a bi-specific chimeric protein may comprise one or more peptide ligands that bind to a first target molecule and one or more peptide ligands that bind to a second target molecule and a tri-specific chimeric protein may comprise one or more peptide ligands that bind to a first target molecule, one or more peptide ligands that bind to a second target molecule and one or more peptide ligands that bind to third target molecule.

A bi-specific chimeric protein may bind to the two different target molecules concurrently. This may be useful in bringing the first and second target molecules into close proximity. When the target molecules are located on different cells, concurrent binding of the target molecules to the chimeric protein may bring the cells into close proximity, for example to promote or enhance the interaction of the cells. For example, a chimeric protein which binds to a tumour specific antigen and a T cell antigen, such as CD3, may be useful in bringing T cells into proximity to tumour cells. When the target molecules are from different biological pathways, this may be may be useful in achieving synergistic effects and also for minimising resistance.

A tri-specific chimeric protein may bind to three different target molecules concurrently. In some embodiments, one of the target molecules may be an E3 ubiquitin ligase. For example, tri-specific chimeric protein may binding to a first target molecule from a first biological pathway and a second target molecule from a second biological pathway as well as an E3 ubiquitin ligase. This may be useful in achieving synergistic effects and also for minimising resistance.

A peptide ligand may be located in an inter-repeat loop of the chimeric protein.

An “inter-repeat binding domain” or “inter-repeat peptide ligand” may comprise 5 to 25 amino acid residues, preferably 8 to 15 amino acids. However, since there is no intrinsic restriction on the size of the inter-loop peptide ligand, longer sequences of more than 25 amino acid residues may be used in some embodiments.

In some embodiments, an unstructured peptide ligand may be inserted into an inter-repeat loop.

One or more, two or more, three or more, four or more or five or more of the inter-repeat loops in the chimeric protein may comprise peptide ligands. The peptide ligands may be located on consecutive inter-repeat loops or may have a different distribution in the inter-repeat loops of the chimeric protein. For example, inter-repeat loops comprising a peptide ligand may be separated in the modular protein by one or more, two or more, three or more or four or more inter-repeat loops which lack a peptide ligand.

A peptide ligand may be connected to an inter-repeat loop directly or via one or more additional residues or linkers. Additional residues or linkers may be useful for example when a peptide ligand requires conformational flexibility in order to bind to a target molecule, or when the amino acid residues that are adjacent to the minimal peptide ligand favourably influence the micro-environment of the binding interface.

Additional residues or linkers may be positioned at the N terminus of the peptide ligand, the C terminus of the peptide ligand, or both. For example, the sequence of an inter-repeat loop containing a peptide ligand may be [X_1-i]-[X_1-n]-[X_1-z], where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue that is also denoted by X, [X_1-n] is the peptide ligand, n is 1 to 100, [X_1-i] is a linker and i is independently any number between 1 to 10. In some embodiments, D may be preferred at the first position of the linker [X_1-i], P may be preferred at the second position of linker [X_1-i], D may be preferred at the last position of the linker [X_1-z] and/or P may be preferred at the penultimate position of linker [X_1-z]. Examples of preferred inter-repeat loop sequences may include DP-[X_1-n]-PX; DPXX-[X_1-n]-XXPX; DPXX-[X_1-n]-XPXX; DPXX-[X_1-n]-PXXX; PXXX-[X_1-i]-[X_1-n]-[X_1-i,]-XXPX, DPXX-[X_1-i]-[X_1-n]-[X_1-i]-XPXX, DPXX-[X_1-i]-[X₁-n]-[X_1-i]-PXXX, DPXX-[X_1-i]-[X_1-n]-XPXX, DPXX-[X_1-i]-[X_1-n]-XPXX, DPXX-[X_1-i]-[X_1-n]-XPXX, DPXX-[X_1-n]-[X_1-i]-XXPX, DPXX-[X_1-n]-[X_1-i]-XPXX and DPXX-[X_1-n]-[X_1-i]-PXXX.

The precise sequence of the residues or linkers used to connect a peptide ligand to an inter-repeat loop depends on the peptide ligand and may be readily determined for any peptide ligand of interest using standard techniques. For example, small, non-hydrophobic amino acids, such as glycine, may be used to provide flexibility and increased spatial sampling, for example when a peptide ligand needs to adopt a specific conformation, or proline residues may be used to increase rigidity, for example, when the peptide ligands are short.

In some preferred embodiments, an inter-repeat peptide ligand may be non-hydrophobic. For example, at least 40% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W). Alternatively, the repeat domains may be modified to accommodate a hydrophobic peptide ligand, for example by replacing aromatic residues with charged or polar residues.

A peptide ligand may be located at one or both termini of the chimeric protein.

A peptide ligand may be located in a helical region of the scaffold in the chimeric protein. A helical region or “helix” is a portion of a scaffold which assumes an α-helical structure.

The precise length of a helical peptide ligand is dependent on the length of the helical region of the scaffold. In general, the helical peptide ligand is no longer than the length of the helical region of the scaffold. However, if the helical region of the scaffold is located at one or other termini or is flanked by unstructured or loosely structured residues, then it may be possible to extend it to accommodate a longer helical peptide ligand.

A helical peptide ligand may comprise 3 to 25 amino acid residues, preferably 8 to 15 amino acids in length. In some embodiments, a helical peptide ligand may comprise 3-10 or 3-12 or 3-15 or 8-10 or 8-12 or 8-13 or 8-14 or 8-15 or 3-18 or 3-20 or 3-21 or 3-22 or 3-24 or 3- 25 amino acids. In some embodiments, a helical peptide ligand may comprise 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 amino acid residues.

In some embodiments, a peptide ligand located at the N or C terminus may comprise an α-helical structure and may comprise all or part of a half-repeat (i.e. all or part of a single α-helix) that stacks against an adjacent repeat domain. The α-helix of the terminal peptide ligand makes stabilising interactions with an adjacent repeat domain and is stable and folded. Only a few of the positions that structurally define an α-helix are required for the correct interfacial interaction with the adjacent repeat domain. The residues in some of these positions are defined (Tyr (i)-Ile (i+4)-Tyr (i+7)-Leu (i+11) for the N-terminal α-helix and Ala (i)-Leu (1+4)-Ala/Val (i+7) for the C-terminal helix), but the remaining positions of the α-helix may be modified to form a helical peptide ligand.

A helical peptide ligand may be located at the N terminus of the protein. The N terminal peptide ligand may be helical and may comprise all or part of the sequence X_n-(X)₁₅-X₁X₂XX, preferably all or part of the sequence X_n-XYXXXIXXYXXXLXX-X₁X₂XX, where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, X₁ is independently any amino acid, preferably D, and X2 is independently any amino acid, preferably P, and n is 0 or any number. In some embodiments, the Y, I, and/or L residues in the N terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).

A helical peptide ligand may be located at the C terminus of the scaffold. The C terminal peptide ligand may be helical and may comprise all or part of the sequence X_n-(X)₁₅-X₁X₂XX, preferably all or part of the sequence X₁X₂XX-XXAXXXLXX[A or V]XXXXX-X_n, where X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, X₁ is independently any amino acid, preferably D, and X2 is independently any amino acid, preferably P, and n is 0 or any number. In some embodiments, the A, L and/or V residues in the C terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).

The minimum length of the terminal peptide ligand is determined by the number of residues required to form a helix that binds to the target molecule. There is no intrinsic maximum length of the terminal peptide ligand and n may be any number.

It is within the skill in the art to graft selected residues of a peptide ligand into a helix portion of a scaffold containing a helix, and the invention contemplates this variation of grafting as an equivalent to grafting a peptide ligand itself. The residues of the peptide ligand that are in contact with the peptide ligand binding partner (the target protein) are those whose side chains are outward facing and are exposed to solvent. These residues are suitable for grafting to a helical portion of a scaffold. The residues of the scaffold helix whose side chains face inwards and pack against the rest of the scaffold should not be substituted, and this way their interactions with the rest of the scaffold are maintained. It is within the skill in the art to visualize the scaffold structure to identify which of the residues of the helix selected for grafting are facing outwards. PDB codes from any protein databank provide three dimensional co-ordinates that allow one of skill in the art to visualize the structure of the domain using programs such as PYMOL®, CHIMERA® and RASMOL®. At the same time, it is well within the skill in the art to identify residues of the helix peptide ligand that face outwards form noncovalent interactions (hydrogen bonds and/or Van der Waals and/or hydrophobic interactions) with its binding partner, using a program such as PYMOL®, CHIMERA® and RASMOL® to visualize a peptide ligand complexed with its binding partner. Helix grafting is performed by selectively replacing the outward-facing residues of the helix with corresponding outward-facing residues of the peptide ligand. The inward-facing residues of the helix are undisturbed, and hence the resultant grafted scaffold will have a grafted helix that comprises a mixture of outward facing residues derived from the helix peptide and the native inward facing residues of the helix that were undisturbed.

For instance, the following example shows a nine-residue helix peptide ligand (X1-X2-X3-X4-X5-X6-X7-X8-X9). A 3-dimensional view of the peptide ligand in complex with the target protein (using one of the above-noted programs) shows that residues X1, X2, X5, X8 and X9 (for example) of the peptide ligand interact with the target protein and thus are outward facing. Similarly, a helical portion of a given scaffold may be thirty amino acids in length (Y1-Y2-Y3- . . . -Y28-Y29-Y30). A 3-dimensional view of the scaffold shows the helical region and that residues Y3, Y4, Y6, Y7, and Y10 (for example) are inward facing and thus interact with the rest of the scaffold. One of skill in the art would recognize Y1, Y2, Y5, Y8 and Y9 as outward facing, thus identifying these residues as scaffold helical residues that may be replaced with peptide ligand outward facing residues. Therefore, peptide ligand residues X1, X2, X5, X8 and X9 are grafted to the scaffold replacing residues Y1, Y2, Y5, Y8 and Y9 with the corresponding outward facing residues peptide ligand residues X1, X2, X5, X8 and X9, thereby creating an isomorphic replacement. The resultant grafted scaffold will have a grafted helix whose sequence would include the following residues:

X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9 (Y10-30)

The resulting grafted helix preserves the native hydrogen bonding within the scaffold and at the same time preserves the noncovalent interactions required for specific binding of the peptide ligand to its target protein.

The “peptide ligand” may also contain more than one consecutive set of outward facing residues to graft into the scaffold, in which case the grafted scaffold may contain invariant scaffold residues between the grafted peptide residues (e, g “X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9”).

A helical peptide ligand may comprise all or part of the sequence C₁X₁X₂C₂X₃X₄C₃X₅X₆C₄, where X₁ to X₆ are independently any amino acid and, C₁, C₂, C₃ and C₄ are A, B, C and D, respectively.

In some embodiments, a helical peptide ligand may be non-hydrophobic. For example, at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W).

In other embodiments, a peptide ligand located at the N or C terminus may comprise a non-helical structure. For example, a peptide ligand that is an obligate N- or C-terminal domain (for example because the terminal amino or carboxylate group mediates the binding interaction) may be located at the beginning or end of the one or more repeat domains.

In some embodiments, one or more positions in a peptide ligand may be diverse or randomised. A chimeric protein comprising one or more diverse or randomised residues may form a library as described below.

In some embodiments, the N and C terminal peptide ligands may be non-hydrophobic. For example, at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W). Alternatively, the helix turn helix scaffold of the repeat domains may be modified, for example by replacing aromatic residues with charged or polar residues in order to accommodate a hydrophobic peptide ligand.

A chimeric protein as described herein may comprise peptide ligands in any arrangement or combination. For example, peptide ligands may be located at both the N and C terminus and optionally one or more inter-repeat loops of a chimeric protein; at the N terminus and optionally one or more loops of a chimeric protein; at the C terminus and optionally one or more loops of a chimeric protein; or in one or more inter-repeat loops of a chimeric protein.

The location of the peptide ligands within a chimeric protein may be determined by rational design, for example using modelling to identify the optimal arrangement for the presentation of two target molecules to each other (e.g. for substrate presentation to an E3 ubiquitin ligase); and/or by screening for example using populations of chimeric proteins with different arrangements of peptide ligands to identify the arrangement which confers the optimal interaction of target molecules.

Target Proteins and Targeting Peptide Ligands

Target proteins and peptide ligands that bind such proteins are described herein and are listed, without limitation, in the tables.

Suitable target molecules for chimeric proteins described herein include biological macromolecules, such as proteins. The target molecule may be a receptor, enzyme, antigen, oligosaccharide, oligonucleotide, integral membrane protein, transcription factor, transcriptional regulator, G protein coupled receptor (GPCR) or any other target of interest. Proteins that are difficult to target with small molecules, such as PPIs, proteins that accumulate in neurodegenerative diseases and proteins overexpressed in disease conditions, such as cancer, may be particularly suitable target molecules. Target molecules may include α-synuclein; β-amyloid; tau; superoxide dismutase; huntingtin; β-catenin; KRAS; components of super-enhancers and other types of transcriptional regulators, such as N-Myc, C-Myc, Notch, aurora A, EWS-FLI1 (Ewing's sarcoma-friend leukemia integration 1), TEL-AML1, TALI (T-cell acute lymphocytic leukemia protein 1) and Sox2 ((sex determining region Y)-box 2); tankyrases; phosphatases such as PP2A; epigenetic writers, readers and erasers, such as histone deacetylases and histone methyltransferases; BRD4 and other bromodomain proteins; and kinases, such as PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog I) and BCR (breakpoint cluster region)-ABL.

In some embodiments, a chimeric protein may neutralise a biological activity of the target molecule, for example by inhibiting or antagonising its activity or binding to another molecule or by tagging it for ubiquitination and proteasomal degradation or for degradation via autophagy. In other embodiments, a chimeric protein may activate a biological activity of the target molecule.

In some embodiments, the target molecule may be β-catenin. Suitable peptide ligands that specifically bind to β-catenin are well-known in the art and include β-catenin-peptide ligands derived from axin (e.g. GAYPEYILDIHVYRVQLEL and variants thereof), Bcl-9 (e.g. SQEQLEHRYRSLITLYDIQLML and variants thereof), TCF7L2 (e.g. QELGDNDELMHFSYESTQD and variants thereof), ICAT (e.g. YAYQRAIVEYMLRLMS and variants thereof), LRH-1 (e.g. YEQAIAAYLDALMC and variants thereof) or APC (e.g. SCSEELEALEALELDE and variants thereof).

In some embodiments, the target molecule may be KRAS. Suitable peptide ligands that specifically bind to KRAS are well-known in the art and include a KRAS-peptide ligand from SOS-1 (e.g. FEGIALTNYLKALEG and variants thereof) and KRAS-peptide ligands identified by phage display (see for example Sakamoto et al. Biochem. Biophys. Res. Comm. (2017) 484 605-611).

In some embodiments, the target molecule may be tankyrase. Suitable peptide ligands that specifically bind to tankyrase are well-known in the art and include tankyrase peptide ligands from Axin (e.g. REAGDGEE and HLQREAGDGEEFRS or variants thereof).

In some embodiments, the target molecule may be EWS-FLI1. Suitable peptide ligands that specifically bind to EWS-FLI1 are well-known in the art and include the ESAP1 peptide TMRGKKKRTRAN and variants thereof. Other suitable sequences may be identified by phage display (see for example Erkizan et al. Cell Cycle (2011) 10, 3397-408).

In some embodiments, the target molecule may be Aurora-A. Suitable peptide ligands that specifically bind to Aurora-A are well-known in the art and include Aurora-A binding sequences from TPX2, such as SYSYDAPSDFINFSS (Bayliss et al. Mol. Cell (2003) 12, 851-62) and Aurora-A binding sequences from N-myc, such as N-myc residues 19-47 or 61-89 (see for example Richards et al. PNAS (2016) 113, 13726-31).

In some embodiments, the target molecule may be N-Myc or C-Myc. Suitable peptide ligands that specifically bind to N-myc or C-myc are well-known in the art and include helical binding sequences from Aurora-A (see for example Richards et al. PNAS (2016) 113, 13726-31).

In some embodiments, the target molecule may be WDR5 (WD repeat-containing protein 5). Suitable peptide ligands that specifically bind to WDR5 are well-known in the art and include the WDR5-interacting motif (WIN) of MLL1 (mixed lineage leukemia protein 1) (see for example Song & Kingston J. Biol. Chem. (2008) 283, 35258-64; Patel et al. J. Biol. Chem. (2008) 283, 32158-61), e.g. EPPLNPHGSARAEVHLRKS and variants thereof.

In some embodiments, the target molecule may be BRD4 or a Bromodomain protein. Suitable peptide ligands that specifically bind to BRD4 are well-known in the art and include sequences derived from histone protein ligands.

In some embodiments, the target molecule may be a HDAC (histone deacetylase). Suitable peptide ligands that specifically bind to HDAC are well-known in the art and include binding sequences derived from SMRT and other proteins that recruit HDACs to specific transcriptional regulatory complexes or binding sequences derived from histone proteins (see for example Watson et al. Nat. Comm. (2016) 7, 11262; Dowling et al. Biochem. (2008) 47, 13554-63).

In some embodiments, the target molecule may be Notch. Suitable peptide ligands that specifically bind to Notch are well-known in the art and include binding sequences from the N-terminus of MAML1 (mastermind like protein 1), e.g. SAVMERLRRRIELCRRHHST and variants thereof (see for example Moellering et al. Nature (2009) 462, 182-8).

In some embodiments, the target molecule may be a Cdk (cyclin-dependent kinase). Suitable peptide ligands that specifically bind to Cdks are well-known in the art and include substrate-based peptides, for example, Cdk2 sequences derived from cyclin A, such as TYTKKQVLRMEHLVLKVLTFDL and variants thereof (see for example Gondeau et al. J. Biol. Chem. (2005) 280, 13793-800; Mendoza et al. Cancer Res. (2003) 63, 1020-4).

In some embodiments, the target molecule may be PLK1 (polo-like kinase 1). Suitable peptide ligands that specifically bind to PLK1 are well-known in the art and include optimised substrate-derived sequences that bind to the substrate-binding PBD (polo-box domain), such as MAGPMQSEPLMGAKK and variants thereof.

In some embodiments, the target molecule may be Tau. Suitable peptide ligands that specifically bind to Tau are well-known in the art and include tau-binding sequences derived from alpha- and beta-tubulin, such as KDYEEVGVDSVE and YQQYQDATADEQG and variants thereof (see for example Maccioni et al. EMBO J. (1988) 7, 1957-63; Rivas et al. PNAS (1988) 85, 6092-6).

In some embodiments, the target molecule may be BCR-ABL. Suitable peptide ligands that specifically bind to BCR-ABL are well-known in the art and include optimized substrate-derived sequences, such as EAIYAAPFAKKK and variants thereof.

In some embodiments, the target molecule may be PP2A (protein phosphatase 2A). Suitable peptide ligands that specifically bind to PP2A are well-known in the art and include sequences that bind the B56 regulatory subunit, such as LQTIQEEE and variants thereof (see for example Hetz et al. Mol. Cell (2016), 63 686-95).

some embodiments, the target molecule may be EED (Embryonic ectoderm development). Suitable peptide ligands that specifically bind to EED are well-known in the art and include helical binding sequences from co-factor EZH2 (enhancer of zeste homolog 2), such as FSSNRQKILERTEILNQEWKQRRIQPV and variants thereof (see for example Kim et al. Nat. Chem. Biol. (2013) 9, 643-50.)

In some embodiments, the target molecule may be MCL-1 (induced myeloid leukemia cell differentiation protein). Suitable peptide ligands that specifically bind to MCL-1 are well-known in the art and include sequences from BCL2, e.g. KALETLRRVGDGVQRNHETAF and variants thereof (see for example Stewart et al. Nat. Chem. Biol. (2010) 6, 595-601).

In some embodiments, the target molecule may be RAS. Suitable RAS peptide ligands are well-known in the art and include RAS-binding peptides identified by phage display, such as RRRRCPLYISYDPVCRRRR and variants thereof (see for example Sakamoto et al. BBRC (2017) 484, 605-11).

In some embodiments, the target molecule may be GSK3 (glycogen synthase kinase 3). Suitable GSK3 peptide ligands are well-known in the art and include substrate-competitive binding sequences such as KEAPPAPPQDP, LSRRPDYR, RREGGMSRPADVDG, and YRRAAVPPSPSLSRHSSPSQDEDEEE and variants thereof (see for example Ilouz et al. J. Biol. Chem. 281 (2006), 30621-30630. Plotkin et al. J. Pharmacol. Exp. Ther. (2003) 305, 974-980).

In some embodiments, the target molecule may be CtBP (C-terminal binding protein). Suitable CtBP peptide ligands are well-known in the art and include sequences identified from a cyclic peptide library screen, such as SGWTVVRMY and variants thereof (see for example Birts et al. Chem. Sci. (2013) 4, 3046-57).

Examples of suitable peptide ligands for target molecules that may be used in a chimeric protein as described herein are shown in Tables 2 and 7.

E3 Ligase Peptide Ligands

In some preferred embodiments, a chimeric protein as described herein may comprise a peptide ligand for an E3 ubiquitin ligase. Examples of suitable E3 ubiquitin ligases include MDM2, SCF^Skp2, BTB-CUL3-RBX1 APC/C, SIAH, CHIP, Cul4-DDB1, SCF-family, β-TrCP, Fbw7 and Fbx4.

E3 Ligase Peptide Ligands

Suitable peptide ligands for E3 ubiquitin ligases (degrons) are well known in the art and may be 5 to 20 amino acids. For example, a suitable peptide ligand for MDM2 may include a peptide ligand from p53 (e.g. FAAYWNLLSAYG) and or a variant thereof. A suitable peptide ligand for SCF^Skp2 may include a peptide ligand from p27 (e.g. AGSNEQEPKKRS) and variants thereof. A suitable peptide ligand for Keap1-Cul3 may include a peptide ligand from Nrf2 (e.g. DPETGEL) or a variant thereof. A suitable peptide ligand for SPOP-Cul3 may be include a peptide ligand from Puc (e.g. LACDEVTSTTSSSTA or a variant thereof. A suitable peptide ligand for APC/C may include the degrons termed ABBA (e.g. SLSSAFHVFEDGNKEN), KEN (e.g. SEDKENVPP), or DBOX (e.g. PRLPLGDVSNN) or a variant thereof. In some instances, a combination of these degrons for may be used (mimicking the bipartite or tripartite degrons found in some natural substrates). A suitable peptide ligand for SIAH may include a peptide ligand from PHYL (e.g. LRPVAMVRPTV) or a variant thereof. A suitable peptide ligand for CHIP (carboxyl terminus of Hsc70-interacting protein) may include peptide sequences such as ASRMEEVD (from Hsp90 C-terminus) and GPTIEEVD (from Hsp70 C-terminus) or a variant thereof. A suitable peptide ligand for beta-TrCP may include a degron sequence motif (including phosphomimetic amino acids), such as DDGYFD or a variant thereof. A suitable peptide ligand for Fbx4 may include sequences derived from TRF1, such as MPIFWKAHRMSKMGTG or a variant thereof (see for example Lee et al. Chembiochem (2013) 14, 445-451). A suitable peptide ligand for FBw7 may include degron sequence motifs (including phosphomimetic amino acids), such as LPSGLLEPPQD. A suitable peptide ligand for DDB1-Cul4 may include sequences derived from HBx (hepatitis B virus X protein) and similar proteins from other viruses and from DCAFs (DDB1-CUL4-associated factors) including helical motifs such as ILPKVLHKRTLGL, NFVSWHANRQLGM, NTVEYFTSQQVTG, and NITRDLIRRQIKE (see for example Li et al. Nat. Struct. Mol. Biol. (2010) 17, 105-111).

E3 Ligases and E3 Ligase Peptide Ligands

Examples of suitable peptide ligands for E3 ubiquitin ligases that may be used in a chimeric protein as described herein are shown in Table 3.

A chimeric protein comprising a peptide ligand for an E3 ubiquitin ligase may also comprise a peptide ligand for a target molecule. Without being bound to any one hypothesis, binding of the chimeric protein to both the target molecule and the E3 ubiquitin ligase may cause the target molecule to be ubiquitinated by the E3 ubiquitin ligase. Ubiquitinylated target molecules may then degraded by the proteasome. This allows the specific targeting of molecules for proteolysis by the chimeric protein. The ubiquitination and subsequent degradation of a target protein has been shown for hetero-bifunctional small molecules (PROTACs; proteolysis targeting chimeras) that bind the target protein and a ubiquitin ligase simultaneously (see for example Bondeson et al. Nat. Chem. Biol. 2015; Deshaies 2015; Lu et al. 2015).

