Patent Application
20080167385
The present invention relates to proliferating cell nuclear antigen (PCNA) and small molecule inhibitors thereof. More specifically, the invention relates to crystals comprising human PCNA, and methods and assays for designing and identifying small molecule PCNA inhibitors using said crystals.
PCNA is an essential auxiliary protein for the processes of both DNA replication and repair. It stimulates the activity of DNA polymerase δ (pol δ) and increases its processivity1 by acting as a clamp platform that slides along the DNA template2. Apart from pol δ, PCNA associates with a host of other proteins, either involved directly in DNA replication and repair, or in the regulation of these processes3. The presence of a common PCNA-binding motif in such proteins suggests that regulation may depend largely on PCNA partner proteins competing with one another for access to PCNA.
Proteins involved in cell-cycle control, particularly the tumour suppressor protein p21 (also known as WAF1, CAP20, Cip1, and Sdi1), also participate in these interactions4. In particular, the induction of p21 after DNA damage leads to inhibition of cell-cycle progression and DNA replication. This effect is not only due to CDK inhibition, but also results from direct binding to PCNA, thereby interfering with PCNA-dependent DNA synthesis5,6, while permitting DNA repair7,8. p21 contains a CDK-binding site in its N-terminal region (residues 53-58), which is distinct from two cyclin-binding sites located in the N— and C-terminal regions respectively9,10. The PCNA-binding motif present in the C-terminus of p21 has been characterized extensively11,12 and is conserved in many other PCNA protein partners3, thus supporting the notion that PCNA plays multiple roles in DNA replication and repair, as well as in cell-cycle regulation13. At the cellular level the competition between p21 and DNA replication factors for binding to PCNA is believed to be the mechanism through which DNA synthesis is inhibited. It is known that in cells PCNA and p21 can participate in quaternary complexes with CDK/cyclin pairs, particularly CDK4/cyclin D114,15 probably contributing to the coordination of cell cycle progression and DNA replication16.
Deregulation of PCNA expression is a hallmark of many proliferative diseases and in the clinic PCNA serves as a general proliferative marker, especially in the prognosis of tumour development17. In fact PCNA expression levels are directly related to the malignancy of various tumours and antisense oligonucleotide-mediated suppression of PCNA expression was demonstrated to selectively inhibit gastric cancer cell proliferation in vitro and in vivo18. Antisense strategies targeting PCNA mRNA have also shown promise in models of other proliferative diseases, including glomerular nephritis19, restenosis20 and rheumatoid arthritis21. The fact that PCNA is required absolutely for cell proliferation indicates that pharmacological modulation of PCNA function should not be able to be circumvented by compensatory pathways. Furthermore, the ablation of PCNA expression or function in cells under proliferative stimuli appears to constitute an apoptotic trigger17, suggesting that effective elimination of hyper-proliferative cells should be possible in a therapeutic setting. Collectively these facts indicate that PCNA may represent an attractive target for intervention in proliferative disease.
Interest in p21 as a starting point for the peptidomimetic design of anticancer agents has stemmed from observations that synthetic peptides derived from the C-terminus of this protein, when rendered cell-permeable22, are capable of arresting and killing cancer cells23-25. Interestingly, the C-terminus of p21 harbours overlapping recognition sites for both cyclins and PCNA. For the purposes of structure-based drug design there is therefore an interest in defining and delineating the relevant recognition sites in PCNA and cyclins responsible for the antiproliferative effects of the p21-derived peptides.
Previous studies have determined the structure-activity relationships of peptides related to p21(141-160) with respect to both PCNA- and cyclin binding26,27. The p21 sequence in question is 141KRRQTSMTDFYHSKRRLIFS160 (determinant residues for PCNA and cyclin interactions are shown in italics and bold face, respectively). Analysis of the motif conserved in many PCNA-binding proteins3 demonstrated that it takes the form QXXhXXaa, where X represents any amino acid, h indicates moderately hydrophobic residues, and a corresponds to aromatic hydrophobic residues; i.e. in the case of p21 144QTSMTDFY151 Ref.11,25. As far as the interaction with G1- and S-phase cyclins (D, E, and A) is concerned, p21(141-160) peptide truncation and substitution studies showed that the sequence 155RRLIF159 was necessary and sufficient for effective inhibition of CDK4- and CDK2-associated kinase activity26,27. Pentapeptides based on this minimal sequence represent a starting point for the development of a new class of selective CDK inhibitor drugs.
Truncation of p21(141-160) peptides from either terminus was not tolerated without a serious diminution of the binding affinity for PCNA. This suggested that it would not be possible to design small tight-binding peptides or peptide analogues that would inhibit PCNA. Furthermore, separation of the cyclin-binding properties from PCNA affinity appeared difficult. On the basis of the sequences of two particular PCNA binding proteins, the Pogo DNA transposase and DNA ligase I (
The present invention seeks to elucidate structural information on the binding interactions between PCNA, p21, and CDK/cyclin. Specifically, the invention seeks to elucidate information on the 3-dimensional structure of the PCNA binding domain and the nature of the binding interactions between PCNA and compounds capable of modulating PCNA. The invention further seeks to provide assays and methods for identifying candidate compounds capable of modulating PCNA.