In some embodiments, the chimeric protein may lack lysine residues, so that it avoids ubiquitination by the E3 ubiquitin ligase.

Examples of chimeric proteins that bind E3 ubiquitin ligase and a target molecule are shown in Tables 1 and 8.

A suitable chimeric protein may comprise an N terminal peptide ligand that binds a target protein, such as β catenin, and a C terminal peptide ligand that binds an E3 ubiquitin ligase. For example, the N terminal peptide ligand may be a β catenin-binding sequence derived from Bcl9 and the C terminal peptide ligand may be an Mdm2-binding sequence derived from p53. Alternatively, a chimeric protein may comprise a C terminal peptide ligand that binds a target protein, such as β catenin, and an N terminal peptide ligand that binds an E3 ubiquitin ligase (see FIG. 10A).

Another suitable chimeric protein may comprise three repeat domains, a peptide ligand located in an inter-repeat loop that binds a target protein, such as β catenin, and a C terminal peptide ligand that binds an E3 ubiquitin ligase. For example, the inter-repeat loop peptide ligand may be derived from the phosphorylated region of APC (adenomentous polyposis coli) and the C terminal peptide ligand may be an Mdm2-binding sequence derived from p53. Alternatively, the chimeric protein may comprise a peptide ligand located in an inter-repeat loop that binds an E3 ubiquitin ligase, and a C terminal peptide ligand that binds a target protein, such as β catenin (See FIG. 10B).

Another suitable chimeric protein may comprise three repeat domains, an N terminal peptide ligand that binds a target protein, such as β catenin, and a peptide ligand located in an inter-module loop that binds an E3 ubiquitin ligase. For example, the N terminal peptide ligand may be a β catenin-binding sequence derived from LRH1 (liver receptor homolog 1) and the inter-module loop peptide ligand may be a sequence derived from the Skp2-targeting region of p27. Alternatively, the chimeric protein may comprise an N terminal peptide ligand that binds an E3 ubiquitin ligase and a peptide ligand located in an inter-module loop that binds a target protein, such as β catenin (see FIG. 10C).

Another suitable chimeric protein may comprise four repeat domains, a first peptide ligand located in an inter-repeat loop that binds an E3 ubiquitin ligase and a second peptide ligand located in an inter-repeat loop that binds a target molecule. The first and second inter-repeat loops may be separate by an inter-repeat loop lacking a peptide ligand. For example, the first peptide ligand may be located in the first inter-repeat loop inter-repeat loop from the N terminus and the second peptide ligand may be located in the third inter-repeat loop from the N terminus or vice versa.

In some preferred embodiments, a chimeric protein as described herein may comprise an amino acid shown in Table 8 or a variant thereof.

In other preferred embodiments, a chimeric protein as described herein may comprise a peptide ligand that binds to a component of a target-selective autophagy pathway, such as chaperone-mediated autophagy (CMA). The chimeric protein and target molecules bound thereto are thus recognised by the autophagy pathway and the target molecules are subsequently degraded. Suitable components of the CMA pathway include heat shock cognate protein of 70 kDa (hsc70, HSPA8, Gene ID: 3312). Suitable peptide ligands are well known in the art (Dice J. F. (1990). Trends Biochem. Sci. 15, 305-309) and include Lys-Phe-Glu-Arg-Gln (KFERQ) and variants thereof, such as CMA_Q and CMA_K, as described herein. These domains have been demonstrated to be capable of targeting heterologous proteins to the autophagy pathway (Fan, X. et al; (2014) Nature Neuroscience 17, 471-480).

In addition to repeat domains and peptide ligands, a chimeric protein may further comprise one or more additional domains which confer additional functionality, such as targeting domains, intracellular transport domains, stabilising domains or oligomerisation domains. Additional domains may for example be located at the N or C terminus of the chimeric protein or in a loop between repeats.

A targeting domain may be useful in targeting the chimeric protein to a particular destination in vivo, such as a target tissue, cell, membrane or intracellular organelle. Suitable targeting domains include chimeric antigen receptors (CARs).

An intracellular transport domain may facilitate the passage of the chimeric protein through the cell membrane into cells, for example to bind intracellular target molecules. Suitable intracellular transfer domains are well known in the art (see for example Bechara et al FEBS Letters 587 1 (2013) 1693-1702) and include cell-penetrating peptides (CPPs), such as Antennapedia (43-58), Tat (48-60), Cadherin (615-632) and poly-Arg.

A stabilising domain may increase the half-life of the chimeric protein in vivo. Suitable stabilising domains are well known in the art and include Fc domains, serum albumin, unstructured peptides such as XTEN⁹⁸ or PAS⁹⁹ and polyethylene glycol (PEG).

An oligomerisation domain may facilitate the formation of multi-protein complexes, for example to increase avidity against multi-valent targets. Suitable oligomerisation domains include the ‘foldon’ domain, the natural trimerisation domain of T4 fibritin (Meier et al., J. Mol. Biol. (2004) 344(4):1051-69).

In addition to repeat domains, peptide ligands and optionally one or more additional domains, a chimeric protein may further comprise a cytotoxic or therapeutic agent and/or or detectable label.

Suitable cytotoxic agents include, for example, chemotherapeutic agents, such as methotrexate, auristatin adriamicin, doxorubicin, melphalan, mitomycin C, ozogamicin, chlorambucil, maytansine, emtansine, daunorubicin or other intercalating agents, enzymatically active toxins of bacterial, fungal, plant, or animal origin, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-amanitin, alpha-sarcin, Aleurites fordii proteins, tubulysins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, pyrrolobenzodiazepines, and the tricothecenes and fragments of any of these. Suitable cytotoxic agents may also include radioisotopes. A variety of radionuclides are available for the production of radioconjugated chimeric proteins including, but not limited to, ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re and ²¹²Bi. Conjugates of a chimeric protein and one or more small anti-cancer molecules, for example toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, may also be used.

Suitable therapeutic agents may include cytokines (e.g. IL2, IL12 and TNF), chemokines, pro-coagulant factors (e.g. tissue factor), enzymes, liposomes, and immune response factors.

A detectable label may be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers. Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. Detectable labels may be attached to chimeric proteins using conventional chemistry known in the art.

There are numerous methods by which the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents. The label can also be bound to another specific binding member that binds the chimeric protein, or to a support.

In some embodiments, a chimeric protein may be configured for display on a particle or molecular complex, such as a cell, ribosome or phage, for example for screening and selection. A suitable chimeric protein may further comprise a display moiety, such as phage coat protein, to facilitate display on a particle or molecular complex. The phage coat protein may be fused or covalently linked to the chimeric protein.

Providing a Chimeric Protein According to the Invention

Chimeric proteins as described herein may be produced by recombinant means. For example, a method of producing a chimeric protein as described herein may comprise expressing a nucleic acid encoding the chimeric protein. A nucleic acid may be expressed in a host cell and the expressed chimeric protein may then be isolated and/or purified from the cell culture.

In some embodiments, the recombinant method may comprise;

• • inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding two or more repeat domains, e.g, a TPR repeat as described herein, e.g., CTPR or RTPR2, to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand. The first nucleic acid may be inserted into an inter-repeat loop (for example, the RTPR2 scaffold contains a 20 amino acid loop and the first peptide ligand may be inserted anywhere between codons encoding two loop amino acids. Alternatively, the first nucleic acid may be inserted into the second nucleic acid at the codon encoding the N-terminus or the C-terminus of the scaffold such that the peptide is in-frame with the scaffold, thereby forming a chimeric nucleic acid encoding a chimeric protein (a grafted scaffold); and,
  • expressing the chimeric nucleic acid to produce the chimeric protein.

Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;

• • providing a nucleic acid encoding two or more repeat domains linked by inter-repeat loops, each repeat domain; and
  • incorporating into the nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising the first and second peptide ligands, wherein the first nucleotide sequence encoding the first peptide ligand is located in an inter-repeat loop or at the N or C terminus of the grafted scaffold and the second nucleotide sequence encoding the second peptide ligand is located in a different inter-repeat loop than the first peptide ligand or is located at the N or C terminus wherein the first peptide ligand is not located; and
  • expressing the nucleic acid to produce the chimeric protein.

One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;

• • providing a nucleic acid encoding two or more repeat domains linked by inter-repeat loops between the repeat domains; and
  • incorporating into the nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising the first and second peptide ligands, wherein the first and second peptide ligands are located (i) in different inter-repeat loops or (ii) the first ligand is located in an inter-repeat loop while the second peptide ligand is located at the N or C terminus of the scaffold, or (iii) the first and second peptide ligands are located at the N and C termini of the scaffold, respectively; and
  • expressing the nucleic acid to produce the protein.

An isolated nucleic acid encoding a chimeric protein as described herein is provided as an aspect of the invention. The nucleic acid may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts such as E. coli and/or in eukaryotic cells.

Many techniques and protocols that are suitable for the expression of recombinant chimeric proteins in cell culture and their subsequent isolation and purification are known in the art (see for example Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed R S Tuan (March 1997) Humana Press Inc).

A host cell comprising a nucleic acid encoding a chimeric protein as described herein or vector containing such a nucleic acid is also provided as an aspect of the invention. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of proteins in prokaryotic cells is well established in the art. A common bacterial host is E. coli. A chimeric protein may also be produced by expression in eukaryotic cells in culture. Mammalian cell lines available in the art for expression of a chimeric protein include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells (e.g. HEK293 cells), human embryonic retina cells (e.g. PerC6 cells) and many others.

The following procedures and assays may be used according to the invention.

Preparation of Grafted Scaffold Protein

Large-Scale Protein Purification (His-Tagged) from E. coli

The pRSET B (His-tag) constructs are transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies are grown in 2TY media containing ampicillin (50 micrograms/mL) at 37° C., 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures are then induced with IPTG (0.5 mM) for 16-20 h at 20° C. or 4 h at 37° C. Cells are pelleted by centrifugation at 3000 g (4° C., 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris is pelleted by centrifugation at 15,000 g at 4° C. for 45 min. Ni-NTA beads 50% bed volume (GE Healthcare) (5 mL) are washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) before the supernatant of the cell lysate is bound to them for 1 hr at 4° C. in batch. The loaded beads are washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non-specific interaction of lysate proteins with the beads. Samples are eluted using phosphate buffer with 300 mM imidazole, and purified by size-exclusion chromatography using a HiLoad 16/60 SuperdexG75 column (GE Life-Science) pre-equilibrated in phosphate buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) and proteins separated in isocratic conditions. Purity is checked on NuPage protein gel (Invitrogen), and fractions found to be over 95% pure are pooled. Purified protein is flash-frozen and stored at −80° C. until further use. Concentrations are determined by measuring absorbance at 280 nm and using a calculated extinction coefficient from ExPASy ProtParam (Gasteiger et al. 2005) for each variant. Molecular weight and purity is confirmed using mass spectrometry (MALDI).

Large-Scale Protein Purification (Heat Treatment) from E. coli

Many of the chimeric proteins described herein are thermally very stable, with melting temperatures above 80° C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65° C. for 20 min. Very few of the E. coli proteins will remain folded at such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins are removed by centrifugation, leaving 80-90% pure sample of the desired protein. Constructs that fold reversibly can be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.

This approach allows the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is potentially scalable to industrial production and bioreactors.

Small-Scale Purification of His-Tagged Proteins for Higher-Throughput Testing

Plasmids are transformed into E. coli C41 cells and plated overnight. 15 mls of 2TY medium (Roche) containing 50 micrograms/ml ampicillin is placed in each one f multiple 50 ml tubes. Several colonies are picked from the plates and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells are grown at 37° C. until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells are pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAse I treatment is used. The lysate is spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.

The supernatant is added to 100 μl bed volume of pre-washed Ni-NTA agarose beads. The subsequent affinity purification is performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used). The first ish contained 10% BugBuster® solution and 30 mM imidazole in the chosen buffer. Here we used 50 mM sodium phosphate buffer pH 6.8, 150 mM NaCl. The three successive ishes had 30 mM of imidazole in the chosen buffer. Beads are washed thoroughly to remove the detergent present in the BugBuster® solution. Protein is eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole. The combination of Bugbuster® and imidazole and the repeat washes in small bead volumes yielded >95% pure protein. Imidazole is removed using a NAP-5 disposable gel-filtration column (GE Healthcare).

Measuring Binding of Grafted Scaffold Protein to Target Protein

Competition Fluorescence Polarisation (FP) Assay

To measure the binding of a grafted scaffold to a target protein, Competition FP can be performed using 384-well black opaque optiplate microplates and a CLARIOstar microplate reader. The grafted scaffold protein is titrated into a solution containing a mixture of FITC-labelled peptide ligand and target binding partner (target protein). The prepared plates are incubated for 30 minutes at room temperature before readings are taken. The grafted scaffold is then titrated into the preformed FITC-peptide-target protein complex. A decrease in polarisation with increasing concentrations of grafted scaffold indicates displacement of FITC-peptide upon binding of the grafted scaffold to its target.

Isothermal Titration Calorimetry (ITC)

ITC can be performed using a VP-ITC instrument (Microcal). Grafted scaffolds are dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl, 0.5 mM TCEP. Dialysed target protein (200 μM) is titrated into the sample cell containing the grafted scaffold at 20 μM. Injections of target protein into the cell are initiated with a 5 μL injection, followed by 29 injections of 10 μL. The reference power is set at 15 μCal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data are fitted using the instrument software a one-site binding model.

Cell Culture and Cell Transfection

HEK293T cells are cultured in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) supplemented with 10% fetal bovine serum and penicillin/streptomycin (LifeTech) at 37° C. with 5% CO₂ air supply.

HEK293T are seeded in 6-well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.

Western Blot Assay of Target-Protein Engagement and of Target-Protein Levels

Plasmid encoding the target protein (1 μg) alone and with plasmid encoding one of various target-specific grafted scaffolds (1 μg) is transfected in HEK293T cells in 6-well plates using Lipofectamine2000. After 48 hours of transfection, the cells are lysed in 200 μL of Laemmli buffer. After sample is boiled at 95° C. for 20 min proteins are resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting is performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies. Changes in target protein levels upon co-transfection with bifunctional grafted scaffolds are evaluated by the densitometry of the bands corresponding to the target protein normalised to actin levels using ImageJ. Co-immunoprecipitation can also be used to show that the grafted scaffold binds to the target protein and/or to the desired component of the degradation machinery.

Liposomal Formulation and Cytotoxicity Assay

To make liposomal formulations of proteins (LFP), lipids (DOTAP (cationic): DOPE (neutral): DiR (aromatic)=1:1:0.1 w/w) are dissolved in chloroform, and solvent is evaporated under vacuum overnight. Resulting mixed lipid cake is hydrated with 10 mM HEPES pH 7.4, containing 27 μM protein, so that the total lipid concentration is 4 mg/ml. This mixture is vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins are stored at 4° C. until further use. To make empty liposomes (EL, empty liposomes without proteins), lipid cake is hydrated with 10 mM HEPES pH 7.4 without proteins.

An ATP assay is used to investigate whether there is any cytotoxicity associated with EL and LFP. In a typical procedure, 2×10⁵HEK 293T cells/well in 500 μL of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum are grown for 24 hours in a 24-well cell culture plate. Cells are incubated with liposome (EL/LFP)-media (DMEM without FBS) mix, having different volumes (0-60 μL) of EL and LFP, for 15 minutes at 37° C. After washing twice with 1×PBS, 500 μL of CellTiter-Glo® Reagent (Promega) is added and luminescence is measured using a microplate reader as par the manufacture's protocol. Untreated cells are used as control. Data are obtained from triplicate samples, and the standard deviations are calculated from two independent experiments.

HiBit Split-Luciferase Assay

An alternative method for measuring target protein levels is the Nano-Glo® HiBiT Lytic Detection System from Promega Corporation. It is based on the split NanoLuc assay, which consists of a large N-terminal fragment (LgBiT) and a small C-terminal region (SmBiT). Five of the SmBiT amino acids have been replaced to produce the HiBiT (VSGWRLFKKIS) fragment, which has greater affinity for the LgBiT fragment and maintains NanoLuc luciferase activity. Either the HiBiT-tagged target DNA can be transient transfected or the endogenous target can be monitored by knock-in of the HiBiT tag sequence using CRISPR/Cas9 technology. Subsequent introduction of the complementary polypeptide, LgBiT, results in spontaneous and high affinity interaction between the HiBiT tag and LgBiT to reconstitute the luminescent NanoBit® enzyme. Detection of tagged protein levels is possible from live or lysed cells.

Protein is introduced into HEK293T cells by either DNA transient transfection or encapsulation within fusogenic liposomes. HEK293T cells are seeded into either 24-well or 96-well plates After 24 hours, DNA encoding the HiBiT-tagged target protein (20 ng for 96-well plate; 100 ng for 24-well plate) is transiently transfected into cells. Chimeric protein DNA (100 ng) is either transiently transfected into cells at the same time as HiBiT-target DNA transfection or encapsulated into liposomes and introduced 24 hours into the cells after transfection. Cells are treated with chimeric protein-containing liposomes for 15 minutes before 2 hours of incubation.

Nano-Glo® HiBiT Lytic Buffer (LgBiT protein (1:100), Nano-Glo® HiBiT Lytic Substrate (1:50) 1×PBS (1:1)) is added to the cells 24 hours after transient transfection or 2 hours after liposomal treatment. The plates are shaken on an orbital shaker (1,000 rpm, 10 min) to ensure homogenous cell lysis and equilibration of LgBiT and HiBiT in the cell lysate. The luminescence measurements are performed in white Nunclon™ Delta 96-well plates at 25° C. using a CLARIOstar plate reader using a 460-480 emission filter.

Determining Properties of a Grafted Scaffold

The biophysical properties of a grafted scaffold may be assessed as follows: The molar ellipticity at 222 nm (a measure of helical structure content) is monitored as a function of increasing temperature. A decrease in the molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. This thermal unfolding experiment is used to determine the melting temperature of the scaffold and thereby to assess whether or not the grafting process has had a detrimental effect on the thermostability of the scaffold.

An alternative method to determine the thermodynamic stability of the proteins is to measure chemical-induced denaturation (either guanidine hydrochloride (GdnHCl) or urea) monitored by intrinsic protein fluorescence (tryptophan and tyrosine residues). Solutions are dispensed into Corning® 96-well, half-area, black polystyrene plates (CLS3993) with a Microlab ML510B dispenser (Hamilton) and measurements are carried out on a CLARIOstar Plate Reader (BMG Labtech). The buffer is added first into the wells, followed by 15 μl aliquots of protein stock. A stock solution of chemical denaturant (either 7 M GdnHCl or 9 M urea) is then dispensed into the wells to create a chemical-denaturant concentration gradient.

Preparation of a Helix-Grafted Scaffold that Binds to a Target Protein

First, the helix of a given protein that interacts with its target binding partner is mapped onto the heptad distribution, and the stapled side of the peptide is set so as to form the hydrophobic interface with the rest of the scaffold protein. The grafted scaffold may then be docked against the target protein using Haddock software (de Vries & Bonvin 2011; de Vries et al. 2010). Haddock is a data-driven docking algorithm that uses known information about the interaction for its calculations. The active (primary interaction residues) and the passive (5 A proximity to active) residues are extracted and inputted into the calculations. Docking is not necessary to validate helical grafted scaffold, and inspection of the structure of the helix-target protein structure and of the scaffold structure may be sufficient: The geometry of alpha-helices permits selection of amino acid positions of the scaffold that accommodate outward facing target binding residues of the peptide ligand.

Preparation of a Grafted Scaffold with a Single Binding Function Grafted onto an Inter-Repeat Loop

First, a peptide ligand that binds to a given target protein is grafted onto the scaffold in a loop. Binding of the loop-grafted scaffold may be tested using ITC. ITC is particularly useful to assess these interactions, as it can measure the stoichiometry (n) of the interaction, and thus inform as to which loops (if there is more than one loop) are more or less accessible to the target protein, and can inform as to whether a multi-loop scaffold affords multivalency. An advantage of a multivalent grafted scaffold is that one may achieve an avidity effect. This is particularly useful where a target molecule has multiple domains that can be bound by a peptide ligand. Binding of a multivalent grafted scaffold to such a target protein would produce an increased binding affinity and a decreased off rate according to the number of repeats in the grafted scaffold, thus achieving an avidity effect.

Introducing Multivalency into a Single Binding Function Scaffold

The function of a multi-valent grafted scaffold containing variable numbers of the peptide ligand binding motif that binds to a given target protein can be tested using the same assays as for the mono-valent grafted scaffold. The results are used to assess whether increased potency can be achieved by increasing the valency.

Preparing a Loop-Grafted Scaffold Using a Peptide Ligand that Binds to an E3 Ubiquitin Ligase

A peptide ligand that is known to bind the substrate recognition subunit of an E3 ligase (see Table 3 for such peptides and ligases) is inserted into the scaffold loop. Immunoprecipitation is used to confirm binding of the grafted scaffold to the E3 ligase. ITC analysis is used to assess the affinity of the interaction.

Preparation of Hetero-Bifunctional Scaffolds that Direct Target Proteins for Ubiquitination and Subsequent Degradation

A bispecific grafted scaffold is constructed using a peptide ligand specific for a target protein (see Table 2) and a peptide ligand specific for an E3 ligase.

To test whether these bispecific grafted scaffolds are capable of directing the target protein for ubiquitination and degradation, a plasmid encoding the hetero-bifunctional scaffold is transfected into HEK293T cells using Lipofectamine2000 together with HA-tagged β-catenin plasmid (using cells transfected with HA-tagged β-catenin plasmid alone as a control). After 48 hours of transfection, the cells are lysed, the sample is boiled and proteins are resolved by SDS-PAGE and immunoblotting is performed using anti-HA and anti-actin antibodies. Changes in target protein levels are evaluated by the densitometry of the bands corresponding to HA-target protein normalised to actin levels. In this way, different combinations of target protein binding peptides and E3 ligase peptide ligands can be compared for their abilities to reduce the levels of target protein.

Delivering a Grafted Scaffold Protein into Cells

A grafted scaffold protein is encapsulated within fusogenic liposomes made from cationic, neutral, and aromatic lipids, and then delivered into cells. Empty liposomes and liposomes encapsulating grafted scaffolds have been determined to be non-toxic to cells.

Libraries

Chimeric proteins as described herein may be used to produce libraries. For example, where a given chimeric protein (grafted scaffold) is demonstrated to binds bispecifically to a target protein and to an E3 ligase may be further optimized by changing amino acid residues of the grafted scaffold and selecting for stronger or weaker binders.

Chimeric proteins which are demonstrated to bind may be further engineered to improve an activity or property or introduce a new activity or property, for example a binding property such as affinity and/or specificity, an in vivo property such as solubility, plasma stability, or cell penetration, or an activity such as increased neutralization of the target molecule and/or modulation of a specific activity of the target molecule or an analytical property. Chimeric proteins may also be engineered to improve stability, solubility or expression level.

Alternatively, a library may be used to screen in order to identify and isolate chimeric proteins with specific binding activity.

A library may comprise chimeric proteins, each chimeric protein in the library comprising:

• • (i) two or more repeat domains,
  • (ii) inter-repeat loops linking the repeat domains; and
  • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
  • wherein at least one amino acid residue in the peptide ligands in the library is diverse.

The residues at one or more positions in the peptide ligand of the chimeric proteins in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.

For example, 1 to 12 positions within a helical peptide ligand at the N or C terminus of the chimeric proteins in the library may be diverse or randomised. In addition, the non-constrained X_n sequence of the peptide ligand may contain additional diversity. Alternatively or additionally, 1 to n positions within an inter-repeat peptide ligand of the chimeric proteins in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.

In some embodiments, peptide ligands may be screened individually and a chimeric protein progressively assembled from repeat domains comprising peptide ligands identified in different rounds of screening. For example, a library may comprise chimeric proteins, each chimeric protein in the library comprising:

• • (i) two or more repeat domains,
  • (ii) inter-repeat loops linking the repeat domains; and
  • (iii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,
  • the peptide ligands being located in an inter-repeat loop or at the N or C terminus of the chimeric protein.

At least one amino acid residue in the diverse peptide ligands in the library may be diverse.

A library may be produced by a method comprising:

• • (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
    • (i) two or more repeat domains,
    • (ii) inter-repeat loops linking the two or more repeat domains; and
    • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
    • wherein one or more residues of a peptide ligand in each chimeric protein is diverse in the library, and
  • (b) expressing the population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

The population of nucleic acids may be provided by a method comprising inserting a first population of nucleic acids encoding a diverse peptide ligand into a second population of nucleic acids encoding the two or more repeat domains linked by inter-repeat loops, optionally wherein the first and second nucleic acids are linked with a third population of nucleic acids encoding linkers of up to 10 amino acids.

The nucleic acids may be contained in vectors, for example expression vectors. Suitable vectors include phage-based or phagemid-based phage display vectors.

The nucleic acids may be recombinantly expressed in a cell or in solution using a cell-free in vitro translation system such as a ribosome, to generate the library. In some preferred embodiments, the library is expressed in a system in which the function of the chimeric protein enables isolation of its encoding nucleic acid. For example, the chimeric protein may be displayed on a particle or molecular complex to enable selection and/or screening. In some embodiments, the library of chimeric proteins may be displayed on beads, cell-free ribosomes, bacteriophage, prokaryotic cells or eukaryotic cells. Alternatively, the encoded chimeric protein may be presented within an emulsion where activity of the chimeric protein causes an identifiable change. Alternatively, the encoded chimeric protein may be expressed within or in proximity of a cell where activity of the chimeric protein causes a phenotypic change or changes in the expression of a reporter gene.

Preferably, the nucleic acids are expressed in a prokaryotic cell, such as E. coli. For example, the nucleic acids may be expressed in a prokaryotic cell to generate a library of recombine binding proteins that is displayed on the surface of bacteriophage. Suitable prokaryotic phage display systems are well known in the art, and are described for example in Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN: 3540413545, WO92/01047, U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, U.S. Pat. Nos. 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404. Phage display systems allow the production of large libraries, for example libraries with 10⁸ or more, 10⁹ or more, or 10¹⁰ or more members.

In other embodiments, the cell may be a eukaryotic cell, such as a yeast, insect, plant or mammalian cell.

A diverse sequence as described herein is a sequence which varies between the members of a population i.e. the sequence is different in different members of the population. A diverse sequence may be random i.e. the identity of the amino acid or nucleotide at each position in the diverse sequence may be randomly selected from the complete set of naturally occurring amino acids or nucleotides or a sub-set thereof. Diversity may be introduced into the peptide ligand using approaches known to those skilled in the art, such as oligonucleotide-directed mutagenesis²², Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein).

Diverse sequences may be contiguous or may be distributed within the peptide ligand. Suitable methods for introducing diverse sequences into peptide ligand are well-described in the art and include oligonucleotide-directed mutagenesis (see Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein). For example, diversification may be generated using oligonucleotide mixes created using partial or complete randomisation of nucleotides or created using codons mixtures, for example using trinucleotides. Alternatively, a population of diverse oligonucleotides may be synthesised using high throughput gene synthesis methods and combined to create a precisely defined and controlled population of peptide ligands. Alternatively, “doping” techniques in which the original nucleotide predominates with alternative nucleotide(s) present at lower frequency may be used.

Preferably, the library is a display library. The chimeric proteins in the library may be displayed on the surface of particles, or molecular complexes such as beads, for example, plastic or resin beads, ribosomes, cells or viruses, including replicable genetic packages, such as yeast, bacteria or bacteriophage (e.g. Fd, M13 or T7) particles, viruses, cells, including mammalian cells, or covalent, ribosomal or other in vitro display systems. Techniques for the production of display libraries, such as phage display libraries are well known in the art. Each particle or molecular complex may comprise nucleic acid that encodes the chimeric protein that is displayed by the particle.

In some preferred embodiments, the chimeric proteins in the library are displayed on the surface of a viral particle such as a bacteriophage. Each chimeric protein in the library may further comprise a phage coat protein to facilitate display. Each viral particle may comprise nucleic acid encoding the chimeric protein displayed on the particle. Suitable viral particles include bacteriophage, for example filamentous bacteriophage such as M13 and Fd.

Suitable methods for the generation and screening of phage display libraries are well known in the art. Phage display is described for example in WO92/01047 and U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404.

Libraries as described herein may be screened for chimeric proteins which display binding activity, for example binding to a target molecule. Binding may be measured directly or may be measured indirectly through agonistic or antagonistic effects resulting from binding. A method of screening may comprise;

• • (a) providing a library of chimeric proteins, each chimeric protein in the library comprising;
    • (i) two or more repeat domains,
    • (ii) inter-repeat loops linking the repeat domains; and
    • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
    • wherein one or more residues of the one or more peptide ligands are diverse in the library,
  • (b) screening the library for chimeric proteins which display a binding activity, and
  • (c) identifying one or more chimeric proteins in the library which display the binding activity.

In some embodiments, the chimeric proteins in the library may comprise one peptide ligand with at least one diverse amino acid residue. Conveniently the chimeric proteins in the library comprise two repeat domains. The library may be screened for peptide ligands that bind to a target molecule. Peptide ligands identified in this fashion can be assembled in a modular fashion to generate a chimeric protein as described herein that is multi-specific.

For example, a first library may be screened for a first peptide ligand that binds to a first target molecule and a second library may be screened for a second peptide ligand that binds to a second target molecule. The first and second peptide ligands are in different locations in the chimeric protein i.e. they are not both N terminal peptide ligands, C terminal peptide ligands or inter-repeat peptide ligands. First and second peptide ligands that bind to the first and second target molecules, respectively, are identified from the first and second libraries. The identified first and second peptide ligands may then be incorporated into a chimeric protein that binds to the first and second target molecules.

A first library may comprise chimeric proteins in the library with a first diverse peptide ligand having at least one diverse amino acid residue. A first peptide ligand that binds to a target molecule may be identified from the first library. Chimeric proteins comprising the first peptide ligand may be used to generate a second library comprising a second diverse peptide ligand having at least one diverse amino acid residue. For example, the chimeric protein from the first library may be modified by addition of a second diverse peptide ligand at the N or C terminal or by the addition of additional repeat domains comprising the second diverse peptide ligand in an inter-repeat loop. A second peptide ligand that binds to the same or a different target molecule may be identified from the second library. Chimeric proteins comprising the first and second peptide ligands may be used to generate a third library comprising a third diverse peptide ligand having at least one diverse amino acid residue. For example, the chimeric protein from the second library may be modified by addition of a third diverse peptide ligand at the N or C terminal or by the addition of additional repeat domains comprising the third diverse peptide ligand in an inter-repeat loop. A third peptide ligand that binds to the same target molecule as the first and/or second peptide ligands or a different target molecule may be identified from the third library. In this way, a chimeric protein containing multiple peptide ligands may be sequentially assembled (see FIG. 16).

The use of separate libraries for each peptide ligand allows large numbers of different variants of each peptide ligand to be screened independently and then combined. For example, a phage library of 10⁸-10¹² first peptide ligand variants may be combined with a phage library of 10⁸-10¹² second peptide ligand variants and a phage library of 10⁸-10¹² third peptide ligand variants. In some embodiments, a phage library of 10⁸-10¹² N terminal peptide ligand variants may be combined with a phage library of 10⁸-10¹² C terminal peptide ligand variants to generate a chimeric protein with N and C terminal peptide ligands.

Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cell phenotype. The one or more identified or selected chimeric proteins may be recovered and subjected to further selection and/or screening.

In other embodiments, the chimeric proteins in the library may comprise a first peptide ligand for a first target molecule, which has at least one diverse amino acid residue, and a second peptide ligand for a second target molecule, which has at least one diverse amino acid residue. The library may be screened for peptide ligands that bind to the first and second target molecules. For example, the library may be screened for chimeric proteins comprising a first peptide ligand that binds to a first target molecule and a second peptide ligand that binds to a second target molecule.

Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cell phenotype. The one or more identified or selected chimeric protein may be recovered and subjected to further selection and/or screening.

Chimeric proteins as described herein may be used to produce libraries comprising different combinations of peptide ligands grafted into an scaffold. The combinations of ligands may comprise first peptide ligands that bind to a members of a protein degradation pathway, such as an E3 ubiquitin ligase, and second peptide ligands that bind to a target molecule. A library may be screened in order to identify and isolate chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule, (ii) causing degradation of the target molecule in a cell through the protein degradation pathway.

A library may comprise chimeric proteins, each chimeric protein in the library comprising:

(i) a scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,

wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, the chimeric proteins in the library comprising different combinations of the first and second peptide ligands.

Suitable chimeric proteins, target molecules and members of protein degradation pathways and examples of peptide ligands thereto are described elsewhere herein.

Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins may comprise:

(i) a scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands.

Different chimeric proteins in the library may comprise a peptide ligand for a different E3 ubiquitin ligase. For example, the chimeric proteins in the library may comprise peptide ligands for a panel of E3 ubiquitin ligases, each chimeric protein in the library comprising a peptide ligand for one of the E3 ubiquitin ligases in the panel.

Numerous E3 ubiquitin ligases are known in the art. A suitable panel of E3 ubiquitin ligases may for example, comprise two, three, four, five or more of Mdm2, SCF(Skp2), Cul3-Keap1, Cul3-SPOP, APC/C, SIAH, SCF^Fbw7, SCF^Fbw8, Cul4-DDB1-Cdt2, DDB1-Cul4, DDB1-Cul5, SOCS box-Cul5-SPSB2, SOCS box-Cul5-SPSB4, CHIP, CRL4(COP1/DET), UBR5, CRL2(KLHDC2), GID4, TRIM21, Nedd4, Elongin C and β-TRP. Examples of peptide ligands for E3 ubiquitin ligases are shown in Table 3.

The target molecule may be a target molecule as described above, for example, β-catenin, KRAS, or myc. The chimeric proteins in the library may comprise different peptide ligands for the target molecule i.e. different chimeric proteins in the library may comprise different peptide ligands for the same target molecule. Each chimeric protein in the library may comprise a different peptide ligand for the target molecule. Examples of peptide ligands target molecules are shown in Table 3. For example, the target molecule may be β-catenin, KRAS, or myc and the chimeric proteins in the library may comprise different peptide ligands for β-catenin, KRAS, or myc, respectively. Examples of different peptide ligands for β-catenin, KRAS, and myc are shown in Table 3.

A method of screening a library of chimeric proteins may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a scaffold;

(ii) a first peptide ligand for a member of a protein degradation pathway and

(iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,

wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,

(c) identifying one or more chimeric proteins in the library which display the activity.

In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins may comprise;

(a) providing a library of chimeric proteins, each chimeric protein in the library comprising:

(i) a scaffold;

(ii) a first peptide ligand for an E3 ubiquitin ligase and

(iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,

wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands,

(b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,

(c) identifying one or more chimeric proteins in the library which display the activity.

A method may further comprise identifying one or more combinations of first and second peptide ligands in chimeric proteins in the library which display the activity.

Determination of Binding of a Chimeric Protein

Binding of a chimeric protein may be determined by any suitable technique, described below and in the examples herein.

Suitable methods for determining binding of a chimeric protein to a target molecule are well known in the art and include ELISA, bead-based binding assays (e.g. using streptavidin-coated beads in conjunction with biotinylated target molecules, surface plasmon resonance, flow cytometry, Western blotting, immunocytochemistry, immunoprecipitation, and affinity chromatography. Alternatively, biochemical or cell-based assays, such as fluorescence-based or luminescence-based reporter assays may be employed. For example, Isothermal Titration calorimetry, Cell transfection followed by assaying for expressed chimeric protein, Liposomal formulation and cytotoxicity assays, a dual-Luciferase Reporter Assay System such as TOPFLASH®, and a competition fluorescence polarisation (FP) assay to measure the binding of a chimeric protein to its targets.

In some embodiments, binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme.

Where a library is in use, the library may be contacted with the target molecule under binding conditions for a time period sufficient for the target molecule to interact with the library and form a binding reaction complex with a least one member thereof. Binding conditions are those conditions compatible with the known natural binding function of the target molecule. Those compatible conditions are buffer, pH and temperature conditions that maintain the biological activity of the target molecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction. Typically, those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the target molecule of interest. The library may be contacted with the target molecule in the form of a heterogeneous or homogeneous admixture. Thus, the members of the library can be in the solid phase with the target molecule present in the liquid phase. Alternatively, the target molecule can be in the solid phase with the members of the library present in the liquid phase. Still further, both the library members and the target molecule can be in the liquid phase.

Multiple rounds of panning may be performed in order to identify chimeric proteins which display the binding activity. For example, a population of chimeric proteins enriched for the binding activity may be recovered or isolated from the library and subjected to one or more further rounds of screening for the binding activity to produce one or further enriched populations. Chimeric proteins which display binding activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.

In some embodiments, binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme. For example, the library may be screened by expressing the library in reporter cells and identifying one or more reporter cells with altered gene expression or phenotype. Suitable functional screening techniques for screening recombinant populations of chimeric proteins are well-known in the art.

Further rounds of screening may be employed to identify chimeric proteins which display the improved property or activity. For example, a population of chimeric proteins enriched for binding to the target molecule may be recovered or isolated from the library and subjected to one or more further rounds of screening for the improved or new property or activity to produce one or further enriched populations. Optionally, this may be repeated one or more times. Chimeric proteins which display the improved property or activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.

A chimeric protein as described herein may be encapsulated in a liposome, for example for delivery into a cell. Preferred liposomes include fusogenic liposomes. Suitable fusogenic liposomes may comprise a cationic lipid, such as 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP), and a neutral lipid, such as dioleoylphosphatidylethanolamine (DOPE) for example in a 1:1 (w/w) ratio. Optionally, a liposome may further comprise an aromatic lipid, such as DiO (3, 3′-dioctadecyloxacarbocyanine perchlorate), DiR (1,1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanine iodide), N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (BODIPY FL-DHPE), and 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diazas-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-C12HPC) for example in a 0.1:1:1 (w/w) ratio relative to the neutral and cationic lipid. Suitable techniques for the encapsulation of proteins in liposomes and their delivery into cells are established in the art (see for example, Kube et al Langmuir (2017) 33 1051-1059; Kolas̆inac et al (2018) Int. J. Mol. Sci. 19 346).

A method described herein may comprise admixing a chimeric protein or encoding nucleic acid as described herein with a solution of lipids, for example in an organic solvent, such as chloroform, and evaporating the solvent to produce liposomes encapsulating the chimeric protein. Liposome encapsulations comprising a chimeric protein as described herein are provided as an aspect of the invention.

A chimeric protein or encoding nucleic acid as described herein may be admixed with a pharmaceutically acceptable excipient. A pharmaceutical composition comprising a chimeric protein or nucleic acid as described herein and a pharmaceutically acceptable excipient is provided as an aspect of the invention.

The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

Pharmaceutical Compositions and Formulations

The pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the chimeric protein into association with a carrier which may constitute one or more accessory ingredients. In general, pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Pharmaceutical compositions may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

Dosage and Mode of Administration

A chimeric protein, encoding nucleic acid or pharmaceutical composition comprising the chimeric protein or encoding nucleic acid may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.

Pharmaceutical compositions suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

Pharmaceutical compositions suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to cells, tissue or organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example, from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

It will be appreciated that appropriate dosages of the chimeric protein, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of diagnostic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the imaging agent, the amount of contrast required, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of imaging agent and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the imaging agent at a site, such as a tumour, a tissue of interest or the whole body, which allow for imaging without causing substantial harmful or deleterious side-effects.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.

Chimeric proteins described herein may be used in methods of diagnosis or treatment in human or animal subjects, e.g. human. Chimeric proteins for a target molecule may be used to treat disorders associated with the target molecule.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

Experiments

1. Methods

1.1 Large-Scale Protein Purification (His-Tagged) from E. coli

The pRSET B (His-tag) constructs were transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies were grown in 2TY media containing ampicillin (50 micrograms/mL) at 37° C., 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures were then induced with IPTG (0.5 mM) for 16-20 h at 20° C. or 4 h at 37° C. Cells were pelleted by centrifugation at 3000 g (4° C., 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris was pelleted by centrifugation at 15,000 g at 4° C. for 45 min. Ni-NTA beads 50% bed volume (GE Healthcare) (5 mL) were washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) before the supernatant of the cell lysate was bound to them for 1 hr at 4° C. in batch. The loaded beads were washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non-specific interaction of lysate proteins with the beads. Samples were eluted using phosphate buffer with 300 mM imidazole, and purified by size-exclusion chromatography using a HiLoad 16/60 SuperdexG75 column (GE Life-Science) pre-equilibrated in phosphate buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) and proteins separated in isocratic conditions. Purity was checked on NuPage protein gel (Invitrogen), and fractions found to be over 95% pure were pooled. Purified protein was flash-frozen and stored at −80° C. until further use. Concentrations were determined by measuring absorbance at 280 nm and using a calculated extinction coefficient from ExPASy ProtParam (Gasteiger et al. 2005) for each variant. Molecular weight and purity was confirmed using mass spectrometry (MALDI.

1.2 Large-Scale Protein Purification (Heat Treatment) from E. coli

All chimeric proteins described herein are thermally very stable, with melting temperatures above 80° C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65° C. for 20 min. Very few of the E. coli proteins survive such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins were removed by centrifugation, leaving 80-90% pure sample of the desired protein. All our constructs folded reversibly, and therefore could be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.

This approach allowed the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is scalable to industrial production and bioreactors.

1.3 Small-Scale Purification of His-Tagged Proteins for Higher-Throughput Testing

Plasmids were transformed into E. coli C41 cells and plated overnight. 15 mls of 2TY medium (Roche) containing 50 micrograms/ml ampicillin was placed in multiple 50 ml tubes. Several colonies were picked and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells were grown at 37° C. until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells were pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAse I treatment was used. The lysate was spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.

The supernatant was added to 100 μl bed volume of pre-washed Ni-NTA agarose beads. The subsequent affinity purification was performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used). The first wash contained 10% BugBuster® solution and 30 mM imidazole in the chosen buffer. Here we used 50 mM sodium phosphate buffer pH 6.8, 150 mM NaCl. The three successive washes had 30 mM of imidazole in the chosen buffer. Beads were washed thoroughly to remove the detergent present in the BugBuster® solution. Protein was eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole. The combination of Bugbuster® and imidazole and the repeat washes in small bead volumes yielded >95% pure protein. Imidazole was removed using a NAP-5 disposable gel-filtration column (GE Healthcare).

1.4 Competition Fluorescence Polarization (FP)

To assay the binding of the designed SOS-TPR protein to KRAS, Competition FP was performed using purified KRAS Q61H mutant and (2′-(or -3′)-O—(N-Methylanthraniloyl) Guanosine 5′-Triphosphate, a fluorescent version of GTP, also known as mant-GTP. SOS-TPR was titrated using a 2-fold serial dilution against a 1:1 complex of KRAS Q61H and mant-GTP (1 μM) in a black 96-well plate (CLS3993 SIGMA). Plates were prepared under reduced light conditions and incubated at room temperature. Readings were taken on the CLARIOstar microplate reader, using an excitation filter at 360 nm and emission filter at 440 nm.

1.5 Isothermal Titration Calorimetry (ITC)

ITC was performed at 25° C. using a VP-ITC (Microcal). 1TBP-CTPR2, 2TBP-CTPR4, 3TBP-CTPR6 and TNKS2 ARC4 were dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl, 0.5 mM TCEP. Dialysed TNKS2 ARC4 (200 μM) was titrated into the sample cell containing 1TBP-CTPR2 at 20 μM. Similar experiments were performed for 2TBP-CTPR4 and 3TBP-CTPR6. Injections of TNKS2 ARC4 into the cell were initiated with a 5 μL injection, followed by 29 injections of 10 μL. The reference power was set at 15 μCal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data were fitted using the instrument software a one-site binding model.

1. 6 Cell Culture

HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) supplemented with 10% fetal bovine serum and penicillin/streptomycin (LifeTech) at 37° C. with 5% CO₂ air supply.

1. 7 Cell Transfection

HEK293T were seeded in 6-well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.

1. 8 β-Catenin Levels Western Blot Assay

HA-β-catenin (1 μg) alone and with various PROTACs (1 μg) was transfected in HEK293T cells in 6-well plates using Lipofectamine2000. After 48 hours of transfection, the cells were lysed in 200 μL of Laemmli buffer. After sample was boiled at 95° C. for 20 min proteins were resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting was performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies. Changes in β-catenin levels were evaluated by the densitometry of the bands corresponding to HA-β-catenin normalised to actin levels using ImageJ.

1.9 Liposomal Formulation and Cytotoxicity Assay

To make liposomal formulations of proteins (LFP), lipids (DOTAP (cationic): DOPE (neutral): DiR (aromatic)=1:1:0.1 w/w) were dissolved in chloroform, and solvent was evaporated under vacuum overnight. Resulting mixed lipid cake was hydrated with 10 mM HEPES pH 7.4, containing 27 μM protein, so that the total lipid concentration is 4 mg/ml. This mixture was vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins were stored at 4° C. until further use. To make empty liposomes (EL, empty liposomes without proteins), lipid cake was hydrated with 10 mM HEPES pH 7.4 without proteins.

An ATP assay was used to investigate whether there is any cytotoxicity associated with EL and LFP. In a typical procedure, 2×10⁵HEK 293T cells/well in 500 μL of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum were grown for 24 hours in a 24-well cell culture plate. Cells were incubated with liposome (EL/LFP)-media (DMEM without FBS) mix, having different volumes (0-60 μL) of EL and LFP, for 15 minutes at 37° C. After washing twice with 1×PBS, 500 μL of CellTiter-Glo® Reagent (Promega) was added and luminescence was measured using a microplate reader as par the manufacture's protocol. Untreated cells were used as control. Data were obtained from triplicate samples, and the standard deviations were calculated from two independent experiments.

1.10 TOPFLASH Assay

The Wnt pathway was activated by treating HEK293T cells with Wnt-conditioned media obtained from L-cells expressing Wnt3A for 8 days. To perform the assay, 10⁵ HEK293T cells/well were seeded on a 24-well plate Nunclon Delta Surface plate (NUNC) and incubated overnight at 37° C., 5% CO2. The following day, cells were transfected with 100 ng of TOPflash TCF7L2-firefly luciferase plasmid, 10 ng of CMV-Renilla plasmid (as internal control) and 100 ng of the corresponding TPR construct. Plasmids were mixed with 0.5 μL of Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (invitrogen). Transfected cells were allowed to recover for 8 h, then they were treated with Wnt-conditioned media (1:2 final concentration) for a further 16 h. The TOPflash assay was performed using the Dual-Luciferase Reporter Assay System (Promega) (Korinek et al., 1997 Science 275(5307):1784-7) following the manufacturer's instructions. The activities of firefly and Renilla luciferases were measured sequentially from a single sample, using the CLARIOstar plate reader. Relative luciferase values were obtained from triplicate samples dividing the firefly luminescence activity by the CMV-induced Renilla activity, and standard deviation was calculated.

1.11 TOPFLASH Assay Using Liposome Encapsulation to Deliver Designed TPR Proteins into the Cell

10⁵HEK 293T cells in 500 μL of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum were grown overnight in each well of a 24-well cell culture plate. For TOPFLASH reporter assays, 100 ng/well of TOPFLASH plasmid and 10 ng/well of CMV-Renilla plasmid (as internal control) were used to transfect cells in 24-well plates. Cells were transfected with the Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (Invitrogen). Transfected cells were allowed to recover for 8 hours, and Wnt signalling was activated by addition of Wnt3A-conditioned media obtained from L-cells. 16 hours post Wnt pathway activation, proteins were delivered into the cells by liposomal treatment. Cells were incubated with liposome (LFP)-media (DMEM without FBS) mix for 15 minutes at 37° C. followed by one PBS wash. Wnt3A conditioned media was replaced and cells were incubated for variable time durations (2-8 hours). Following incubation, TOPFLASH assays were performed using the Dual-Luciferase Reporter Assay System (Promega) (Korinek et al., 1997) following the manufacturer's instructions. Relative luciferase values were obtained from triplicate samples (from two independent experiments) by dividing the firefly luciferase values (from TOPFLASH) by the Renilla luciferase values (from CMV renilla), and standard deviations were calculated.

1.12. Competition Fluorescence Polarisation (FP) Assay to Measure the Binding of Designed Nrf-TPR Proteins to Keap1

To measure the binding of the designed Nrf-TPR proteins to Keap1, Competition FP was performed using 384-well black opaque optiplate microplates and a CLARIOstar microplate reader. Nrf-TPR proteins were titrated into a solution containing a mixture of FITC-labelled Nrf2 peptide and Keap1 protein. The prepared plates were incubated for 30 minutes at room temperature before readings were taken.

2. Results

Tetratricopeptide repeat (TPR) is a 34-residue motif that can be repeated in tandem to generate modular proteins. TPRs are used here as an example of helix-turn-helix tandem-repeats arrays, but any tandem repeat array may be used.