The present invention relates to various crystal structures comprising human PCNA, and their use in the identification of compounds capable of binding to and/or modulating PCNA.
Specifically, binding studies carried out in the context of the present invention suggest the formation of a quaternary complex between PCNA, p21, and CDK/cyclin, in which a 20mer peptide is sufficient to mimic the assembly role of full-length p21. A structural model of the complex shows how p21 can act like double-sided tape to bind to both PCNA and cyclin/CDK. The invention also provides a complex structure of PCNA and the CM peptide, as well as the first X-ray structures of free human PCNA. These X-ray and model structures delineate a well-defined surface binding-pocket in PCNA that can be used for the design of inhibitors of PCNA-dependent DNA replication.
Aspects of the invention are presented in the accompanying claims and are further described in the following paragraphs.
A first aspect of the invention relates to a crystal comprising human proliferating cell nuclear antigen (PCNA).
In one preferred embodiment, the crystal of the invention is human PCNA.
In one particularly preferred embodiment, the crystal is monoclinic. More preferably, the crystal is of space group C121.
In one preferred embodiment, the crystal comprises a unit cell having the following unit dimensions: a=136.6 Å, b=83.26 Å, c=71.63 Å.
In a highly preferred embodiment, the crystal comprises the atomic coordinates set forth in Table 3.
In another preferred embodiment of the invention, the crystal is trigonal. More preferably, the crystal is of space group P3.
In one preferred embodiment, the crystal comprises a unit cell having the following unit dimensions: a=82.89 Å, b=82.89 Å, c=70.86 Å.
In a highly preferred embodiment, the crystal comprises the atomic coordinates set forth in Table 4.
In another preferred embodiment, the crystal of the invention comprises human PCNA and a ligand.
Preferably, the ligand is a peptide structurally related to p21.
In one particularly preferred embodiment, the ligand is a peptide of formula I
In one especially preferred embodiment, the crystal comprising human PCNA and a peptide of formula I comprises a unit cell having the following unit dimensions: a=119.1 Å, b=119.1 Å, c=305.82 Å.
Even more preferably, the crystal comprising human PCNA and a peptide of formula I comprises the structural coordinates set forth in Table 5.
More preferably still, the crystal comprises one or more of the following interactions between human PCNA and residues 3 to 15 of said peptide of formula I: V3(N)-I255(O), L4(N)-I255(O), L4(O)-I255(N), Q5(OE1)-W227(O), Q5(NE2)-A252(O), Q5(NE2)-P253(O), K6(N)-P253(O), K6(CG)-I255(CG1), I8(N)-H44(O), I8(CD1)-P234(CB), I8(CD1)-Y250(CB), I8(CD1)-Y250(C), I8(CG2)-L47(CD1), I8(CG1)-V45(C), Y11(CG)-P234(CD), Y11(CD1)-P234(CD), Y11(CZ)-P234(CD), Y11(CZ)-P234(CG), P12(CD2)-P129(CD), P12(CE2)-P129(CD), P12(CZ)-P234(CG), P12(CZ)-Y250(CD2), P12(CE1)-Y250(CD2), P12(CE1)-W364(O), H13(O)-G127(N), P14(CA)-L126(CD2) and K15(N)-Q125(O).
In one preferred embodiment, the peptide of formula I comprises one or more of the following intramolecular H-bonds: Q5(NE2)-K6(O), K7(O)-D10(N), I8(O)-Y11(N), I8(O)-F12(N) and D10(N)-D10(OD1).
In another preferred embodiment, the crystal of the invention as described above comprises a ligand binding domain.
Another aspect of the invention relates to a crystal comprising a human PCNA ligand binding domain. Preferably, the crystal has a ligand associated therewith.
Preferably, the ligand binding domain comprises amino acid residues selected from one or more of the following amino acid residues: I255, P253, A252, Y250, P234, P129, G127, L126, Q125, L47, V45, H44, W227 and W364.