RTPR proteins comprising TPRs were derived from the consensus TPR sequence (CTPR). Two repeats were found to be sufficient to generate a highly stable mini-protein of 68 amino acids (RTPR2). The biophysical properties of two types of engineering strategy; loop insertions and terminal helix grafting, were assessed. The molar ellipticity at 222 nm (a measure of helical secondary structure content) of three different RTPR modules was monitored as a function of increasing temperature. A decrease in the absolute molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. Even at the highest temperature recorded (85° C.), the RTPR2 protein without insertion was not fully denatured (FIG. 1). RTPR2 with a 20-residue unstructured loop between the two repeats showed a small shift to a lower melting temperature (FIG. 1), but the protein remains fully folded up to 55° C. This is well above physiologically relevant temperatures. RTPR2 with an additional N-terminal helix showed an increase in absolute molar ellipticity, indicating that the additional helical domain is folded. Moreover, unlike the loop insertion, the helix domain was capable of stabilising the RTPR2 module, shifting the transition midpoint to above 90° C. (FIG. 1). These results showed that the two engineering strategies generated folded and stable modular mini-proteins capable of withstanding high temperatures.

A key feature of the TPR scaffold was its modular nature. This modularity allowed display any number of binding modules in tandem to obtain bi- and multi-valent and multi-functional molecules against one, two or more targets. The stability of these proteins was shown to be modular. The stabilities of proteins comprising TBP-CTPR2 (a two-repeat CTPR with a loop insertion that binds to the protein tankyrase (Guettler et al. 2011)) repeated in tandem were measured. The TBP-CTPR2-containing proteins had two, four, six, and eight repeats, and they displayed one, two, three and four binding loops, respectively. The helical content of the proteins, monitored by molar ellipticity at 222 nm, was found to increase in proportion to the number of repeats, as did the stability, indicating that they were behaving like classic helical repeat proteins (FIG. 2). These results demonstrate that bi- or multi-functional chimeric proteins have a high thermostability.

2.1. Demonstration of Proteins with a Single Binding Function Grafted onto an Alpha-Helix

2.1.1 SOS1-TPR, a Helix-Grafted Binding Module Designed to Bind to Oncoprotein KRAS

First, we mapped the helix of SOS1 that interacts with KRAS (Margarit et al. 2003 Cell 112 5 685-695) onto the heptad distribution. We matched the heptad positions with the stapled SOS1 helical peptide produced by Leshchiner et al. (PNAS 2015 112 (6) 1761-1′766) and set the stapled side of the peptide to form the hydrophobic interface with the rest of the TPR protein (FIG. 3A). The length of the helix is important. An N-terminal solvating CTPR helix ends in the sequence DPNN, which forms a short loop that leads into the next repeat. CTPR-mediated “stapling” (constraining) of binding helices therefore occurred through residues Tyr (i)-Ile (1+4)-Tyr (i+7)-Leu (i+11), fully stapling a 15-residue helix.

We created a hydrophobic interface between the grafted helix and the adjacent repeat and allowed the formation of the DPNN loop at the C-terminal end of the grafted helix. We then grafted the final sequence onto the crystal structure of a CTPR B helix for further validation of the interaction. Our designed KRAS-binding protein, SOS1-TPR, was docked against KRAS using the Haddock software (de Vries & Bonvin 2011; de Vries et al. 2010). Haddock is a data-driven docking algorithm that uses known information about the interaction for its calculations. The crystal structure of SOS1-KRAS (PDB: 1NVU) (Margarit et al. 2003) was originally used to design the stapled peptide. The active (primary interaction residues) and the passive (5 A proximity to active) residues were extracted and inputted into the calculations.

Docking is not necessary to validate helical grafted scaffold. The geometry of α-helices permits selection of amino acid positions of the scaffold that accommodate outward facing target binding residues of the peptide ligand. TPR repeat scaffolds are exceptional for display of binding helices, as they grow linearly in the opposite direction of the helix, thereby avoiding steric clashes with the target protein.

KRAS binding of the grafted scaffold can be assessed using the change in fluorescence polarisation of mant-GTP (2′-/3′-O—(N′-Methylanthraniloyl) guanosine-5′-O-triphosphate), a fluorescent analog of GTP (FIG. 3B). The fluorescence of mant-GTP is dependent on the hydrophobicity of its environment (excitation at 360 nm, emission at 440 nm). An increase in fluorescence intensity and fluorescence polarization was observed previously upon binding to KRAS (Leshchiner et al. 2015). SOS-TPR2 was then titrated into the preformed mant-GTP-KRAS complex. There was a clear decrease in polarisation with increasing concentrations of SOS-TPR2, indicating displacement of mant-GTP upon binding of SOS-TRP2 to KRAS (FIG. 3B). Fitting the data gave an EC50 of 3.4 μM. In contrast, a blank protein, CTPR3, had no effect on the fluorescence polarisation.

2.1.2 p53-TPR, a Helix-Grafted Binding Module Designed to Bind to Mdm2

Many degrons (region within the substrate that is recognized by the E3 ubiquitin ligase) are unstructured. However, p53 binds to the Mdm2 E3 through an alpha helix (FIG. 4A). Stapled versions of the p53 helix, as well as circular peptides and grafted coiled coils, have been developed by many groups, and the sequences have been optimised to give nanomolar affinities in some cases (see for example, Ji et al. 2013; Lee et al. 2014; Kritzer et al. 2006). The p53 helix has a favourable geometry to be grafted onto the C-terminal solvating helix of the CTPR scaffold, and moreover the two helices have 30% sequence identity.

Proof of binding of p53-CTPR2 to Mdm2 (N-terminal domain) was obtained using isothermal titration calorimetry (ITC). Mdm2 was titrated into a solution containing 10 μM of p53-TPR2. ITC measures the heat released upon binding. A high-affinity interaction was observed with a dissociation constant of approximately 50 nM (FIG. 4B).

2.2. Demonstration of Proteins with a Single Binding Function Grafted onto an Inter-Repeat Loop

2.2.1 TPB2-TPR, a Loop Module Designed to Bind to Oncoprotein Tankyrase

First, we introduced the SLiM “3BP2”, a sequence that binds to the substrate-binding ankyrin-repeat clusters (ARC) of the protein tankyrase, a multi-domain poly ADP-ribose polymerase that is upregulated in many cancers (Guettler et al. 2011) onto the CTPR scaffold. Grafting SLiMs in folded domains led to an increase of proteolysis resistance; showing the potential to expand the interaction surface through further rational engineering, in silico methods and/or directed evolution; controlled geometric arrangement; and bi- or multivalency of interactions.

We tested the binding of 1TBP-CTPR2, 2TBP-CTPR4 and 3TBP-CTPR6 to the ARC4 domain of tankyrase using ITC (Figure. 5A). This technique is particularly useful for these interactions, as it can measure the stoichiometry (n) of the interaction. We showed that n increased with the number of binding loops, meaning that there were as many tankyrase molecules bound to one TBP-CTPR as loops in the protein. Thus, all loops are accessible to the binding partner. Moreover, the binding affinity increases and the off rate decreases with the number of repeats indicative of an avidity effect. This type of multivalent molecule would be particularly useful for full-length tankyrase, as it has four ARC domains capable of binding the 3BP2 peptide.

Multivalency in this system was increased further via oligomerisation of the binding modules by fusing them to the foldon domain of T4 fibritin (FIG. 5B). This trimerisation domain comprises of a C-terminal helix, such as that of p53-CTPR, ending with the foldon domain, a short β-sheet peptide capable of homo-trimerising. The foldon domain has been shown to be highly stable and independently folded (Boudko et al 2002; Meier et al. 2004). In this way, multiple binding modules can be arranged with specified geometries to inhibit complex multivalent molecules that cannot be targeted with monovalent interactions due to their natural tendency to interact with other multivalent networks with high avidity.

2.2.2 Effect of Introducing Multivalency into a Single Binding Function TPR

We tested the function of multi-valent CTPR proteins containing variable numbers of the “3BP2” motif that binds to the protein tankyrase. (1TBP-CTPR2, 2TBP-CTPR4 and 3TBP-CTPR6 etc.). Multi-valency was increased further via oligomerisation of the TPRs by fusing them to the foldon domain of T4 fibritin (1TBP-CTPR2-Foldon, 2TBP-CTPR4-Foldon etc.). Tankyrase is upregulated in many cancers and exerts its effect by downregulating beta-catenin. Therefore, the inhibitory effect of the TBP-grafted TPRs was assayed using a beta-catenin reporter gene assay (TOPFLASH assay). Increasing the number of functional units increased the inhibitory effect of the proteins, as mentioned using a Wnt signalling assay (FIG. 17).

2.2.3 Skp2-RTPR, a Loop Module Designed to Bind to E3 Ubiquitin Ligase SCF^Skp2

Skp2 is the substrate recognition subunit of the SCF^Skp2ubiquitin ligase. The Skp2-binding sequence that we inserted into the RTPR loop was based on the previously published degron peptide sequence derived from the substrate p27 that binds to Skp2 in complex with Cks1 (an accessory protein) (Hao et al. 2005). We used only 10 residues of this peptide. Although ideally the Skp2-binding sequence would include a phospho-threonine (as this residues makes some key contacts with Skp2 and Cks1), we instead explored whether we could replace the phospho-threonine with a phosphomimetic (glutamate) without affecting binding affinity. We found using co-immunoprecipitation that the resulting p27-TPR protein was able to bind to Skp2 (FIG. 6A) and that it was able to inhibit the ubiquitination of p27 in vitro with a high efficiency indicating a dissociation constant of the order of 30 nM (FIG. 6B). As the peptide adopts a turn-like conformation in its Skp2/Cks1-bound state, constraining it within the RTPR scaffold leads to a large enhancement in binding affinity that outweighs any loss in affinity arising from replacing the phosphothreonine with a phosphomimetic.

2.2.4 Nrf-TPR, a Loop Module Designed to Bind to E3 Ubiquitin Ligase Keap1-Cul3

Keap1 is the substrate recognition subunit of the Keap1-Cul3 ubiquitin ligase. A Keap1-binding sequence that we inserted into the CTPR loop was based on the previously published degron peptide sequence derived from the Keap1 substrate Nrf2. We found using co-immunoprecipitation that the resulting Nrf-TPR protein was able to bind to Keap1 (FIG. 7A) and that the interaction had a high affinity in the low nanomolar range as measured by ITC analysis (FIG. 7B).

2.3. Engineering the RTPR Scaffold for Delivery into the Cell

Combining our RTPR sequences with an alternative consensus TPR sequence (Parmeggiani et al. 2015) we included additional solvent-exposed Arginine residues, as such ‘resurfacing’ or ‘supercharging’ has been shown previously to facilitate the entry of proteins into cells (Chapman & McNaughton 2016; Thompson et al. 2012). FIG. 8 shows that this approach was successful in delivering a fluorescent-labelled resurfaced TBP-RTPR2 protein into two different cell lines.

2.4. Design of Hetero-Bifunctional TPRs to Direct Proteins for Ubiquitination and Subsequent Degradation

The Wnt/β-catenin signalling pathway is deregulated in many cancers and in neurodegenerative diseases, and therefore β-catenin is an important drug target. There are a large number of known binding sequences (both helical and non-helical) for β-catenin that appear suitable for grafting onto the TPR scaffold, and therefore we chose it as the first target for our design of hetero-bifunctional TPRs to induce protein degradation. We selected Mdm2 and SCF^Skp2 to test as E3 ubiquitin ligases, as we had successfully generated single-function TPRs to bind to them (FIGS. 4 and 6). We generated structural models of some of the hetero-bifunctional molecules and used these as a crude assessment of whether the resulting presentation of β-catenin to the E3 looked appropriate. We then generated a small library of plasmids encoding proteins comprising three or four TPRs functionalized with different combinations of the β-catenin-binding module and the two E3 ligase-binding modules.

We transfected HA-tagged β-catenin plasmid alone or HA-tagged β-catenin plasmid together with one of the various hetero-bifunctional TPR plasmids in HEK293T cells using Lipofectamine2000. After 48 hours of transfection, the cells were lysed, the sample was boiled and proteins were resolved by SDS-PAGE and immunoblotting was performed using anti-HA and anti-actin antibodies. Changes in β-catenin levels were evaluated by the densitometry of the bands corresponding to HA-β-catenin normalised to actin levels (FIG. 9). The results show that a number of the hetero-bifunctional molecules are capable of reducing β-catenin levels by up to 70%. In contrast, neither a blank TPR nor single-function TPRs have any effect on β-catenin levels.

A range of different factors contribute to efficient ubiquitination and target degradation by these hetero-bifunctional molecules, hence the power of screening different combinations of single-function modules and potentially also different lengths of intervening blank modules.

2.5 Using a Delivery Vehicle to Introduce the Modular TPR Proteins into Cells

We encapsulated the designed TPR proteins within fusogenic liposomes made from cationic, neutral, and aromatic lipids, and we showed that they were thereby delivered into cells (FIGS. 18 and 19). Empty liposomes and liposomes encapsulating TPR proteins are not toxic to the cell (FIG. 20).

2.6 Further Examples of Hetero-Bifunctional TPRs to Direct Proteins for Ubiquitination and Subsequent Degradation

Hetero-bifunctional TPR proteins were designed to target either tankyrase (FIG. 21), beta-catenin (FIG. 22) or KRAS (FIG. 23) for ubiquitination and degradation. TPR proteins targeting tankyrase or beta-catenin were delivered into cells using liposome encapsulation, and the effect on Wnt signalling was assayed using a TOPFLASH assay. The results show that the designed hetero-bifunctional TPR proteins are able to inhibit Wnt signalling. For KRAS, we transfected KRAS plasmid alone or KRAS plasmid together with one of the TPR plasmids in HEK293T cells using Lipofectamine2000. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. The results show that the designed hetero-bifunctional TPR is capable of reducing KRAS levels.

2.7 Hetero-Bifunctional TPRs to Direct KRAS for Degradation Via Chaperone-Mediated Autophagy (CMA)

Hetero-bifunctional TPR proteins were designed to target endogenous KRAS for degradation via CMA (FIG. 24). TPR constructs or empty vector (light grey) were transiently transfected into either HEK293T or DLD1 (colorectal cancer cell line) using Lipofectamine2000. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. The designed hetero-bifunctional TPRs that resulted in reduction of KRAS levels compared to the empty vector control are shown in white.

2.8 Variations in the Linker Sequence Connecting a Peptide Ligand to an Inter-Repeat Loop

The linker sequence connecting a peptide ligand to an inter-repeat loop was varied in order to optimise the binding affinity for the target for Nrf-TPR, a TPR protein designed to bind to the protein Keap1 (see FIG. 7). Glycine residues were introduced into the linker to provide flexibility and increased spatial sampling. The introduction of this more flexible linker sequence was found to increase the binding affinity of the Nrf-TPR protein (labelled ‘Flexible’) when compared with the consensus-like linker sequence altering the charge content of the linker sequence (‘labelled ‘Charged’) and altering the conformational properties (based on the predictions of the program CIDER (Holehouse et al. Biophys. J. 112, 16-21 (2017)) of the loop by changing the amino acid composition of the linker sequence (labelled ‘CIDER-optimised’) also affected the Keap1-binding affinity (FIG. 25).

TABLE 1
	Degron		β-catenin-
	sequence	Targeted	binding
Targeted	derived	protein for	sequence
Ubiquitin Ligase	from	Degradation	derived from:	Scaffold
Mdm2	p53	β-catenin	axin	RTPR
Mdm2	p53	β-catenin	Bcl-9	RTPR
Mdm2	p53	β-catenin	TCF-4	RTPR
Mdm2	p53	β-catenin	ICAT	RTPR
Mdm2	p53	β-catenin	LRH-1	RTPR
Mdm2	p53	β-catenin	APC	RTPR
SCFskp2	p27	β-catenin	axin	RTPR
SCFskp2	p27	β-catenin	Bcl-9	RTPR
SCFskp2	p27	β-catenin	TCF-4	RTPR
SCFskp2	p27	β-catenin	ICAT	RTPR
SCFskp2	p27	β-catenin	LRH-1	RTPR
SCFskp2	p27	β-catenin	APC	RTPR
BTB-CUL3-RBX1	Nrf2	β-catenin	Bcl-9	RTPR
BTB-CUL3-RBX1	SPOP	β-catenin	Bcl-9	RTPR
APC/C	ABBA	β-catenin	Bcl-9	RTPR
APC/C	KEN	β-catenin	Bcl-9	RTPR
APC/C	DBOX	β-catenin	Bcl-9	RTPR
SIAH	PHYL	β-catenin	Bcl-9	RTPR
BTB-CUL3-RBX1	Nrf2	β-catenin	axin	RTPR
BTB-CUL3-RBX1	SPOP	β-catenin	axin	RTPR
APC/C	ABBA	β-catenin	axin	RTPR
APC/C	KEN	β-catenin	axin	RTPR
APC/C	DBOX	β-catenin	axin	RTPR
SIAH	PHYL	β-catenin	axin	RTPR
BTB-CUL3-RBX1	Nrf2	β-catenin	TCF-4	RTPR
BTB-CUL3-RBX1	Nrf2	β-catenin	APC	RTPR

TABLE 2
	Grafting
Target protein and	site in
binding partner	scaffold	Amino acid sequence	DNA sequence optimised for E. coli expression
β-catenin
axin	helix	GAYPEYILDIHVYRVQLEL	GGTGCATATCCGGAATACATCCTGGATATTCATGTTTATCGTGTTCAGCTGGAACTG
Bcl-9	helix	SQEQLEHRYRSLITLYDIQLML	AGCCAAGAACAGCTGGAACATCGTTATCGTAGCCTGATTACCCTGTATGATATTCAGCTGATGCTG
TCF-4	loop	QELGDNDELMHFSYESTQD	CAAGAACTGGGCGATAATGATGAACTGATGCACTTTAGCTATGAAAGCACCCAGGAT
ICAT	helix	YAYQRAIVEYMLRLMS	TATGCATATCAGCGTGCCATCGTTGAATATATGCTGCGTCTGATGAGC
LRH-1	helix	YEQAIAAYLDALMC	TATGAACAGGCAATTGCAGCATATCTGGATGCACTGATGTGT
APC	loop	SCSEELEALEALELDE	AGCTGTAGCGAAGAACTGGAAGCCCTGGAAGCATTAGAACTGGATGAA
α-catenin	helix	RSKKAHVLAASVEQATQNFL	CGCAGCAAAAAAGCGCATGTGCTGGCGGCGAGCGTGGAACAGGCGACCCAGAACTTTCTGGAAAAAGGCGAACAGA
		EKGEQIAKESQ	TTGCGAAAGAAAGCCAG
α-catenin	helix	RTLTVERLLEPLVTQVTTLV	CGCACCCTGACCGTGGAACGCCTGCTGGAACCGCTGGTGACCCAGGTGACCACCCTGGTG
APC Membrance	loop	RREQLEAQEARAREAHAREA	CGCCGCGAACAGCTGGAAGCGCAGGAAGCGCGCGCGCGCGAAGCGCATGCGCGCGAAGCGCATGCGCGCGA
recruitment protein		HAREAYTREAYGREAYAREA	AGCGTATACCCGCGAAGCGTATGGCCGCGAAGCGTATGCGCGCGAAGCGCATACCTGGGAAGCGCATGGCCGCGAA
		HTWEAHGREARTREAQA	GCGCGCACCCGCGAAGCGCAGGCG
SOX	loop	D..EFDQYL	GATNNNNNNGAATTTGATCAGTATCTG
kindlin 2	loop	QALLDKAKINQ	CAGGCGCTGCTGGATAAAGCGAAAATTAACCAGGGCTGGCTGGATAGCAGCCGCAGCCTGATGGAACAGGATAAAG
		GWLDSSRSLMEQDKENEALL	AAAACGAAGCGCTGCTGCGCTTT
		RF
KRAS
SOS1	helix	FEGIALTNYLKALEG	TTTGAAGGTATTGCACTGACCAATTATCTGAAAGCACTGGAAGGT
phage-display library	loop	PLYISY	CCCCTGTACATCAGCTAC
peptide KR-pep1
Synthetic peptide 225-1	helix	SIEDLHEYWARLWNYLYVA	AGCATTGAAGATCTGCATGAATATTGGGCGCGCCTGTGGAACTATCTGTATGTGGCG
Synthetic peptide 225-	helix	QASLEELHEYWARLWNYRVA	CAGGCGAGCCTGGAAGAACTGCATGAATATTGGGCGCGCCTGTGGAACTATCGCGTGGCG
15a
Synthetic peptide 225-	helix	NASIKQLHAYWQRLYAYLAA	AACGCGAGCATTAAACAGCTGCATGCGTATTGGCAGCGCCTGTATGCGTATCTGGCGGCGGTGGCG
15b		VA
phage-display library	loop	CMWWREICPVWW	TGCATGTGGTGGCGCGAAATTTGCCCGGTGTGGTGG
peptide KR-pep3
Raf-S	loop	FARKTFLKLAF	TTTGCGCGCAAAACCTTTCTGAAACTGGCGTTT
NF1	loop	ARRFFLDIAD	GCGCGCCGCTTCTTTCTGGATATTGCGGAT
Rasin peptide 2	loop	FRWP..RL..	TTTCGCTGGCCGNNNNNNCGCCTGNNNNNN
Rasin peptide 1	loop	t.VFXh.p	AGCATTGTGTTTGGCGCGCATGAT
NF1 monobody peptide	loop	YGHGQVYYY	TATGGCCATGGCCAGGTGTATTATTAT
(74-84)
farnesyl transferase 1	loop	ENPKQN	GAAAACCCGAAACAGAAC
farnesyl transferase 2	loop	DAYECLDASRPW	GATGCGTATGAATGCCTGGATGCGAGCCGCCCGTGG
farnesyl transferase 3	loop	KSRDFYH	AAATCCCGCGATTTCTATCAT
c-Myc
Aurora A	helix	AGVEHQLRREVEIQSH	GCGGGCGTGGAACATCAGCTGCGCCGCGAAGTGGAAATTCAGAGCCAT
Aurora A	loop	WSVHAPSSRRTTpLAGTLDYLPPEMI	TGGAGCGTGCATGCGCCGAGCAGCCGCCGCACCGAACTGGCGGGCACCCTGGATTATCTGCCGCCGGAAATGATT
Aurora A	helix	TYQETY	ACCTATCAGGAAACCTAT
Omomyc	helix	QAEEQKLSEEDLLRKRREQLKHKLEQLRNSCA	CAGGCGGAAGAACAGAAACTGAGCGAAGAAGATCTGCTGCGCAAACGCCGCGAACAGCTGAAACATAAACTGGAACA
			GCTGCGCAACAGCT
Myc H1 F8A		NELKRSFAALRDQI	AACGAACTGAAACGCAGCTTTGCGGCGCTGCGCGATCAGATT
Myc H1 F8A S6A		NELKRAFAALRDQI	AACGAACTGAAACGCGCGTTTGCGGCGCTGCGCGATCAGATT
MIP	helix	IREKNHYHRQEVDDLRRQNALLEQQVRAL	ATTCGCGAAAAAAACCATTATCATCGCCAGGAAGTGGATGATCTGCGCCGCCAGAACGCGCTGCTGGAACAGCAGGT
			GCGCGCGCTG
PIN1	loop	FNHITNASQWE	TTTAACCATATTACCAACGCGAGCCAGTGGGAA
PIN2	loop	GDLGAFSRGQM	GGCGATCTGGGCGCGTTTAGCCGCGGCCAGATG
9E10 paratope	loop	RSEFYYYGNTYYYSAMD	CGCAGCGAATTTTATTATTATGGCAACACCTATTATTATAGCGCGATGGAT
BIN1	loop	QHDYTATDE	CAGCATGATTATACCGCGACCGATGAA
BIN1	loop	QNPEEQDEGW	CAGAACCCGGAAGAACAGGATGAAGGCTGG
BIN1	loop	EKCRGVFPENF	GAAAAGTGCCGCGGCGTGTTTCCGGAAAACTTT
BRD4
JMJD6	loop	KWTLERLKRKYRN	AAATGGACCCTGGAACGTCTGAAACGTAAATACCGTAAC
murine leukemia virus	loop	TWRVQRSQNPLKIRLTR	ACCTGGCGTGTTCAGCGTTCTCAGAACCCGCTGAAAATCCGTCTGACCCGT
integrase
EWS-FLJ1
ESAP1	loop	TMRGKKKRTRAN	ACCATGCGCGGCAAAAAAAAACGCACCCGCGCGAAC
Aurora A
TPX2	loop	SYSYDAPSDFINFSS	AGCTATAGCTATGATGCGCCGAGCGATTTTATTAACTTTAGCAGC
TPX2	loop	SYSYDAPSDFINFSSLDDEGDTQNIDSWF	AGCTATAGCTATGATGCGCCGAGCGATTTTATTAACTTTAGCAGCCTGGATGATGAAGGCGATACCCAGAACA
		EEKANLEN	TTGATAGCTGGTTTGAAGAA
TPX3	loop	MSQVKSSYSYDAPSDFINFSSLDD	ATGAGCCAGGTGAAGTCATCTTATTCCTATGATGCCCCCAGCGATTTCATCAATTTTTCATCCTTGGATGATGAA
N-myc	helix	MALSPSRGFAEHSSEPPSWVTTMLYENEL	ATGGCGCTGAGCCCGAGCCGCGGCTTTGCGGAACATAGCAGCGAACCGCCGAGCTGGGTGACCATTATGCTGTATG
		WI	AAAACGAACTGTGGATT
N-myc	loop	LEFDSLQPCFYPDEDDFYFGGPDSTPPGE	CTGGAATTTGATAGCCTGCAGCCGTGCTTTTATCCGGATGAAGATGATTTTTATTTTGGCGGCCCGGATAGCACCCCG
			CCGGGCGAA
CK2alpha
CK2beta	loop	RLYGFKIHPMAYQLQ	CGCCTGTATGGCTTTAAAATTCATCCGATGGCGTATCAGCTGCAG
WDRS
MLL1	loop	EPPLNPHGSARAEVHLRKS	GAACCGCCGCTGAACCCGCATGGCAGCGCGCGCGCGGAAGTGCATCTGCGCAAAAGC
Notch
MAML1	helix	SAVMERLRRRIELCRRHHST	AGCGCGGTGATGGAACGCCTGCGCCGCCGCATTGAACTGTGCCGCCGCCATCATAGCACC
Cdk2
cyclin A	helix	TYTKKQVLRMEHLVLKVLTFDL	ACCTATACCAAAAAACAGGTGCTGCGCATGGAACATCTGGTGCTGAAAGTGCTGACCTTT
aptmer library		LVCKSYRLDWEAGALFRSLF	CTGGTGTGCAAAAGCTATCGCCTGGATTGGGAAGCGGGCGCGCTGTTTCGCAGCCTGTTT
aptmer library		YSFVHHGFFNFRVSWREMLA	TATAGCTTTGTGCATCATGGCTTTTTTAACTTTCGCGTGAGCTGGCGCGAAATGCTGGCG
peptide	loop	TAALS	ACCGCGGCGCTGAGC
peptide	loop	TALLS	ACCGCGCTGCTGAGC
peptide	loop	LAALS	CTGGCGGCGCTGAGC
peptide	loop	DAALT	GATGCGGCGCTGACC
peptide	loop	YAALQ	TATGCGGCGCTGCAG
peptide	loop	SKL.RFTGCSC	AGCAAACTGNNNCGCTTTACCGGCTGCAGCTGC
RXL peptide	loop	PVKRRLFL	CCGGTGAAACGCCGCCTGTTTCTG
p21	loop	GRKRRQTSMTDFYHSKRRLIFSKRKP	GGCCGCAAACGCCGCCAGACCAGCATGACCGATTTTTATCATAGCAAACGCCGCCTGATTTTTAGCAAACGCAAACCG
PLK1
peptide	loop	MAGPMQTSpTPKNAGKK	ATGGCGGGCCCGATGCAGACCAGCACCCCGAAAAACGCGGGCAAAAAA
PBIP1	loop	FDPPLHSpTA	TTTGATCCGCCGCTGCATAGCACCGCG
designed peptide	loop	PLHSpTAI	CCGCTGCATAGCACCGCGATT
designed peptide	loop	MDSpTPL	ATGGATAGCACCCCGCTG
Emi2	loop	FSQHKpTI	TTTAGCCAGCATAAAACCAGCATT
HEF1	loop	LHYPSpTTALQE	CTGCATTATCCGAGCACCACCGCGCTGCAGGAA
cdc-25	loop	LLCSpTPNGL	CTGCTGTGCAGCACCCCGAACGGCCTG
BCR-ABL
optimised substrate	loop	EAIYAAPFAKKK	GAAGCGATTTATGCGGCGCCGTTTGCGAAAAAAAAA
peptide
proline-rich peptide	helix	APSYPPPPP	GCGCCGAGCTATCCGCCGCCGCCGCCG
PP2A
optimised substate	loop	LQTIQEEE	CTGCAGACCATTCAGGAAGAAGAA
peptide
PP1c
consensus sequence	loop	RV.F	CGCGTGNNNTTT
consensus sequence	loop	SILK	AGCATTCTGAAA
KNL1	loop	SRRVSFADTIKVFQT	AGCCGCCGCGTGAGCTTTGCGGATACCATTAAAGTGTTTCAGACC
EED (Embryonic
ectoderm
development)
EZH2	helix	FSSNRQKILERTEILNQEWKQ	TTTAGCAGCAACCGCCAGAAAATTCTGGAACGCACCGAAATTCTGAACCAGGAATGGAAACAGCGCCGCATTCAGC
		RRIQPV	CGGTG
MCL-1
EZH2	helix	KALETLRRVGDGVQRNHETAF	AAAGCGCTGGAAACCCTGCGCCGCGTGGGCGATGGCGTGCAGCGCAACCATGAAACCGCGTTT
NOXA BH3	helix	AELEVESATQLRRFGDKLNFRQKLL	GCGGAACTGGAAGTGGAAAGCGCGACCCAGCTGCGCCGCTTTGGCGATAAACTGAACTTTCGCCAGAAA
			CTGCTG
MCL-1 BH3	helix	KALETLR.VGD.VQRNHETAF	AAAGCGCTGGAAACCCTGCGCNNNGTGGGCGATNNNGTGCAGCGCAACCATGAAACCGCGTTT
GSK3
Substrate-competitive	loop	KEAPPAPPQDP	AAAGAAGCGCCGCCGGCGCCGCCGCAGGATCCG
binding peptide
Substrate-competitive	loop	LSRRPDYR	CTGAGCCGCCGCCCGGATTATCGC
binding peptide
Substrate-competitive	loop	RREGGMSRPADVDG	CGCCGCGAAGGCGGCATGAGCCGCCCGGCGGATGTGGATGGC
binding peptide
Substrate-competitive	loop	YRRAAVPPSPSLSRHSSPSQD EDEEE	TATCGCCGCGCGGCGGTGCCGCCGAGCCCGAGCCTGAGCCGCCATAGCAGCCCGAGCCAGGAT
binding peptide			GAAGATGAAGAAGAA
CtBP
From cyclic peptide	loop	SGWTVVRMY	AGCGGCTGGACCGTGGTGCGCATGTAT
library
tankyrase
consensus substrate	loop	REAGDGEE	CGTGAAGCCGGTGATGGTGAAGAA
peptide
consensus substrate	loop	HLQREAGDGEEFRS	CATCTGCAGCGTGAAGCCGGTGATGGTGAAGAATTTCGTAGC
peptide
Bcl-2 and BCL-XL
Bim BH3	helix	IWIAQELRRIGDEFNAYYARR	ATTTGGATTGCGCAGGAACTGCGCCGCATTGGCGATGAATTTAACGCGTATTATGCGCGCCGC
Bak BH3	helix	GQVGRQLAIIGDDINR	GGCCAGGTGGGCCGCCAGCTGGCGATTATTGGCGATGATATTAACCGC
Bad BH3	helix	NLWAAQRYGRELRRMSDEFVDSFKK	AACCTGTGGGCGGCGCAGCGCTATGGCCGCGAACTGCGCCGCATGAGCGATGAATTTGTGGATAGCTTTAAAAAA
Jun
library-selected peptide	helix	SIAATLEKEEANLEKMNKKLAAEIESLLKEK	AGCATCGCCGCCACCCTGGAGAAGGAGGAGGCCAACCTGGAGAAGATGAACAAGAAGCTGGCCGCCGAGATCGAGAG
		DKLESVLNYHE	CCTGCTGAAGGAGAAGGACAAGCTGGAGAGCGTGCTGAACTACCACGAG
library-selected peptide	helix	VQEIEQEIQELEKRIKQIQQEFQEIEQQIALL	GTTCAGGAAATCGAACAGGAAATCCAGGAACTGGAAAAACGTATCAAACAGATCCAGCAGGAATTCCAGGAAATC
			GAACAGCAGATCGCGCT
BFL1
NOXA BH3	helix	ATQLRRFGDKLNFRQ	GCGACCCAGCTGCGCCGCTTTGGCGATAAACTGAACTTTCGCCAG
BAX
Bcl-2 BH3	helix	EIVAKYIHYKLSQRGYEWDA	GAAATTGTGGCGAAATATATTCATTATAAACTGAGCCAGCGCGGCTATGAATGGGATGCG
	and loop
elF4E
elF4G	helix	KKRYDREFLLGFQF	AAAAAACGCTATGATCGCGAATTTCTGCTGGGCTTTCAGTTT
elF4G	helix	GKKRYDREFLLGFQFIFASMQKPEGLPHIS	GGCAAAAAACGCTATGATCGCGAATTTCTGCTGGGCTTTCAGTTTATTTTTGCGAGCATGCAGAAACCGGAAGGCCTG
	and loop	DVVL	CCGCATATTAGCGATG TGGTGCTG
optimised peptide	helix	TKLIYERAFMKNLRGSPLSQTPPSNVPSCL	ACCAAACTGATTTATGAACGCGCGTTTATGAAAAACCTGCGCGGCAGCCCGCTGAGCCAGACCCCGC
	and loop	LRGT	CGAGCAACGTGCCGAGCTGCCTGCTG CGCGGCACC
Fos
library-selected peptide	helix	AIARLEERVKTLKAEIYELRSKANMLREQAQ	GCGATTGCGCGCCTGGAAGAACGCGTGAAAACCCTGAAAGCGGAAATTTATGAACTGCGCAGCAAAGC
		LGAP	CGCGAACAGATTGC
library-selected peptide	helix	AIARLEERVKTLKAEIYELQSEANMLREQ1AQ	GCGATTGCGCGCCTGGAAGAACGCGTGAAAACCCTGAAAGCGGAAATTTATGAACTGCGCAGCAAAGC
		GCGAACAGALGAP	GAACATGCTGCGAACATGCTGTTGC
HDAC4
SMRT corepressor	loop	HIRGSITQGIPRSYV	CACATCCGTGGTTCTATCACCCAGGGTATCCCGCGTTCTTACGTT
BCL6
SMRT and N-CoR	loop	GRSIHEIPR	GGCCGCAGCATTCATGAAATTCCGCGC
corepressors
SMRT and N-CoR	loop	GLVATVKEAGRSIHEIPREEL	GGCCTGGTGGCGACCGTGAAAGAAGCGGGCCGCAGCATTCATGAAATTCCGCGCGAAGAACTG
corepressors
Tau
alpha-tubulin	loop	KDYEEVGVDSVE	AAAGATTATGAAGAAGTGGGCGTGGATAGCGTGGAA
beta-tubulin	loop	YQQYQDATADEQG	TATCAGCAGTATCAGGATGCGACCGCGGATGAACAGGGC
PD-L1
HIP1R	loop	DAVRRIEDMMNQARHASSGV	GATGCGGTGCGCCGCATTGAAGATATGATGAACCAGGCGCGCCATGCGAGCAGCGGCGTG
KDM4A
library-selected peptide	loop	YVYNTRSGWRWYT	TACGTTTACAACACCCGTTCTGGTTGGCGTTGGTACACC
EGFR
EGFR (juxtamembrane	helix	VRKRTLRRLLQERELVE	GTGCGCAAACGCACCCTGCGCCGCCTGCTGCAGGAACGCGAACTGGTGGAA
coiled-coil domain)
RA825
RFIP1	helix	RQVRELENYIDRLVRVMEETPNILRIPR	CGCCAGGTGCGCGAACTGGAAAACTATATTGATCGCCTGGTGCGCGTGATGGAAGAAACCCCGAACATTCTGCG
			CATTCCGCGC
GPCRs and other
transmembrane
proteins
PAR1 pepducin	N-term	pal-KKSRALF-NH2	synthetic peptide
		(pal—= palmitoyl; —NH2 = amino group)
PAR1 pepducin	N-term	pal-RCLSSSAVANRS-NH2	synthetic peptide
PAR1 pepducin	N-term	pal-RSLSSSAVANRS-NH2	synthetic peptide
PAR1 pepducin	N-term	pal-AVANRSKKSRALF-NH2	synthetic peptide
PAR1 pepducin	N-term	pal-RCESSSAEANRSKKERELF-NH2	synthetic peptide
PAR1 pepducin	N-term	pal-ASSESQRYVYSIL-NH2	synthetic peptide
PAR1 pepducin	N-term	pal-ASSASQEYVYSIL-NH2	synthetic peptide
PAR2 pepducin	N-term	pal-RSSAMDENSEKKRKSAIK-NH2	synthetic peptide
PAR2 pepducin	N-term	pal-GDENSEKKRKQAIK-NH2	synthetic peptide
PAR4 pepducin	N-term	pal-SGRRYGHALR-NH2	synthetic peptide
PAR4 pepducin	N-term	pal-ATGAPRLPST-NH2	synthetic peptide
PAR4 pepducin	N-term	pal-RLAHGYRRGS-NH2	synthetic peptide
CXCR1/2 pepducin	N-term	pal-RTLFKAHMGQKHR-NH2	synthetic peptide
CXCR1/2 pepducin	N-term	pal-LCA-YSRVGRSVTD-NH2	synthetic peptide
		(LCA—= lithocholic acid)
CXCR4 pepducin	N-term	pal-HSKGHQKRKALK-NH2	synthetic peptide
CXCR4 pepducin	N-term	pal-MGYQKKLRSMTD-NH2	synthetic peptide
CXCR4 pepducin	N-term	pal-MGYQKKLRSMTDKYRL-NH2	synthetic peptide
S1P3 pepducin	N-term	myristoyl-GMRPYDANKR-NH2	synthetic peptide
S1P3 pepducin	N-term	myristoyl-GRPYDAN-NH2	synthetic peptide
FRP2 pepducin	N-term	pal-KIHKKGMIKSSRPLRV-NH2	synthetic peptide
FRP2 pepducin	N-term	pal-KIHKKGMIKS-NH2	synthetic peptide
FRP2 pepducin	N-term	pal-KIHKKGMIKSSR-NH2	synthetic peptide
LGR7 pepducin	N-term	pal-KRKALKALILNEKKVQ-H	synthetic peptide
		(—H = hydrogen)
SMO pepducin	N-term	pal-TFVADWRNSNRY-H	synthetic peptide
SMO pepducin	N-term	pal-TWAWHTSFKALGTTYQPLSG KTS-H	synthetic peptide
SMO pepducin	N-term	pal-	synthetic peptide
		RGVMTLFSIKSNHPGLLSEKAASKINETMLR-H
IGF1R pepducin	N-term	pal-RNNSRLGNGVLY-NH2	synthetic peptide
CD226 pepducin	N-term	pal-RRERRDLFTE-NH2	synthetic peptide
TRPV1 TRPducin	N-term	pal-MGETVNKIAQES-NH2	synthetic peptide
Nrp1/2
paratope	loop	RASQYFSSYLA	CGCGCGAGCCAGTATTTTAGCAGCTATCTGGCG
paratope	helix	AREDFRNRRLWYVMDY	GCGCGCGAAGATTTTCGCAACCGCCGCCTGTGGTATGTGATGGATTAT
IL18
paratope	helix	KASGYSFTDYFIY	AAAGCGAGCGGCTATAGCTTTACCGATTATTTTATTTAT
IL15
paratope	loop	YRDRRRPS	TATCGCGATCGCCGCCGCCCGAGC
Thyroid stimulating
hormone receptor
paratope	loop	SGSSSDIGSNYVS	AGCGGCAGCAGCAGCGATATTGGCAGCAACTATGTGAGC
EGF receptor
paratope	loop	QQWSSHIFT	CAGCAGTGGAGCAGCCATATTTTTACC
paratope	helix	ASRDYDYAGRYFDY	GCGAGCCGCGATTATGATTATGCGGGCCGCTATTTTGATTAT
IL23
paratope	loop	QNGHSFPFT	CAGAACGGCCATAGCTTTCCGTTTACC
paratope	helix	YINPYNDGTK	TATATTAACCCGTATAACGATGGCACCAAA
paratope	helix	ARNWDVAY	GCGCGCAACTGGGATGTGGCGTAT
Lymphocyte function
associated antigen 1
(LFA-1
paratope	helix	SGYSFTGHWMN	AGCGGCTATAGCTTTACCGGCCATTGGATGAAC
paratope	helix	MIHPSDSETR	ATGATTCATCCGAGCGATAGCGAAACCCGC
paratope	helix	ARGIYFYGTTYFDY	GCGCGCGGCATTTATTTTTATGGCACCACCTATTTTGATTAT
C3b
paratope	helix	SGFSFTSSVS	AGCGGCTTTAGCTTTACCAGCAGCGTGAGC
paratope	helix	LIYPYNGFN	CTGATTTATCCGTATAACGGCTTTAAC
FGF receptor
paratope	helix	AASGFTFTSTGIS	GCGGCGAGCGGCTTTACCTTTACCAGCACCGGCATTAGC
paratope	helix	ARTYGIYDLYVDYTE	GCGCGCACCTATGGCATTTATGATCTGTATGTGGATTATACCGAA
IL2
paratope	helix	SRDYGYYFD	AGCCGCGATTATGGCTATTATTTTGAT
paratope	helix	GYSFTRYWMH	GGCTATAGCTTTACCCGCTATTGGATGCAT
HER2
paratope	loop	QWWWWPST	CAGTGGTGGTGGTGGCCGAGCACC
paratope	helix	ASGFSIWWSWIH	GCGAGCGGCTTTAGCATTTGGTGGAGCTGGATTCAT
membrane-type serine
protease 1
paratope	loop	YDNNQRPS	TATGATAACAACCAGCGCCCGAGC
paratope	helix	TFHIRRYRSGYYDKMDH	ACCTTTCATATTCGCCGCTATCGCAGCGGCTATTATGATAAAATGGATCAT
beta-secretase
paratope	helix	ARGPFSPWVMDY	GCGCGCGGCCCGTTTAGCCCGTGGGTGATGGATTAT
VEGF-R
paratope	helix	TRHDGTNFD	ACCCGCCATGATGGCACCAACTTTGAT
paratope	helix	QQAKAFPPT	CAGCAGGCGAAAGCGTTTCCGCCGACC
Irp5/6 receptor
paratope	helix	SGHVNAVKNYGY	AGCGGCCATGTGAACGCGGTGAAAAACTATGGCTAT
hepsin protease
paratope	helix	WINTETGS	TGGATTAACACCGAAACCGGCAGC
Factor D
paratope	helix	WINTYTGE	TGGATTAACACCTATACCGGCGAA
paratope	helix	GYTFTNYGMN	GGCTATACCTTTACCAACTATGGCATGAAC

TABLE 3
Ubiquitin Ligase	Degron sequence derived	Grafting	Amino acid sequence	DNA sequence E. Coli codon optimised 5′ to 3′
Mdm2	Consensus	helix	F[∨P]{3}W[∨P]{2,3}[VIL]	TTT[NNNNNNNN(EXCEPT CCN)]TGG[NNN{2,3}(∨CCN)][GTG/
				ATT/CTG]
Mdm2	p53	helix	FAAYWNLLSAYG	TTTGCAGCCTATTGGAATCTGCTGAGCGCATATGGT
Mdm2	p53	helix	RFMDYWEGL	CGCTTCATGGATTATTGGGAAGGTCTG
Mdm2	p53	helix	TSFAEYWALLAENL	ACCAGCTTTGCCGAGTATTGGGCCCTGCTGGCCGAGAATCTG
Mdm2	p53	helix	EAQWAAL	GAAGCGCAGTGGGCGGCGCTG
Mdm2	p53	helix	FEAQWAAL	TTTGAAGCGCAGTGGGCGGCGCTG
Mdm2	p63	helix	FQHIWDFL	TTTCAGCATATTTGGGATTTTCTG
Mdm2	p73	helix	FEHLWSSL	TTTGAACATCTGTGGAGCAGCCTG
SCF(Skp2)	Consensus	loop	.[DE].pTP.K	NNN[GAT/GAA]NNNACCCCGNNNAAA
SCF(Skp2)	p27	loop	AGSNEQEPKKRS	GCAGGTAGCAATGAACAAGAACCGAAAAAACGTAGT
Cul3-Keap1	Consensus	loop	[DNS].[DES][TNS] GE	[GAC/AAC/AGC]NNN[GAC/GAA/AGC][ACC/AAC/AGC]GGCGAA
Cul3-Keap1	Nrf2	loop	DPETGEL	GATCCGGAAACCGGTGAACTG
Cul3-Keap1	Sequestosome-1	loop	DPSTGEL	GATCCGAGCACCGGCGAACTG
Cul3-Keap1	IKKB	loop	NQETGE	AACCAGGAAACCGGCGAA
Cul3-KEAP1	APC membrane recruitment	loop	SPETGE	AGCCCGGAAACCGGCGAA
	protein 1
Cul3-KEAP1	Prothymosin alpha	loop	NEENGE	AACGAAGAAAACGGCGAA
Cul3-KEAP1	Nucleosome-remodeling	oop	DPENGE	GATCCGGAAAACGGCGAA
	factor subunit
Cul3-KEAP1	Serine/threonine-protein	loop	NVESGE	AACGTGGAAAGCGGCGAA
	phosphatase PGAM5,
Cul3-KEAP1	Nuclear factor erythroid 2-	loop	DEETGE	GATGAAGAAACCGGCGAA
	related
Cul3-KEAP1	Partner and localizer of	loop	DEETGE	GATGAAGAAACCGGCGAA
Cul3-KEAP1_2	Consensus	loop	QD.DLGV	CAGGATNNNGATCTGGGTGTG
Cul3-SPOP	Consensus	loop	[AVP].[ST][ST][ST]	[GCG/GTG/CCG]NNN[AGC/ACC][AGC/ACC][AGC/ACC]
Cul3-SPOP	Map kinase phosphatase	loop	ELDSPSSTSSSS	GAACTGGATAGCCCGAGCAGCACCAGCAGCAGCAGC
Cul3-SPOP	SBC	loop	LACDEVTSTTSSSTA	CTGGCATGTGATGAAGTTACCAGCACCACCAGTAGCAGCACCGCA
Cul3-SPOP	Androgen receptor	loop	ASSTT	GCGAGCAGCACCACC
Cul3-SPOP	Map kinase phosphatase	loop	DEVTSTTSSST	GATGAAGTGACCAGCACCACCAGCAGCAGCACC
Cul3-KELCH	Consensus	loop	E.EE.E[AV]DQH	GAANNNGAAGAANNNGAA[GCG/GTG]GATAACCAT
Cul3-KELCH	Serine/threonine-protein	helix/loop	EPEEPEADQH	GAACCGGAAGAACCGGAAGCGGATCAGCAT
	kinase WNK1
Cul3-KELCH	Serine/threonine-protein	helix/loop	ECEETEVDQH	GAATGCGAAGAAACCGAAGTGGATCAGCAT
Cul3-KELCH	Serine/threonine-protein	helix/lo	EPEEPEADQH	GAACCGGAAGAACCGGAAGCGGATCAGCAT
Cul3-KELCH	Nuclear factor erythroid 2-	helix/lo	ILWRQDIDLGV	ATTCTGTGGCGCCAGGATATTGATCTGGGCGTG
	related
KELCH actinfilin	Consensus	loop	[AP]P[MV][IM]V	[GCG/CCG]CCG[ATG/GTG][ATT/ATG]GTG
APC/C	ABBA	loop	[FIVL].[ILMVP][FHY].[DE].{O,3}{DEST}	[TTT/ATT/GTG/CTG]NNN[ATT/CTG/ATG/GTG/CCG][TTT/
				CAT/TAT][NNN{0,3}] [GAT/GAA/AGC/ACC]
APC/C	ABBA	loop	SLSSAFHVFEDGNKEN	AGCCTGAGCAGCGCGTTTCATGTGTTTGAAGATGGCAACAAAGAAAAC
APC/C	Cyclin-A2: ABBA	loop	FTIHVD	TTTACCATTCATGTGGAT
APC/C	ABOX	loop	QRVL	CAGCGTGTTCTG
APC/C	Consensus	loop	.KEN.	NNNAAAGAAAACNNN
APC/C	KEN	loop	SEDKENVPP	AGCGAGGATAAAGAAAATGTTCCGCCT
APC/C	DBOX consensus	loop	.R..L..[LIVM].	NNNCGTNNNNNNCTGNNNNNN[CTG/ATT/GTG/ATG]NNN
APC/C	Shugoshin 1: DBOX	loop	RLSLSPKKN	CGCCTGAGCCTGAGCCCGAAAAAAAAC
APC/C	Shugoshin 1: DBOX	loop	RSSLKKHCN	CGCAGCAGCCTGAAAAAACATTGCAAC
APC/C	Shugoshin 1: DBOX	loop	HLSLKDITN	CATCTGAGCCTGAAAGATATTACCAAC
APC/C	Bcl-2-like protein 11:	loop	RSPLFIF	CGCAGCCCGCTGTTTATTTTT
APC/C	Bcl-2-like protein 11:	loop	RSSLLSR	CGCAGCAGCCTGCTGAGCCGC
APC/C	Securin: DBOX	loop	RKALGTV	CGCAAAGCGCTGGGCACCGTG
APC/C	Securin-2: DBOX	loop	RKALGTV	CGCAAAGCGCTGGGCACCGTG
APC/C	Ski-like protein: DBOX	loop	RLCLPQV	CGCCTGTGCCTGCCGCAGGTG
APC/C	Aurora kinase B: DBOX	loop	RLPLAQV	CGCCTGCCGCTGGCGCAGGTG
APC/C	Serine/threonine-protein	loop	NRKPLTVLN	AACAGGAAGCCCCTGACCGTGCTGAAC
	kinase PLK1
APC/C	Cyclin-A2: DBOX	loop	RAALAVL	CGCGCGGCGCTGGCGGTGCTG
APC/C	G2/mitotic-specific cyclin-B1:	loop	PRTALGDIG	CCGCGCACCGCGCTGGGCGATATTGGC
	DBOX
APC/C	G2/mitotic-specific cyclin-B3	loop	RSAFEDLTN	CGCAGCGCGTTTGAAGATCTGACCAAC
APC/C	S-phase kinase-associated	loop	HRKHLQEIP	CATCGCAAACATCTGCAGGAAATTCCG
	protein 2:
APC/C	Nuclear autoantigen Sp-100:	loop	RSGLQLS	CGCAGCGGCCTGCAGCTGAGC
APC/C	Nucleolar and spindle-	loop	RRGLILA	CGCCGCGGCCTGATTCTGGCG
	associated
APC/C	BRCA1-A complex subunit	loop	RHCLPTL	CGCCATTGCCTGCCGACCCTG
	RAP80:
APC/C	BARD1: DBOX	loop	RNLLHDN	CGCAACCTGCTGCATGATAAC
APC/C	BARD1: DBOX	loop	RAALDRL	CGCGCGGCGCTGGATCGCCTG
APC/C	E3 Ubiquitin ligase RNF157:	loop	RKKL	CGCAAAAAACTG
	DBOX
APC/C	E3 Ubiquitin ligase RNF157:	loop	RRRL	CGCCGCCGCCTG
	DBOX
APC/C	Nuclear-interacting partner of	loop	RARLCSS	CGCGCGCGCCTGTGCAGCAGC
	ALK:
APC/C	Nuclear-interacting partner of	loop	RLPLVPE	CGCCTGCCGCTGGTGCCGGAA
	ALK:
APC/C	Tribbles homolog 3:	loop	RKKLVLE	CGCAAAAAACTGGTGCTGGAA
APC/C	Anillin: DBOX	loop	RENLQRK	CGCGAAAACCTGCAGCGCAAA
APC/C	Anillin: DBOX	loop	RQPLSEA	CGCCAGCCGCTGAGCGAAGCG
APC/C	Ninein-like protein: DBOX	loop	RTQLETK	CGCACCCAGCTGGAAACCAAA
APC/C	Dual specificity protein	loop	RNSLRQT	CGCAACAGCCTGCGCCAGACC
APC/C	Inactive serine/threonine-	loop	RYGLHPD	CGCTATGGCCTGCATCCGGAT
	protein
APC/C	DBOX	loop	PRLPLGDVSNN	CCGCGTCTGCCGCTGGGTGATGTTAGCAATAAT
APC/C	Bub1b	loop	AKENE	GCGAAAGAAAACGAA
APC/C	Bub1b	loop	SKENV	AGCAAAGAAAACGTG
APC/C TPR1	Consensus	loop	.[ILM]R$	NNN[ATT/CTG/ATG]CTG
SCFFbw7_1	Consensus	loop	[LIVMP].{0,2}pTP..[pSpT]	[CTG/ATT/GTT/ATG/CCG][NNN{0,2}]ACCCCGNNNNNN[AGC/ACC]
SCFFbw7_1	Neurogenic locus notch	loop	PFLpTPpSPE	CCGTTTCTGACCCCGAGCCCGGAA
	homolog
SCFFbw7_1	Uracil-DNA glycosylase	loop	PGpTPPSpS	CCGGGCACCCCGCCGAGCAGC
SCFFbw7_1	G1/S-specific cyclin-E1	loop	LLpTPPQpS	CTGCTGACCCCGCCGCAGAGC
SCFFbw7_2	Consensus	loop	[LIVMP].{0,2}pTP..E	[CTG/ATT/GTT/ATG/CCG][NNN{0,2}]ACCCCGNNNNNNGAA
SCFFbw7_2	Neurogenic locus notch	loop	PFLpTPSPE	CCGTTTCTGACCCCGAGCCCGGAA
	homolog
SCFFbw7	G1/S-specific cyclin-E1	loop	SLIPpTPDK	AGCCTGATTCCGACCCCGGATAAA
SCFFbw7	cyclin-D3	loop	PEQTSEPTDVAI	CCGGAACAGACCAGCGAACCGACCGATGTTGCAATT
SCFFbw7	Sterol regulatory element-	loop	SDSEPD	AGCGATAGCGAACCGGAT
SCFFbw7	SV40	loop	TPxxE	ACCCCGNNNNNNGAA
SCFFbw7	cyclin E1	loop	SLIPEPDR	AGCCTGATTCCGGAACCGGATCGT
SCFFbw7	Nuclear factor NF-kappa-B	loop	pSGVETpSF	AGCGGCGTGGAAACCAGCTTT
	p105
SCFFbw7	E3 Ubiquitin ligase	loop	LKLKKSL	CTGAAACTGAAAAAAAGCCTG
SCFFbw7	NF-kappa-B inhibitor	loop	pSGLDpS	AGCGGCCTGGATAGC
SCFFbw7	NF-kappa-B inhibitor	loop	DpSGIEpS	GATAGCGGCATTGAAAGC
SCFFbw7	Programmed cell death	loop	pSSRDSGRGDS	AGCAGCCGCGATAGCGGCCGCGGCGATAGC
SCFFbw7	NF-kappa-B inhibitor	loop	DpSGLGpS	GATAGCGGCCTGGGCAGC
SCFFbw8	myc	loop	EPPLEP	GAACCGCCTCTGGAACCG
SCF_TIR1	Consensus	loop	.[VLIA][VLI]GWPP[VLI]...R.	NNN[GTG/CTG/ATT/GCG][GTG/CTG/ATT]GGTTATCCGCCG[GTG/
				CTG/ATT] NNNNNNCGTNNN
Cul4-DDB1-	Consensus	loop	[NQ]{0,1}..[ILMV]	[AAC/CAG{0,1}]NNNNNN[ATT/CTG/ATG/GTG][AGC/
Cdt2_1			[ST][DEN][FY][FY].{2,3}	ACC][GAC/GAA/AAC][TTT/TAT] [TTT/
			[KR]{2,3}[{circumflex over ( )}DE]	TAT][NNN{0,3}][AAA/CGT{2,3}][NNN (∨GAA/GAT)]
Cul4-DDB1-	Consensus	loop	[NQ]{0,1}..[ILMV]T[DEN][HMFY][F	[AAC/CAG{0,1}]NNNNNN[ATT/CTG/ATG/GTG][ACC][GAC/
Cdt2_2			MY].{2,3}[KR]{2,3}[{circumflex over ( )}DE]	GAA/AAC][CAT/ATG/TTT/TAT] [TTT/TAT/ATG][NNN{2,3}]
				[AAA/CGT{2,3}][NNN (∨GAA/GAT)]
Cul4-DDB1-Cdt2	PIP	loop	QRRMTDFYARRR	CAGCGTCGTATGACCGATTTTTATGCACGTCGTCGT
DDB1-CUL4	paramoxyvirus SV5-V	helix	TVAYFTLQQVYG	ACCGTTGCATATTTTACCCTGCAGCAGGTTTATGGT
DDB1-CUL4	Hepatitis B virus X	helix	ILPAVLHLRTVYG	ATTCTGCCTGCAGTTCTGCATCTGCGTACCGTTTATGGT
DDB1-CUL4	Woodchuck Hepatitis virus X	helix	NFVAWHALRQVYG	AATTTTGTTG CATGGCATGCACTGCGTCAG GTTTATGGT
DDB1-CUL5	DCAF9	helix	NITADLILRQVYG	AACATTACCGCAGATCTGATTCTGCGTCAGGTTTATGGT
Unknown	Bonger	loop	RRRG	CGTCGTCGTGGT
SOCS box-Cul5-	iNOS	loop	DINN	GACATCAACAAC
SPSB2
SCF_TRCP1_1	Consensus	loop	DpSG.{2,3}[pSpt]	GATAGCGGC[NNN{2,3}][AGC/ACC]
SCF_TRCP1_1	SETBP1	loop	DSGIGT	GATAGCGGCATTGGCACC
β-TRCP	β-catenin	loop	DEGNYE	GATGAAGGCAACTATGAA
β-TRCP	Vpu	loop	DSGxxS	GATAGCGGCNNNNNNAGC
β-TRCP	RE1-silencing transcription	loop	SEGSDDSGL	AGCGAAGGCAGCGATGATAGCGGCCTG
	factor
β-TRCP	Prolactin receptor	loop	TDSGRGS	ACCGATAGCGGCCGCGGCAGC
β-TRCP	Protein aurora borealis	loop	DSGYNT	GATAGCGGCTATAACACC
β-TRCP	Vaccinia virus	loop	YSGNLEpS	TATAGCGGCAACCTGGAAAGC
SCFFbw2	G1/S-specific cyclin-D3	loop	pSQTSTPTDVTAIHL	AGCCAGACCAGCACCCCGACCGATGTGACCGCGATTCATCTG
SCFFbw3	G1/S-specific cyclin-D3	loop	PTDVTAI	CCGACCGATGTGACCGCGATT
SCFFbw4	cyclin D1	loop	EEEVSLASEPTDVRD	GAAGAAGAAGTTAGCCTGGCAAGCGAACCGACCGATGTTCGTGAT
OPDH VHL 1	consensus	loop	[IL]ApT.{6,8}[FLIVM].[FLIVM]	[ATT/CTG]GCGACC[NNN{6,8}][TTT/CTG/ATT/
				GTG/ATG]NNN[TTT/CTG/ATT/GTG/ATG]
SCF coil	consensus	loop	[RK][RK].SL.F[FLM].[RK]R[HRK].	[CGT/AAA][CGT/AAA]NNNAGCCTGNNNTTT[TTT/
				CTA/ATG]NNN[CGT/AAA]CGT[CGT/AAA/CAT]NNN[CGT/AAA]
CHIP	Hsp90	loop or	ASRMEEVD	GCAAGCCGTATGGAAGAAGTTGAT
		C-
		terminus
CHIP	Hsp70	loop or	GPTIEEVD	GGTCCGACCATTGAAGAAGTTGAT
		C-
		terminus
SOCS box-	VASA	loop	DINNNNNIVEDVERKREFYI	GACATCAACAACAACAACAACATCGTTGAAGACGTTGAACGTAAAC
				GTGAATTCTACATC
UBR5	PAM2	loop	SKLSVNAPEFYPSG	TCTAAACTGTCTGTTAACGCGCCGGAATTCTACCCGTCTGGT
CRL2(KLHDC2)	Usp1	C-	IGLLGG	ATCGGTCTGCTGGGTGGT
		terminu
CID4	Pro/N-degron	N-	PGLW	CCGGGTCTGTGG
		terminu
TRIM21	Fc fragment	loop	WxW	TGGNNNTGG
TRIM21	Fc fragment	loop	HNH	CATAACCAT
Nedd4	PPxY motif	loop	TAPPPAYATLG	ACCGCGCCGCCGCCGGCGTATGCGACCCTGGGC
Elongin C	Vif	loop	SLSH3LSH3I	AGCCTGNNNNNNNNNCTGNNNNNNNNNATT
Unknown	ID2	loop	SRTPLTTLN	AGCCGCACCCCGCTGACCACCCTGAAC
Unknown	ZAP70	loop	DGYTPEP	GATGGCTATACCCCGGAACCG
Unknown	SH3R1	loop	RPTAAVTPI	CGCCCGACCGCGGCGGTGACCCCGATT
Unknown	ETV1	loop	DEQFVPD	GATGAACAGTTTGTGCCGGAT
Unknown	EPAS1	loop	LAPYIPMDGEDFQL	CTGGCGCCGTATATTCCGATGGATGGCGAAGATTTTCAGCTG
Unknown	hantavirus	loop	YVGLVWGVLLTTELIVWAASA	TATGTGGGCCTGGTGTGGGGCGTGCTGCTGACCACCGAACTGATTGT
				GTGGGCGGCGAGCGCG
CRL4_CDT2_1	SETD8	loop	PKTPPSSCDSTN	CCGAAAACCCCGCCGAGCAGCTGCGATAGCACCAAC
CBL (PTK)	Consensus	loop	[DN].pY[ST].P	[GAT/AAC]NNNTAT[AGC/ACC]NNNCCG
CBL (met)	Consensus	loop	DpYR	GATTATCGT
CBL	SH2B adapter protein 3	loop	RAIDNQYTPL	CGCGCGATTGATAACCAGTATACCCCGCTG
CBL	Protein sprouty homolog 1	loop	IRGSNEYTEGPS	ATTCGCGGCAGCAACGAATATACCGAAGGCCCGAGC
CBL	Protein sprouty homolog 2	loop	IRNTNEYTEGPT	ATTCGCAACACCAACGAATATACCGAAGGCCCGACC
CBL	Protein sprouty	loop	HVENDYIDNPS	CATGTGGAAAACGATTATATTGATAACCCGAGC
CBL	Tyrosine-protein	loop	SFNPYEPELA	AGCTTTAACCCGTATGAACCGGAACTGGCG
CBL	Tyrosine-protein kinase	loop	TLNSDGpYTPEPA	ACCCTGAACAGCGATGGCTATACCCCGGAACCGGCG
CBL	Plexin-A3	loop	IPFLDYRTYAV	ATTCCGTTTCTGGATTATCGCACCTATGCGGTG
CBL	Plexin-A1	loop	IPFLDYRTYAM	ATTCCGTTTCTGGATTATCGCACCTATGCGATG
CBL	Platelet-derived growth	loop	SIFDNLYTTLSD	AGCATTTTTAACAGCCTGTATACCACCCTGAGCGAT
	factor
CBL	Platelet-derived growth	loop	SIFNSLYTTLSD	AGCATTTTTAACAGCCTGTATACCACCCTGAGCGAT
	factor
CBL	Tumor necrosis factor	loop	KGDGGLYSSLPP	AAAGGCGATGGCGGCCTGTATAGCAGCCTGCCGCCG
	receptor
	superfamily member 16
CRL4(COP1/DET	Trib1	loop	SDQIVPEY	TCTGACCAGATCGTTCCGGAATAC
SH3RF1	E3 Ubiquitin-protein	loop	RPTAAVTPI	CGCCCGACCGCGGCGGTGACCCCGATT
COP-1	Consensus	loop	[D,E][D,E].{2,3}VP[DE]	[GAA/GAC][GAA/GACHNNNNNN/NNNNNNNNN]GTGCCG[GAA/GAC]
COP-1	Tribbles homolog 1	loop	SDQIVPEY	AGCGATCAGATTGTGCCGGAATAT
SIAH	Consensus	loop	.P.A.V.P[{circumflex over ( )}P]	NNNCCGNNNGCGNNNGTGNNNCCG[NNN EXCEPT CCN]
SIAH	AF4/FMR2 family	loop (beta	KPTAYVRPM	AAACCGACCGCGTATGTGCGCCCGATG
		sttrand)
SIAH	calcyclin-binding	loop	KPAAVVAPI	AAACCGGCGGCGGTGGTGGCGCCGATT
SIAH	POU domain class 2-	loop	APTAVVLPH	GCGCCGACCGCGGTGGTGCTGCCGCAT
	associating factor
SIAH	Retrotransposon-derived	loop	PPRALVLPH	CCGCCGCGCGCGCTGGTGCTGCCGCAT
	protein
ERAD-C	CL1	amphipathic	ACKNWFSSLSHFVIHL	GCGTGCAAAAACTGGTTTAGCAGCCTGAGCCATTTTGTGATTCATCTG
		helix
		extension
UBR	Nend_Nbox 2	N-terminal	ΛM{0,1}[FLYIW][∨P]	[ATG{0,1}][TTT/CTG/TAT/TGG/ATT][NNN∨CCG]
		extension
UBR	Nend_UBRbox 1	N-terminal	ΛM{0,1}[RK][∨P]	[ATG{0,1}][AAA/CGT][NNN∨CCG]
		extension
UBR	Nend_UBRbox 2	N-terminal	ΛM{0,1}[ED]	[ATG{0,1}][GAT/GAA]
		extension
UBR	Nend_UBRbox 3	N-terminal	ΛM{0,1}[NQ]	[ATG{0,1}][CAG/AAC]
		extension
UBR	Nend_UBRbox 4	N-terminal	ΛM{0,1}[C]	[ATG{0,1}][TGC]
		extension
Other
degradation
pathways:	Degron sequence derived	Grafting
Pathway;	from	site in	Amino acid sequence	DNA sequence E. Coli codon optimised 5′ to 3′
ESRCT; ALIX	Consensus, e.g. HIV Gag	loop	LYP...L, e.g. ELYPLTSLRS	GAACTGTACCCGCTGACCTCTCTGCGTTCT
ESRCT; ALIX	SIV(mac239) Gag	loop	SREKPYKEVTEDLLHLNSLF	AGCCGCGAAAAACCGTATAAAGAAGTGACCGAAGATCTGCTGCATCT
		insertio	GAACAGCCTGTTT
ESRCT; ALIX	SIV(agmTan-1) Gag	loop	AAGAYDPARKLLEQY	GCGGCGGGCGCGTATGATCCGGCGCGCAAACTGCTGGAACAGTAT
		insertio
ESCRT; AP-1	Nef	loop	ESH3LL	GAANNNNNNNNNCTGCTG
		insertio
ESCRT; AP-2	Env	loop	YxxL	TATNNNNNNCTG
		insertio
ESCRT; AP-1	HIPR1	loop	MDFSGLSLIKLKKQ	ATGGATTTTAGCGGCCTGAGCCTGATTAAACTGAAAAAACAG
		insertio
ESCRT; AP-2	consensus	loop	D(E)SH3LL(I)	GAT(GAC)NNNNNNNNNCTGCTG(ATT)
		insertion
ESCRT; AP	viral adaptor	loop	SREKPYKEVTEDLLHLNSLF	AGCCGCGAAAAACCGTATAAAGAAGTGACCGAAGATCTGCTGCATCTG
				AACAGCCTGTTT
ESCRT; AP	viral adaptor	loop	AAGAYDPARKLLEQYAKK	GCGGCGGGCGCGTATGATCCGGCGCGCAAACTGCTGGAACAGTATGCGAA
				AAAA
CMA; Hsc70	Consensus	loop	KFERQ	AAATTTGAACGCCAG
CMA; Hsc70	Consensus	loop	QRFFE	CAGCGCTTTTTTGAA
CMA; Hsc70	repeat consensus	loop	KFERQQKILDQRFFE	AAATTTGAACGCCAGCAGAAAATTCTGGATCAGCGCTTTTTTGAA
Autophagy;	Consensus LIR (LC3-	loop	[W/F/Y]..[L/I/V]	(TGG/TTC/TAT)NNNNNN(CTG/ATC/GTG)
LC3/Atg8	interacting)/AIM (Atg8
family	family-interacting) motif
Autophagy;	CCPG1	loop	TASDDSDIVTLEPPK	ACCGCGTCTGACGACTCTGACATCGTTACCCTGGAACCGCCGAAA
Autophagy; LC3	DVL	loop	EVRDRMWLKITI	GAAGTTCGTGACCGTATGTGGCTGAAAATCACCATC
Autophagy;	Ankyrin G	loop	PEDDWIEFSSEEIREARQQAAAS	CCGGAAGATGATTGGATTGAATTTAGCAGCGAAGAAATTCGCGAAGCGCGCC
GABARAP		insertion	QSPS	AGCAGGCGGCGGCGAGCCAGAGCCCGAGC
Key
. Any amino acid
[X] Allowed amino acid at the position
p Phosphorylated amino acid
$ C terminal of chain
ΛX N-terminal of chain
X{x, y} where x & y are the minimum, maximum of X amino acids at the position
[∨X] Amino acid not allowed at the position
NNN Any codon
[NNN/NNN/NNN] Any one of these codons at the position
[∨NNN] Any codon except this
[NNN{x, y}] codon, where x & y are the maximum & minimum of codons
indicates data missing or illegible when filed

TABLE 4
Multiple Alignment of DNA sequences of all CTPR and RTPR used in hetero-
bifunctional CTPRs and RTPRs
CLUSTAL multiple sequence alignment by MUSCLE (3.8)
RTPRc       GCAGAAGCACTGCGTAATCTGGGTCGTGTTTATCGTCGTCAGGGTCGTTATCAGCGTGCA
RTPRa-ii-H  GCCGAAGCTTGGTATAATCTGGGGAATGCCTATTACAGACAGGGGGATTATCAGCGCGCC
RTPRa-i-E   GCAGAAGCATGGTATAATCTGGGTAATGCATATTATCGCCAGGGTGATTATCAGCGTGCC
RTPRa-iii-E GCAGAAGCATGGTATAATCTGGGCAATGCATATTATCGTCAGGGTGATTATCAGCGTGCC
CTPRa-E     GCAGAAGCATGGTATAATCTGGGTAATGCATATTACAAACAGGGCGATTATCAGAAAGCC
CTPRb-E     GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGATTATCAGAAAGCC
RTPRb-E     GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGATTATCAGCGTGCC
RTPRa-ii-E  GCCGAGGCCTGGTATAACCTTGGCAACGCCTATTATCGTCAAGGCGACTACCAGAGAGCA
RTPRc-H     GCCGAGGCTCTGAGAAATCTGGGCAGAGTGTACAGACGGCAGGGCAGATACCAGCGGGCC
CTPRb-H     GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGACTACCAGAAGGCC
RTPRb-H     GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGACTACCAGCGGGCC
RTPRa-iv-E  GCCGAGGCCTGGTACAACCTGGGTAACGCCTATTATCGCCAAGGCGACTACCAGCGTGCA
CTPRa-H     GCCGAGGCCTGGTACAATCTGGGCAACGCCTACTACAAGCAGGGCGACTACCAGAAGGCC
RTPRa-i-H   GCCGAGGCCTGGTACAACCTGGGCAACGCCTACTACCGGCAGGGCGACTACCAGCGGGCC
            ** ** **   *   ** ** **    *  **       ** **    ** ***   **
RTPRc       ATTGAATATTATCGTCGCGCACTGGAATTAGATCCGNNNNNN
RTPRa-ii-H  ATTGAATATTATCAGCGGGCTCTGGAACTGGATCCTNNNNNN
RTPRa-i-E   ATTGAATATTATCAACGTGCACTGGAACTGGACCCGNNNNNN
RTPRa-iii-E ATCGAATATTATCAACGTGCACTGGAACTGGACCCGNNNNNN
CTPRa-E     ATCGAGTATTATCAAAAAGCACTGGAACTGGACCCGNNNNNN
CTPRb-E     ATCGAATATTATCAAAAAGCGCTGGAACTGGACCCGNNNNNN
RTPRb-E     ATTGAATATTATCAACGTGCGCTGGAATTAGATCCGNNNNNN
RTPRa-ii-E  ATCGAATATTACCAGCGTGCGTTAGAATTAGATCCGNNNNNN
RTPRc-H     ATCGAGTATTACCGCAGAGCCCTGGAACTGGACCCCNNNNNN
CTPRb-H     ATCGAGTATTATCAGAAGGCCCTGGAACTGGACCCCNNNNNN
RTPRb-H     ATCGAGTATTATCAGAGAGCCCTGGAACTGGACCCCNNNNNN
RTPRa-iv-E  ATTGAGTACTACCAACGTGCCCTGGAACTGGACCCTNNNNNN
CTPRa-H     ATCGAGTATTATCAGAAGGCCCTGGAACTGGACCCCNNNNNN
RTPRa-i-H   ATCGAGTACTACCAGAGAGCCCTGGAACTGGACCCTNNNNNN
            ** ** ** ** *     **  * *** * ** ** ******

TABLE 5
089	AEAYSNLGNVYKERGQLQEAIEHYRHALRL	118	NP_858058.1
191	AVAWSNLGCVFNAQGEIWLAIHHFEKAVTL	220
327	ADSLNNLANIKREQGNIEEAVRLYRKALEV	356
264	NLACVYYEQGLIDLAIDTYRRAIEL	288
079	AEAYSNLGNVYKERGQLQEAIEHYRHALRL	108	NP_858059.1
181	AVAWSNLGCVFNAQGEIWLAIHHFEKAVTL	210
317	ADSLNNLANIKREQGNIEEAVRLYRKALEV	346
254	NLACVYYEQGLIDLAIDTYRRAIEL	278
079	AEAYSNLGNVYKERGQLQEAIEHYRHALRL	108	NP_003596.2
812	ESFYNLGRGLHQLGLIHLAIHYYQKALEL	840	NP_036218.1
637	AWYGLGMIYYKQEKFSLAEMHFQKALDI	664	NP_001247.2
568	EAWCAAGNCFSLQREHDIAIKFFQRAIQV	596
275	AQSCYSLGNTYTLLQDYEKAIDYHLKHLAI	304
058	YSQLGNAYFYLHDYAKALEYHHHDLTL	084
315	GRACWSLGNAYTALGNHDQAMHFAEKHLEI	344
247	NMGNIYLKQRNYSKAIKFYRMALD	270	NP_783195.2
495	ALTNKGNTVFANGDYEKAAEFYKEAL	520
238	NMGNIYLKQRNYSKAIKFYRMALD	261	NP_006522.2
486	ALTNKGNTVFANGDYEKAAEFYKEAL	511
715	AQAWMNMGGIQHIKGKYVSARAYYERALQL	744	NP_787057.2
575	AEILSPLGALYYNTGRYEEALQIYQEAAAL	604
114	AQAAKNKGNKYFKAGKYEQAIQCYTEAISL	143	NP_055635.3
610	YNLGKLYHEQGHYEEALSVYKEAIQ	634	NP_689801.1
586	AQAWMNMGGIQHIKGKYVSARAYYERALQL	615	NP_114126.2
446	AEILSPLGALYYNTGRYEEALQIYQEA	472
018	AETFKEQGNAYYAKKDYNEAYNYYTKAIDM	47	NP_003306.1
300	AKAYARIGNSYFKEEKYKDAIHFYNKSL	327	NP_006810.1
231	LGNDAYKKKDFDTALKHYDKAKEL	254
365	NKGNECFQKGDYPQAMKHYTEAI	387
028	AETFKEQGNAYYAKKDYNEAYNYYTKAIDM	057	AAH11837.2
228	AYSNLGNAHVFLGRFDVAAEYYKKTLQL	255	NP_056412.2
266	AQACYSLGNTYTLLQDYERAAEYHLRHL	293
28	AEELKTQANDYFKAKDYENAIKFYSQAIEL	57	NP_006238.1
318	DAYKSLGQAYRELGNFEAATESFQKALLL	346	NP_078801.2
1262	ETLKNLAVLSYEGGDFEKAAELYKRAMEI	1290	NP_694972.3
140	GNKYFKQGKYDEAIDCYTKGMD	161	NP_078880.1
289	GNGFFKEGKYERAIECYTRGI	309
600	CWESLGEAYLSRGGYTTALKSFTKASEL	627	NP_055454.1
172	KATYRAGIAFYHLGDYARALRYLQEA	197	NP_689692.2
174	LGKIHLLEGDLDKAIEVYKKAVE	196	NP_149017.2
158	LGDLFSKAGDFPRAAEAYQKQLRF	181	NP_038460.3
384	AYFNAGNIYFHHRQFSQASDYFSKALKF	411	NP_001007796.1
104	EAWNQLGEVYWKKGDVAAAHTCFSGAL	130	NP_612385.1
814	EAWQGLGEVLQAQGQNEAAVDCFLTALEL	842	NP_065191.2
446	AKLWNNVGHALENEKNFERALKYFLQA	472	NP_861448.1
597	ADLWYNLAIVHIELKEPNEALKNFNRALEL	626
251	YRRKGDLDKAIELFQRVLE	269	NP_001540.2
251	YRRKGDLDKAIELFQRVLE	269	NP_001026853.1
079	AKTYKDEGNDYFKEKDYKKAVISYTEGL	106	NP_004614.2
501	AKVHYNIGKNLADKGNQTAAIRYYREAVRL	530	NP_116202.2
482	AKVHYNIGKNLADKGNQTAAIRYYREAVRL	511	NP_001073137.1
200	GNELVKKGNHKKAIEKYSESL	220	NP_006800.2
123	GNEQFKKGDYIEAESSYSRALEM	145	NP_003305.1
438	ESLSLLGFVYKLEGNMNEALEYYERALRL	466	NP_001001887.1
438	ESLSLLGFVYKLEGNMNEALEYYERALRL	466	NP_001539.3
375	AKTKNNLASAYLKQNKYQQAEELYKEIL	402	NP_803136.2
564	WFSLGCAYLALEDYQGSAKAFQRCVTL	590	NP_060205.3
306	AESCYQLARSFHVQEDYDQAFQYYYQATQF	335	NP_055448.1

TABLE 6
CTPRa E. coli expression codon optimised
GCAGAAGCATGGTATAATCTGGGTAATGCATATTACAAACAGGGCGATTA
TCAGAAAGCCATCGAGTATTATCAAAAAGCACTGGAACTGGACCCGNNNN
NNAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPXX
CTPRa H. Sapiens expression codon optimised
GCCGAGGCCTGGTACAATCTGGGCAACGCCTACTACAAGCAGGGCGACTA
CCAGAAGGCCATCGAGTATTATCAGAAGGCCCTGGAACTGGACCCCNNNN
NNAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPXX
RTPRa-i H. sapiens expression codon optimised
GCCGAGGCCTGGTACAACCTGGGCAACGCCTACTACCGGCAGGGCGACTA
CCAGCGGGCCATCGAGTACTACCAGAGAGCCCTGGAACTGGACCCTNNNN
NNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
RTPRa-ii H. sapiens expression codon optimised
GCCGAAGCTTGGTATAATCTGGGGAATGCCTATTACAGACAGGGGGATTA
TCAGCGCGCCATTGAATATTATCAGCGGGCTCTGGAACTGGATCCTNNNN
NNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
RTPRa-i E. coli expression codon optimised
GCAGAAGCATGGTATAATCTGGGTAATGCATATTATCGCCAGGGTGATTA
TCAGCGTGCCATTGAATATTATCAACGTGCACTGGAACTGGACCCGNNNN
NNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
RTPRa-ii E. coli expression codon optimised
GCCGAGGCCTGGTATAACCTTGGCAACGCCTATTATCGTCAAGGCGACTA
CCAGAGAGCAATCGAATATTACCAGCGTGCGTTAGAATTAGATCCGNNNN
NNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
RTPRa-iii E. coli expression codon optimised
GCAGAAGCATGGTATAATCTGGGCAATGCATATTATCGTCAGGGTGATTA
TCAGCGTGCCATCGAATATTATCAACGTGCACTGGAACTGGACCCGNNNN
NNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
RTPRa-iv E. coli expression codon optimised
GCCGAGGCCTGGTACAACCTGGGTAACGCCTATTATCGCCAAGGCGACTA
CCAGCGTGCAATTGAGTACTACCAACGTGCCCTGGAACTGGACCCTNNNN
NNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
CTPRb E. coli expression codon optimised
GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGATTA
TCAGAAAGCCATCGAATATTATCAAAAAGCGCTGGAACTGGACCCGNNNN
NNAEALNNLGNVYREQGDYQKAIEYYQKALEL-DPXX
CTPRb H. sapiens expression codon optimised
GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGACTA
CCAGAAGGCCATCGAGTATTATCAGAAGGCCCTGGAACTGGACCCCNNNN
NNAEALNNLGNVYREQGDYQKAIEYYQKALEL-DPXX
RTPRb E. coli expression codon optimised
GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGATTA
TCAGCGTGCCATTGAATATTATCAACGTGCGCTGGAATTAGATCCGNNNN
NNAEALNNLGNVYREQGDYQRAIEYYQRALEL-DPXX
RTPRb H. sapiens expression codon optimised
GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGACTA
CCAGCGGGCCATCGAGTATTATCAGAGAGCCCTGGAACTGGACCCCNNNN
NNAEALNNLGNVYREQGDYQRAIEYYQRALELDPXX
RTPRc E. Coli expression codon optimised
GCAGAAGCACTGCGTAATCTGGGTCGTGTTTATCGTCGTCAGGGTCGTTA
TCAGCGTGCAATTGAATATTATCGTCGCGCACTGGAATTAGATCCGNNNN
NNAEALRNLGRVYRRQGRYQRAIEYYRRALELDPXX
RTPRc H. Sapiens expression codon optimised
GCCGAGGCTCTGAGAAATCTGGGCAGAGTGTACAGACGGCAGGGCAGATA
CCAGCGGGCCATCGAGTATTACCGCAGAGCCCTGGAACTGGACCCCNNNN
NNAEALRNLGRVYRRQGRYQRAIEYYRRALELDPXX

TABLE 7
Protein Target	Paratope	struct	RSCB no	therapeutic area
Nrp½	RASQYFSSYLA	loop	2qqn	anti-angiogenic
Nrp½	AREDFRNRRLWYVMDY	helix	2qql	anti-angiogenic
IL18	KASGYSFTDYFIY	helix	2yxt	anti-inflammatory
IL15	YRDRRRPS	loop	2xqb	anti-inflammatory
Thyroid stimulating	SGSSSDIGSNYVS	loop	2xwt
hormone receptor
EGF receptor	QQWSSHIFT	loop	3C09	cancer
EGF receptor	ASRDYDYAGRYFDY	helix	3C09	cancer
IL23	QNGHSFPFT	loop	3d85	anti-inflammatory
IL23	YINPYNDGTK	helix	3d85	anti-inflammatory
IL23	ARNWDVAY	helix	3d85	anti-inflammatory
Lymphocyte function-	SGYSFTGHWMN	helix	3eoa	auto-immune
associated antigen 1 (LFA-1)
Lymphocyte function-	MIHPSDSETR	helix	3eoa	auto-immune
associated antigen 1 (LFA-1)
Lymphocyte function-	ARGIYFYGTTYFDY	helix	3eoa	auto-immune
associated antigen 1 (LFA-1)
C3b	SGFSFTSSVS	helix	3g6j	anti-inflammatory
C3b	LIYPYNGFN	helix	3g6j	anti-inflammatory
FGF receptor 3	AASGFTFTSTGIS	helix	3grw	multiple myeloma
FGF receptor 3	ARTYGIYDLYVDYTE	helix	3grw	multiple myeloma
IL2	SRDYGYYFD	helix	3iu3	anti-inflammatory
IL2	GYSFTRYWMH	helix	3iu3	anti-inflammatory
HER2	QWWWWPST	loop	3n85	breast cancer
HER2	ASGFSIWWSWIH	helix	3n85	breast cancer
membrane-type serine protease 1	YDNNQRPS	loop	3nps	metastasis of carcinomas
membrane-type serine protease 1	TFHIRRYRSGYYDKMDH	helix	3nps	metastasis of carcinomas
beta-secretase	ARGPFSPWVMDY	helix	3r1g	Alzheimer's disease
VEGF-R	TRHDGTNFD	helix	3s35	anti-angiogenic
VEGF-R	QQAKAFPPT	loop	3s37	anti-angiogenic
Irp5/6 receptor	SGHVNAVKNYGY	helix	3sob	bone-loss
hepsin protease	WINTETGS	Helix	3t2n	prostrate cancer
Factor D	WINTYTGE	helix	4d9r	anti-inflammatory
Factor D	GYTFTNYGMN	helix	4d9r	anti-inflammatory

TABLE 8
1. Axin-RTPR-ABBA
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSLSSAF
HVFEDGNKENGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
2. Axin-RTPR-DBOX
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGPRLPLG
DVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
3. Axin-RTPR-KEN
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGPRLPLG
DVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
4. Axin-RTPR-Nrf2
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGPRLPLG
DVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
5. Axin-RTPR-SIAH
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGLRPVAM
VRPTVGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
6. Axin-RTPR-SPOP
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPGGLACDEVTSTTSSSTAGGPNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNN
7. Axin-RTPR-p27
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQ
EPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
8. Axin-RTPR-p53
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWN
LLSAYG
10. Bcl9-RTPR-ABBA
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGDPE
TGELGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
11. Bcl9-RTPR-DBOX-v1
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGPRL
PLGDVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
12. Bcl9-RTPR-DBOX-v2
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPGGPRLPLGDVSNNGGPNAEAWYNLGNAYYRQGDYQRAIE
YYQRALELDPNN
13. Bcl9-RTPR-KEN
MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYY
QRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSLSSAF
HVFEDGNKENGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
14. Bcl9-RTPR-Nrf2
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGDPE
TGELGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
15. Bcl9-RTPR-p27
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGS
NEQEPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
16. Bcl9-RTPR-p53
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAA
YWNLLSAYG
17. Bcl9-RTPR-SIAH
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGLRP
VAMVRPTVGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
18. Bcl9-RTPR-SPOP
MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPGGLACDEVTSTTSSSTAGGPNAEAWYNLGNAYYRQGDYQ
RAIEYYQRALELDPNN
19. TCF7L2-RTPR-Nrf2
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFS
YESTQDGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYN
LGNAYYRQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAY
YRQGDYQRAIEYYQRALELDPNN
20. TCF7L2-RTPR-p27
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFS
YESTQDGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYN
LGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLG
NAYYRQGDYQRAIEYYQRALELDPNN
21. p27-RTPR-TCF7L2
MRGSHHHHHHGLVPRGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPN
NAGSNEQEPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSA
EAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYEST
QDGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRS
22. TCF7L2-RTPR-p53
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFS
YESTQDGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYN
LGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLSAYG
23. ICAT-RTPR-p27
MGSYAYQRAIVEYMLRLMSDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRA
LELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPK
KRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
24. ICAT-RTPR-p53
MGSYAYQRAIVEYMLRLMSDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRA
LELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLS
AYG
25. LRH1-RTPR-ABBA
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSEDKENVPPGG
PNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
26. LRH1-RTPR-DBOX
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGPRLPLGDVSNN
GGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
27. LRH1-RTPR-KEN
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSEDKENVPPGG
PNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
28. LRH1-RTPR-Nrf2
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGDPETGELGGPN
AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
29. LRH1-RTPR-p27
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKR
SPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
30. LRH1-RTPR-p53
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLS
AYG
31. LRH1-RTPR-SIAH
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGLRPVAMVRPTV
GGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
32. LRH1-RTPR-SPOP
MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALE
LDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGLACDEVTSTTS
SSTAGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
33. APC-RTPR-Nrf2
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSCSEELEALEALE
LDEGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGN
AYYRQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAYYRQ
GDYQRAIEYYQRALELDPNN
34. APC-RTPR-p27
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSCSEELEALEALE
LDEGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
35. p27-RTPR-APC
MRGSHHHHHHGLVPRGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPN
NAGSNEQEPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSA
EAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSCSEELEALEALELDEG
GPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRS
36. APC-RTPR-p53
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFS
YESTQDGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYN
LGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLSAYG
37. 1TBP-CTPR2
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRS
38. 2TBP-CTPR4
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDY
QKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
EYYQKALELDPRS
39. 3TBP-CTPR6
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDY
QKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREA
GDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPRS
40. 4TBP-CTPR8
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDY
QKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREA
GDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNL
GNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAY
YKQGDYQKAIEYYQKALELDPRS
41. 1TBP-CTPR2-Foldon (Foldon sequence in bold)
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAKASLNLANADIKTIQE
AGYIPEAPRDGQAYVRKDGEWVLLSTFLRS
42. 2TBP-CTPR4-Foldon (Foldon sequence in bold)
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDY
QKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
EYYQKALELDPRSAKASLNLANADIKTIQEAGYIPEAPRDGQAYVRKDGE
WVLLSTFLRS
43. 3TBP-CTPR6-Foldon (Foldon sequence in bold)
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDY
QKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREA
GDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAKASLNL
ANADIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLRS
44. 4TBP-CTPR8-Foldon (Foldon sequence in bold)
MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSA
EAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDY
QKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREA
GDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPRSAEAWYNL
GNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAY
YKQGDYQKAIEYYQKALELDPRSAKASLNLANADIKTIQEAGYIPEAPRD
GQAYVRKDGEWVLLSTFLRS
45. KBL-RTPR-CMA_Q
MGSIPNPLLGLDGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPL
YISYDPAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNA
YYRQGDYQRAIEYYQRALELDPNNQRFFE
46. CMA_Q-KBL-RTPR
MGSQRFFEGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISY
DPAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNAYYRQ
GDYQRAIEYYQRALELDPNN
47. CMA_K-KBL-RTPR
MGSKFERQGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISY
DPAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNAYYRQ
GDYQRAIEYYQRALELDPNN
48. SOS-RTPR-CMA_K
MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRAL
ELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSKFERQ
49. SOS-RTPR-CMA_Q
MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRAL
ELDPRSIPNPLLGLDKFERQ
50. SOS-RTPR-p27
MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRAL
ELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNA
YYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLGNAYY
RQGDYQRAIEYYQRALELDPRS
51. KBL-RTPR-p27
MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISYDPAEAWY
NLGNAYYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNAYYRQGDYQRAI
EYYQRALELDPNNAGSNEQEPKKRSAEAWYNLGNAYYRQGDYQRAIEYYQ
RALELDPNN

TABLE 9
Alignment of TPR Repeat Sequences
S75991
2407639-54
YDAB_MY
GSIA_BA
E64417
A56519
DMUNKNOWN_1-96
SPAC6B12_12-516
NUC2_SC
CELZK320
NFRA_EC
CCU2886
S766850
CELT19A
S766851
S75601
YHBM_EC
HSU4203
YKD1_CA
CELZK32
S56658
G02540
CELC55B
CELF30H
B55508
SMU5458
BSTHRZ1
PSEPILF
CELF38B0
JT06030
JT06031
S76422
CC23_YE0
CELF38B1
CC23_YE1
CELF38B2
MEU8731
CELF38B3
S75615
CELF38B4
CC27_YE0
S74806
CELF38B5
CC27_YE1
CELC56C
F643990
F643991
MTC1_CO
MM284231_1-255
F643992
KNLC_CA
ATAF0
S585440
S585441
A645120
A645121
AB001
I49564
OM70_NE
ATU6213
S75709
CELF32A
CELT09B0
CELT09B1
YCF3_OD
YHR7_YE
SSN6_YE
FLBA_CAUCR-226
BIMA_EM
CUT9_SC0
CC27_HU
MMUTY0
KNLC_ST
S757090
C48583
S757091
S759910
S566581
S759911
S566582
LMU73840
S759912
S566583
LMU73841
S759913
LMU73842
S759914
TPRD_HU
LMU73843
S759915
S60905
YHBM_HA
S759916
LMU73844
S75633
CET12D8
S58544
C485830
BSZ9404
Y366_HA
C485831
NPRA_BA0
YC37_PO
N358_HU
S42210
CC27_YE
HIBN_XE0
STI1_YE0
STI1_YE1
CELF32A0
STI1_YE2
LACALS
CELR05F
G025400
G025401
H64467
G025402
G025403
PTSR_HU0
G025404
PTSR_HU1
G025405
YCA1_PL0
PQ01800
CEC34C61
CEC34C62
SSD900_122-109
STI1_YE
YCV0_YE
HSAB2370_1-631
S75648
CELT19A0
CELT25F0
CEF10B5
CELT25F1
BSAB617_17-292
CEM720
YCV0_YE0
YCV0_YE1
CELC55B0
CELC55B1
HSU46570
HSU46571
BSU5504
HSU46572
HSU46573
HSU46574
LJGLN12
HSU46575
D504537
MMUTY
CELD2020
HSU46576
YB05_YE
HUMFKBP0
HSU46577
CEM72
S75578
PSEPILF0
S450640
BBCDG5
I40554
ACU89981
HSU20361
S668420
HSU20362
ACU89983
S748530
HSU20363
ACU89984
S748531
ACU89985
D63875
ACU89986
YCOA_SY
D644170
YCIM_HA
D644171
YLU28151
S74853
HGV2_HA
CELR05F0
YC37_CY
NCF2_HU
HUMFKBP
JC47510
I40567
CEZK856
S756480
S756481
LEPLIPL
S756482
CUT9_SC
PS43_TO0
RNU76551
S66842
HSU58970
RNU76552
JC4751
HSU58971
RNU76553
HSU58972
RNU76554
YQGP_BA
D907664
AB0010
KNLC_HU
S756010
G02058
YQCH_BA0
S756011
S756012
S760710
CC23_YE
S756013
S760711
CEC34C6
PTSR_PI0
PTSR_PI1
INI6_HU
S75202
YO91_CA
CC16_YE0
CC16_YE1
CC16_YE2
CC16_YE3
ACSC_AC
HSU5251
CC16_YE4
NCU89985_1-41
CC16_YE5
CYP6_YE
D638750
S75685
S76576
D638751
D638752
H644670
RLU3940
H644671
MMU1695
D638753
I495640
OM70_NE0
H644672
D638754
I495641
H644673
OM70_NE1
D638755
CELF38B
JT0603
YHBM_EC0
OM70_NE2
I495642
OM70_NE3
PQ0180
2408032-184
HSU5897
TAFKBP7
YC37_CY0
YC37_PO0
D643710
ATAC01
HSU2036
CELK04G1
ACU8998
ATAC02
YHE3_PS
CELK04G2
SC72_YE
CELK04G3
CELK04G4
CELK04G6
JC4775
ATU62130
ATU62131
ATU62132
ATU62133
YCA1_PL
ATU62134
ATU62135
RNU7655
ATU62136
PS43_TO
D82942
YPU2283
CEUC55B6_3-59
S755780
D86980
D86981
H643320
S55383
YAD5_CL
PPT1_YE
CELC33H
CELC17G
TPRD_HU0
D64417
DMU18291
MXU7705
RSMGPGN0
CEF52H3
CYP4_BO
IEFS_HU0
S76202
IEFS_HU2
F64399
IEFS_HU3
SSN6_YE0
YHR7_YE0
IEFS_HU4
SSN6_YE1
SSN6_YE2
SSN6_YE3
HEMY_EC
SSN6_YE4
SSN6_YE5
RSMGPGN
CYP7_YE
S76685
A55346
CELC18H
S761560
S761561
NASP_HU
S761562
CELF10C0
S761563
C64478
CEF10B50
ATAC98_24-445
A565190
E644170
E644171
E644172
CELF10C
E644173
CYP4_BO0
CYP4_BO1
MMU2783
S752020
ENAC0
PFCYT12
CC16_YE
BSATPC1
ECAE00
ECAE01
ECAE02
YAD5_CL0
PR06_YEAST-701
YAD5_CL1
YPIA_BA
NCF2_HU0
TPU93844_2-211
SKI3_YE
YHJL_EC
ECAE0
C644780
OM70_YE
C644781
C644782
C644783
A64512
LMU7384
C644784
PTSR_HU
HSU4657
CELK04G
YKD1_CA0
YKD1_CA1
S619910
S619911
YKD1_CA2
SR72_CA
CELT09B
MXU77058_2-100
NRFG_EC
NRFF_HA
CELT25F
S45064
S76315
CEAF16427_7-686
BIMA_EM0
BIMA_EM1
S76156
BIMA_EM2
BIMA_EM3
NUC2_SC1
JC4348
LBAPREL
YO91_CA0
PTSR_YE
A60088
BSU55040
ATAC0
D64371
H64332
S748060
S748061
YREC_SY0
YREC_SY1
YREC_SY2
NPRA_BA
JC47750
G020580
PPP5_RA
YQCH_BA
CC27_HU2
S61991
YREC_SY
CC27_HU3
CC27_HU4
Consensus/60% spsbhpbG.h abpbscbppA lphapcAlpl sspp

TABLE 10
Alignment of Ankyrin Repeat Sequences
O04242/1-30
093130/1-30
VB18_VARV/1-32
Q25328/1-30
Q14349_1/1-30
BCL3_HUMAN/1-30
Q24241_4/1-31
O43150/1-30
G3790744/1-30
Q25328_3/1-30
GLP1_CAEEL/1-30
YA2A_SCHPO/1-31
VB04_VACCC/1-30
HT16_HYDAT/1-33
O04703_1/1-30
G3790744_1/1-30
Q25338_1/1-30
Q21920/1-30
O16568/1-30
Q21920_1/1-30
O82630/1-30
O00542/1-31
Q25338_2/1-30
Q86916/1-32
Q23595/1-30
Q21587/1-30
P87621/1-34
YD57_SCHPO/1-33
O04097_1/1-30
P87621_2/1-30
Q84644_1/1-30
O17055_1/1-31
VB18_VARV_1/1-3
HRG_COWPX/1-32
O55222_1/1-30
Q28282/1-30
G3929219_3/1-30
BCL3_HUMAN_1/1-
BCL3_HUMAN_2/1-
O73630/1-30
Q18297_2/1-30
Q94527/1-31
O90757/1-30
HT16_HYDAT_2/1-
P87609_1/1-31
O90757_1/1-30
Q20109_1/1-31
SWI6_YEAST_1/1-
P93755/1-31
Q19995/1-31
O48738/1-30
Q89202/1-30
G3927831_1/1-30
FEM1_CAEEL_3/1-
Q18297_3/1-30
Q38898_1/1-30
HRG_COWPX_2/1-3
O43988/1-30
O43988_1/1-30
AKR1_YEAST/1-30
Q21920_5/1-30
P87621_3/1-32
O18270_1/1-30
VC17_VACCC/1-33
VC09_VACCC/1-30
YIA1_YEAST/1-30
Q89540_1/1-32
Q25338_5/1-31
O17055_3/1-30
O44872/1-30
1790447/1-30
HRG_COWPX_3/1-3
Q21587_2/1-30
VB18_VARV_2/1-3
VB04_VACCC_1/1-
O75407/1-30
P87621_4/1-31
O54807_1/1-31
FEM1_CAEEL_4/1-
Q84566/1-33
Q27105_1/1-28
O13075/1-30
G3930527_4/1-30
G3925387_5/1-30
O88849_6/1-30
O88849_7/1-30
O17055_4/1-30
Q18297_5/1-30
FEM1_CAEEL_5/1-
O75762_4/1-30
O14586_1/1-30
P90784/1-30
Q94527_1/1-30
O54807_2/1-31
O75762_5/1-30
157038_1/1-31
Q02989_4/1-30
O60736_1/1-32
ANK1_MOUSE_18/1
O61222_1/1-33
Q62422/1-31
Q24241_15/1-30
YD57_SCHPO_2/1-
G3927831_2/1-32
P70770_1/1-30
Q94527_2/1-30
P72763_4/1-30
O60733_2/1-30
KBF1_MOUSE/1-30
O88849_9/1-31
O82630_1/1-30
YAHD_ECOLI/1-30
Q84644_3/1-30
157038_2/1-30
1790447_1/1-34
O75762_6/1-31
LI12_CAEEL_2/1-
G3970962_1/1-30
VC09_VACCC_2/1-
O83807_2/1-31
O23296/1-30
G3927831_3/1-30
YB07_FOWPM_3/1-
RN5A_MOUSE_2/1-
O54910_1/1-30
O44997_4/1-32
O00306/1-30
O61222_2/1-30
O74205/1-30
VB18_VARV_3/1-3
P87603_1/1-33
O82630_2/1-30
Q25328_4/1-30
JC4356/1-30
O50999/1-30
Q20109_3/1-30
GLP1_CAEEL_2/1-
Q86916_1/1-33
Q92527_1/1-30
O90757_4/1-30
I50404_2/1-32
Q94400_1/1-31
Q83730/1-32
O44997_5/1-30
Q25338_6/1-30
O90760_3/1-30
391941_1/1-30
1790447_2/1-34
Q02979_2/1-30
O74205_1/1-30
Q86916_2/1-32
Q94527_3/1-31
O15084_16/1-30
YG4X_YEAST_1/1-
O18270_5/1-30
Q14349_4/1-30
JQ1744_2/1-34
PLU_DROME/1-32
O54807_4/1-31
O15084_17/1-30
Q93203_1/1-30
O88849_11/1-31
Q17643/1-30
O48738_2/1-30
1790447_3/1-30
Q02989_9/1-31
Q19995_1/1-31
O90760_4/1-30
Q89340_5/1-31
O90757_5/1-31
Q20313_1/1-30
Q89202_2/1-29
YMV8_YEAST_2/1-
157038_3/1-30
AKR1_YEAST_2/1-
Q18297_6/1-30
O68219/1-30
Q20109_5/1-30
Q25328_7/1-30
O61222_4/1-30
Q21587_5/1-30
O61222_5/1-30
Q21920_13/1-30
Q83730_1/1-30
Q18297_7/1-30
Q18663_1/1-31
Q25328_8/1-31
O18152_4/1-30
O54807_6/1-30
O73630_3/1-30
35040_3/1-30
Q19995_2/1-33
DAPK_HUMAN_3/1-
Q25328_9/1-30
O43150_1/1-33
YAR1_YEAST_1/1-
Q84566_2/1-31
S57237/1-30
35040_4/1-33
Q18587_1/1-30
Q02989_10/1-30
Q09493_1/1-32
O15084_18/1-30
Q21920_15/1-33
O83807_6/1-30
D1037943_4/1-30
YB07_FOWPM_4/1-
O45398_1/1-30
O49409_1/1-30
O83807_8/1-30
S57237_1/1-31
JQ1744_3/1-30
E1350345_1/1-30
P87611_1/1-35
Q23595_2/1-30
P90902_2/1-30
VC17_VACCC_1/1-
JQ1744_4/1-32
Q93203_2/1-30
O45398_2/1-30
O00522_1/1-31
Q02989_11/1-30
YMV8_YEAST_3/1-
Q07045_4/1-35
O15084_20/1-30
Q18297_8/1-30
O24538_2/1-32
O55014_3/1-30
O45398_3/1-33
O17055_5/1-30
O15084_21/1-30
O88849_14/1-33
Q02989_13/1-30
O23295_1/1-30
Q14349_5/1-30
Q12013_1/1-30
Q40785_2/1-30
DAPK_HUMAN_5/1-
O18270_6/1-34
Q86916_3/1-30
AKR_ARATH_2/1-3
O16004_2/1-31
O16004_3/1-41
YA2A_SCHPO_4/1-
Q18297_9/1-30
O35433_1/1-31
O54807_8/1-30
P87603_3/1-40
O73579_3/1-30
Q21587_6/1-33
A53950_3/1-30
Q18663_3/1-31
Q25328_11/1-31
O72760_2/1-36
Q89342_2/1-30
O83807_9/1-30
Q83730_2/1-33
O04242_3/1-32
O82490_1/1-30
O45398_4/1-30
P87600_4/1-35
O73560_2/1-48
P87603_5/1-33
Q14678_3/1-29
Q93318_1/1-38
G3930525_6/1-30
YB07_FOWPM_5/1-
JQ1744_5/1-31
VB04_VACCC_2/1-
O41154_3/1-35
P87600_5/1-36
Q21920_17/1-31
O44997_6/1-30
P72763_6/1-30
Q09493_2/1-31
Q24145_1/1-30
Q17583_2/1-31
O90757_6/1-30
MBP1_YEAST_1/1-
O83807_10/1-30
G4103857_2/1-32
O18270_7/1-30
O68219_2/1-31
Q19995_4/1-30
O45398_5/1-29
Q12013_3/1-30
O18152_5/1-31
Q89540_4/1-32
Q17643_1/1-30
Q63618_3/1-31
O14586_2/1-30
Q17583_4/1-31
O68219_3/1-32
Q93318_2/1-31
O54807_9/1-30
Q01317_4/1-30
O82490_2/1-30
YAHD_ECOLI_2/1-
Q63618_4/1-30
Q89440_2/1-44
P87601_1/1-38
O18152_8/1-31
1AP7_1/1-30
O17055_6/1-32
Q18297_10/1-32
157038_4/1-32
O75762_9/1-30
O23296_4/1-31
TRI9_HUMAN_3/1-
Q23595_3/1-32
VC17_VACCC_2/1-
Q18297_11/1-30
NTC4_MOUSE_4/1-
O54807_10/1-30
O68219_4/1-31
O18270_8/1-32
O18152_9/1-31
Q18297_12/1-30
VC09_VACCC_4/1-
1AP7_2/1-29
P87600_6/1-35
O75762_10/1-30
P87603_6/1-35
O48738_5/1-30
P87611_2/1-30
Q25328_13/1-31
Q02979_3/1-31
Q01317_5/1-30
Q17343_21/1-30
Q19995_6/1-31
P90902_3/1-30
O68219_5/1-34
Q83730_3/1-30
E1344043_2/1-30
O45398_8/1-31
Q12013_4/1-31
O24382_4/1-31
4151809_3/1-30
O73579_4/1-36
O15084_23/1-32
Q20109_8/1-31
O04703_2/1-33
Q18970_1/1-31
Q18663_4/1-30
O72755_3/1-40
Q25338_10/1-30
O54807_12/1-30
O45398_10/1-30
Q21920_18/1-31
Q21587_7/1-29
VC09_VACCC_5/1-
VC17_VACCC_3/1-
VB04_VACCC_3/1-
Q18970_2/1-33
Q23859_3/1-30
Q25328_14/1-30
Q19995_7/1-30
VB04_VACCC_4/1-
O88202_1/1-31
E1350208_1/1-30
O54807_13/1-32
Q23859_4/1-34
O04242_4/1-31
Q93203_3/1-30
Q17583_5/1-30
YIA1_YEAST_1/1-
O35433_2/1-30
O83515_2/1-30
O23295_4/1-30
AKR_ARATH_3/1-3
Q83730_4/1-35
O72760_5/1-43
Q63618_7/1-32
YG4X_YEAST_4/1-
G3786431_4/1-34
O73579_5/1-32
Q01317_6/1-32
O73579_6/1-33
O83807_13/1-31
LI12_CAEEL_4/1-
Q89202_3/1-30
Q24241_22/1-34
Q93318_4/1-31
Q25328_17/1-32
P70770_2/1-30
VB04_VACCC_5/1-
VB04_VACCC_6/1-
O04704_2/1-34
O45398_12/1-31
TRPL_DROME_2/1-
JQ1744_6/1-32
Q84566_5/1-31
O49409_2/1-30
JQ1744_7/1-35
Q02979_4/1-30
O24538_3/1-27
Q89202_4/1-30
O83807_14/1-30
G3786431_5/1-31
O62398_2/1-27
Q25338_11/1-30
Q23595_4/1-31
Q25328_18/1-30
Q28282_5/1-31
Q94527_4/1-29
AKR1_YEAST_5/1-
391941_4/1-31
Q23595_5/1-30
P87603_8/1-29
O72755_4/1-42
O18270_9/1-31
Q90623_5/1-31
O61222_7/1-30
JQ1744_9/1-33
P87621_5/1-32
O16229_1/1-32
VB18_VARV_5/1-3
O90757_7/1-30
Q93318_5/1-32
Q21920_19/1-31
Q02989_18/1-31
A55839_4/1-31
O60733_5/1-31
O83807_16/1-31
O75762_11/1-30
391941_5/1-30
Q94447_2/1-30
Q18104_2/1-33
O48738_6/1-31
O61222_8/1-46
O73579_8/1-30
O23296_6/1-31
Q09493_4/1-30
Q83730_5/1-34
Q91974_4/1-34
O61240_6/1-32
Consensus/60% psp*sLabAs pp.....spb chlcbLlpps s....shsh

TABLE 11
Alignment of Armadillo Repeat Sequences
IMO2HUMANb
MMU34228c
cATAF00130
cATKAPAPRO
ATU69533D
AB002533c
HSSRP11a
HSSRP1Bc
CELF32E10c
SRP1YEASTd
IMO1HUMANc
bATAF00130
bATKAPAPRO
SLU96718b
ATU69533b
IMO2HUMANc
MMU34228a
AB002533b
HSSRP1Bb
CELF32E10b
IMO1HUMANa
SRP1YEASTb
CEF53B24
CELF26B13b
AB002533a
HSSRP1Ba
CELF32E10a
CELF26B13a
aATAF00130
aATKAPAPRO
ATU69533a
SLU96718a
IMO2HUMANa
SRP1YEASTa
IMO1HUMAN1
AB002533e
HSSRP11c
CELF32E10e
IMO2HUMANf
MMU34228e
eATAF00130
ATU69533f
SRP1YEASTf
IMO1HUMANe
CELF26B13e
AB002533d
HSSRP11b
CELF32E10d
IMO1HUMANd
IMO2HUMANe
MMU34228d
dATAF00130
ATU69533e
SRP1YEASTe
CELF26B13d
CRU40057b
CRU40057e
CRU40057d
CRU40057c
CTNBMOUSEk
JC4835h
CTNBMOUSEj
HSPLGLNj
JC4835g
HSP0071a
HSU96136a
HSU51269a
P120MOUSEa
S60712a
HSRNAUa
CTNBMOUSEh
JC4835e
YEB3YEASTb
YEB3YEASTf
P120MOUSEb
HSU51269b
HSP0071b
HSU96136b
S60712b
HSRNAUb
HSU96136c
HSP0071c
HSU51269c
P120MOUSEc
CTNBMOUSEi
JC4835f
CTNBMOUSEc
JC4835a
JC6161b
HSU59919b
SPU38655b
CTNBMOUSEf
JC4835d
CELC54D15
APCHUMAc
XLU64442c
DMU77947c
JC6161a
HSU59919a
SPU38655a
CTNBMOUSEd
JC4835b
CTNBMOUSEe
JC4835c
HSP0071d
HSU96136d
IMO1HUMANb
ATU69533c
CELF32E10-
AB002533z
IMO2HUMAN-
MMU34228b
SRP1YEASTc
IMOB_RAT
HUMNTF9
APCHUMANe
XLU64442d
DMU77947d
APCHUMANb
XLU64442b
DMU77947b
fATAF00130
dATKAPAPRO
ATU69533g
IMO2HUMANg
MMU34228f
SRP1YEASTg
IMO1HUMANf
AB002533f
HSSRP11d
CELF32E10g
APCHUMANa
XLU64442a
DMU77947a
CTNBMOUSE1
JC4835i
CELF08F8
GDS1HUMANc
AT81KBGEN4
S51350
CELF26B13c
HSU51269d
CELF26B13-
YEB3YEASTd
GDS1HUMANa
YEB3YEASTe
CTNBMOUSEa
ADBHUMAN
S60712c
HSRNAUd
YEB3YEASTa
HSRNAUc
CTNBMOUSEb
CET19B106
YSPPAA1B
CRU40057a
D87671
CELM01E114
HSZYGHOMO
YLK3CAEELa
GDS1HUMANb
COPBYEASTa
CEC48D1
YEB3YEASTc
GDS1BOVINc
APCHUMANd
P115_BOVIN
YD71_SCHPO
CELB033611
Consensus/60% ssp.pbphlb pss..slshL lpLLp..... .p.s.plbp. tshslpNls.

REFERENCES

• Bondeson, D. P., Mares, A., Smith, I. E. D., Ko, E., Campos, S., Miah, A. H., Mulholland, K. E., Routly, N., Buckley, D. L., Gustafson, J. L., et al. (2015). Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611-617.
• Boudko, S. P., Londer, Y. Y., Letarov, A. V, Sernova, N. V, Engel, J., and Mesyanzhinov, V. V (2002). Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled-coil protein. Eur. J. Biochem. 269, 833-841.
• Brunette, T. J., Parmeggiani, F., Huang, P.-S., Bhabha, G., Ekiert, D. C., Tsutakawa, S. E., Hura, G. L., Tainer, J. A., Baker, D. (2015) Exploring the repeat protein universe through computational protein design. Nature 528, 580-584.
• Chapman & McNaughton, B. R. (2016). Scratching the surface: Resurfacing proteins to endow new properties and function. Cell Chem. Biol. 23, 543-553.
• D'Andrea, L. D., and Regan, L. (2003). TPR proteins: the versatile helix. Trends Biochem. Sci. 28, 655-662.
• Deshaies, R. J. (2015). Protein degradation: Prime time for PROTACs. Nat. Chem. Biol. 11, 634-635.
• de Vries, S. J., and Bonvin, A. M. J. J. (2011). CPORT: a consensus interface predictor and its performance in prediction-driven docking with HADDOCK. PLoS One 6, e17695.
• de Vries, S. J., van Dijk, M., and Bonvin, A. M. J. J. (2010). The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 5, 883-897.
• Guettler, S., LaRose, J., Petsalaki, E., Gish, G., Scatter, A., Pawson, T., Rottapel, R., and Sicheri, F. (2011). Structural basis and sequence rules for substrate recognition by Tankyrase explain the basis for cherubism disease. Cell 147, 1340-1354.
• Güthe, S., Kapinos, L., Moglich, A., Meier, S., Grzesiek, S., and Kiefhaber, T. (2004). Very Fast Folding and Association of a Trimerization Domain from Bacteriophage T4 Fibritin. J. Mol. Biol. 337, 905-915.
• Hao, B., Zheng, N., Schulman, B. A., Wu, G., Miller, J. J., Pagano, M., Pavletich, N. P. (2005). Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase. Mol. Cell 20, 9-19.
• Kobe, B. & Kajava, A. V. (2000). When protein folding is simplified to protein coiling: the continuum of solenoid protein structures. Trends in Biochem. Sci. 25, 509-515.
• Lee, J.-H., Kang, E., Lee, J., Kim, J., Lee, K. H., Han, J., Kang, H. Y., Ahn, S., Oh, Y., Shin, D., et al. (2014). Protein grafting of p53TAD onto a leucine zipper scaffold generates a potent HDM dual inhibitor. Nat. Commun. 5, 3814.
• Leshchiner, E. S., Parkhitko, A., Bird, G. H., Luccarelli, J., Bellairs, J. A., Escudero, S., Opoku-Nsiah, K., Godes, M., Perrimon, N., and Walensky, L. D. (2015). Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. Proc. Natl. Acad. Sci. U.S.A 112, 1761-1766.
• Longo, L. M. & Blaber, M. (2014). Symmetric protein architecture in protein design: to-down symmetric deconstruction. Methods Mol. Biol. 1216, 161-82.
• Lu, J., Qian, Y., Altieri, M., Dong, H., Wang, J., Raina, K., Hines, J., Winkler, J. D., Crew, A. P., Coleman, K., et al. (2015). Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem. Biol. 22, 755-763.
• Margarit, S. M., Sondermann, H., Hall, B. E., Nagar, B., Hoelz, A., Pirruccello, M., Bar-Sagi, D., and Kuriyan, J. (2003). Structural evidence for feedback activation by Ras.GTP of the Ras-specific nucleotide exchange factor SOS. Cell 112, 685-695.
• Meier, S., Guthe, S., Kiefhaber, T. and Grzesiek, S. (2004). Foldon, the natural trimerization domain of T4 fibritin, dissociates into a monomeric A-state form containing a stable beta-hairpin: atomic details of trimer dissociation and local beta-hairpin stability from residual dipolar couplings. J. Mol. Biol 344, 1051-1069.
• Parmeggiani, F., Huang, P.-S., Vorobiev, S., Xiao, R., Park, K., Caprari, S., Su, M., Seetharaman, J., Mao, L., Janjua, H., Montelione, G. T., Hunt, J., Baker, D. (2015) A general computational approach for repeat protein design. J. Mol. Biol. 427, 563-575.
• Rowling, P. J., Sivertssson, E. M., Perez-Riba, A., Main, E. R., Itzhaki, L. S. (2015) Biochem. Soc. Trans. 43 881-888.
• Tamaskovic, R., Simon, Stefan, N., Scwhill, Pluckthun, A. (2012). Designed ankyrin repeat proteins (DARPins): From research to therapy. Methods in Enzym. 503, 101-134.
• Thompson, D. B., Cronican, J. J., Liu, D. R. (2012). Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells. Methods Enzymol. 503, 293-319.

Claims

1. A chimeric protein comprising two or more repeat domains linked by inter-repeat loops; and one or more heterologous peptide ligands that bind to a target molecule, each said peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein.

2. A chimeric protein according to claim 1 wherein the repeat domains are helix-turn-helix repeat domains.

3. A chimeric protein according to claim 2 wherein the repeat domains are tetratricopeptide (TPR) repeat domains.

4. A chimeric protein according to claim 3 wherein the repeat domains have the amino acid sequence Y-X1X2X3X4; wherein Y is an amino acid sequence shown in any of Tables 4 to 6 or a variant thereof and X1, X2, X3, X4 are independently any amino acid.

5. A chimeric protein according to claim 4 wherein the repeat domains have the amino acid sequence;

6. A chimeric protein according to claim 1 comprising 2-5 repeat domains.

7. A chimeric protein according to claim 1 wherein the peptide ligands are located in one or more inter-repeat loops, and optionally are connected to the inter-repeat loops by a linker, and optionally wherein the peptide ligands are non-hydrophobic.

8. A chimeric protein according to claim 1 wherein a peptide ligand is located at the N terminus, the C terminus or at both the N and C termini, thereby to provide an N terminal peptide ligand and/or a C terminal peptide ligand.

9. A chimeric protein according to claim 8 wherein the N terminal peptide ligand, the C terminal peptide ligand or both of the N and C terminal peptide ligands comprises an α helix.

10. A chimeric protein according to claim 9 wherein the N terminal peptide ligand comprises the sequence Xn-XYXXXIXXYXXXLXX-X1X2XX, where residues denoted by X are independently any amino acid, X1, and X2 are independently any amino acid and n is 0 or any number, and optionally wherein X1 is D and/or wherein X2 is P.

11. A chimeric protein according to claim 9 wherein the C terminal peptide ligand comprises the sequence X1X2XX-XXAXXXLXX[AV]-XXXXX-Xn, where residues denoted by X are independently any amino acid, X1, and X2 are independently any amino acid and n is 0 or any number, and optionally wherein X1 is D and/or wherein X2 is P.

10. A chimeric protein according to claim 1 wherein the target molecule is β-catenin, KRAS, tankyrase, c-myc, n-myc, ras, notch and aurora A, α-synuclein, β-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, or oncogenic histone methyltransferase.

11. A chimeric protein according to claim 1 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds an E3 ubiquitin ligase.

12. A chimeric protein according to claim 11 comprising (i) an N terminal peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or(ii) an inter-repeat peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or(iii) an inter-repeat peptide ligand that binds a target protein and an N terminal peptide ligand that binds an E3 ubiquitin ligase, or(iv) a C terminal domain that binds a target protein and an N terminal peptide ligand that binds an E3 ubiquitin ligase, or(v) an inter-repeat binding domain that binds an E3 ubiquitin ligase and an N terminal binding domain that binds a target protein, or(vi) an inter-repeat binding domain that binds an E3 ubiquitin ligase and a C terminal binding domain that binds a target protein.

13. A method of producing a chimeric protein comprising: (a) inserting a first nucleic acid encoding a binding domain into a second nucleic acid encoding two or more repeat domains linked by inter-repeat loops to produce a chimeric nucleic acid encoding a chimeric protein according to claim 1; andexpressing said chimeric nucleic acid to produce the chimeric protein; or(b) providing a nucleic acid encoding two or more repeat domains linked by inter-repeat loops; andincorporating into said nucleic acid a first nucleotide sequence encoding a first binding domain that binds to a first target molecule and a second nucleotide sequence encoding a second binding domain that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein according to claim 1 comprising said first and second binding domains, wherein said binding domains are located in an inter-repeat loop or at the N or C terminus of the chimeric protein; andexpressing said chimeric nucleic acid to produce the chimeric protein.

14. A method of producing a library of chimeric proteins comprising; (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising(i) two or more repeat domains(ii) inter-repeat loops linking said repeat domains; and(iii) one or more binding domains, each said binding domain being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,wherein the binding domains in said population are diverse, and(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.

15. A method of screening a library comprising; (a) providing a library produced according to the method of claim 14,wherein at least one amino acid residue in the binding domains in said library is diverse,(b) screening the library for chimeric proteins which display a binding activity, and(c) identifying one or more chimeric proteins in the library which display the binding activity.