Details of data collection and refinement for the X-ray structures of the CM peptide-PCNA complex and the two free forms of human PCNA are given in Table 1. Together they provide structural information on 11 crystallographically independent PCNA molecules. The PCNA-CM peptide complex structure (
The two native PCNA crystal structures (Table 1) offer the first published information on the conformations of free human PCNA. These crystals belong to space groups C2 and P3 and together they contain a total of five crystallographically independent PCNA monomer structures: the C2 form has one trimer in the asymmetric unit, whereas the P3 form has two independent (crystallographically exact) trimers in the unit cell. A root-mean-square (RMS) fit of 0.16 Å for the P3 trimer Cα atoms shows that they are essentially identical. The overall fit of the P3 and C2 trimer rings, however, shows a buckling of the C2 trimer compared with the crystallographically constrained planar rings in the P3 structure: a fit of Cα atoms from any one subunit of the C2 structure onto the P3 structure, excluding the two regions of high mobility (residues 115-133 and the protruding loop 184-194), gives a RMS fit for the 218 Cα atoms of around 0.7 Å. However, the average RMS fit of Cα atoms of the other two subunits are near 1 Å and 2 Å, respectively. The individual atomic anisotropic temperature factors for each of the 11 PCNA monomers show a very similar pattern. Regions with high temperature factors correspond to exposed flexible loops on the PCNA surface (residue numbers 62-66, 92-95, 120-130, 161-165, and 184-189). The distribution of high B-factors associated with the flexible loops is always similar and is independent of the three different crystal packing arrangements presented here, indicating that the loop regions are inherently flexible. The conserved flexible regions are highlighted in
Despite substantial differences in amino acid composition, the N-terminal octapeptide helical sequence of CM peptide and the PCNA-bound 22mer fragment of p2111 show similar conformations and interactions with PCNA (
The conformations of the bound CM and p21 peptide structures diverge significantly at the C-terminal end of the helix; H13 (H152) adopt different conformations in the two complexes, with the side chains pointing in different directions. The C-terminal 8 residues of the p21 peptide fold down onto the extended linker residues (L126 to M129), forming a stretch of antiparallel β-sheet. In contrast the PKK terminal residues of the CM peptide straddle the linker strand with P14 providing a bridge clamped by two main chain H-bonds H13(O)-G127(N) and K15(N)-Q125(O). The conformation of the linker sequence (residues 119-134) is clearly different in the p21 22mer and CM 16mer peptide complexes (
p21 interacts with both cyclin/CDK complexes and also with PCNA and the results presented here suggest that a region of p21 can act like double-sided tape to glue both cyclin/CDK and PCNA together to form a quaternary complex. It has previously been shown that both the CM peptide and a C-terminal p21 peptide (residues 141-160) bind PCNA with nanomolar affinity28. Furthermore, the CM peptide inhibits in vitro SV40 DNA replication with a potency very similar to that of p21(141-160). However, whereas the CM peptide is devoid of CDK/cyclin inhibitory activity, the p21(141-160) peptide inhibits CDK4/cyclin D1 kinase activity with an IC50 of 24 μM (
The direct effect of PCNA on CDK4/cyclin D1 kinase activity was studied. At up to 10 μM PCNA the kinase activity of 1 μM CDK4/cyclin D1 was undiminished (
X-ray crystal structures of CDK/cyclin complexes with inhibitory peptides bound in the so-called cyclin binding groove are known27. The cyclin binding motif present in substrates and other protein partners of A-, D-, and E-type cyclin/CDK complexes has been defined as the sequence ZRXLYY′, where Z and X are basic residues, and Y and Y′ are hydrophobic34. The minimal sequence in p21 that binds cyclins is 155RRLIF159. Intriguingly, this pentapeptide corresponds to the C-terminal extension in the PCNA/p21 fragment complex that forms an exposed antiparallel sheet with a section of the interdomain linker (PCNA residues 122-132). As discussed above, the CM peptide does not inhibit CDK function, whereas the p21 peptide with the additional 5 amino acid cyclin-binding motif sequence is an effective inhibitor. It is also striking that the conformation of the RRLIF sequence in the PCNA X-ray structure11 is very similar to that found in our CDK2/cyclin A/RRLIF peptide complex. Furthermore, the side chains protrude in a way that allows the p21 peptide to act like double-sided sticky tape, with one face forming contacts with PCNA and the other face forming complementary contacts with the cyclin groove. It was possible to simply combine the two available X-ray structures and overlay the backbone of the RRLIF structure to give the large quaternary complex of PCNA/p21/CDK2/cyclin A shown in
The RMS fit of the backbone atoms in the RRLIF peptide for this docked structure is less than 0.5 Å. Surprisingly, even without further modelling or refinement, and despite the tight complementary fit between RRLIF and the two proteins, the docked configuration introduces fewer than 10 direct non-bonded contacts under 3.5 Å between PCNA and cyclin; most of theses involve the side chains D283 and I213 on cyclin A, interacting with side chains from N95, D120, D122, and Q125 on PCNA. Restrained molecular mechanics, keeping all main-chain atoms fixed, was used to optimize side chain contacts. Using this model the calculated buried surface areas for cyclin (in the context of RRLIF and PCNA) and PCNA (in the context of RRLIF and cyclin) are 440 Å2 and 340 Å2, respectively, giving a total buried surface of 780 Å2. The total buried surface calculated using a similar model of cyclin docked onto PCNA, but without the RRLIF peptide present, is only 270 Å2, which would be insufficient for stable complex formation. This suggests that for this docking site on PCNA the p21 peptide can act as a genuine adaptor molecule and would be required before cyclin/CDK could be recruited to the PCNA complex.
Initial binding experiments showed that it was not possible to truncate either terminus of the p21(141-160) peptide without significant loss of affinity and it was only the use of different amino acid sequences, less related to p21, that led to shorter peptides with appreciable binding affinity to PCNA28. Without the benefit of the CM peptide structure presented here, it was not obvious which features of the PCNA interaction are most important for the binding. In particular, it was not clear whether the contacts formed by the 9 C-terminal peptide residues 152HSKRRLIFS160 in the p21 peptide/PCNA complex11 played a key role. The present studies show that this interaction, along with the short- and long-range electrostatic interactions involving 155RR156 and 140RKRR143 at the C and N-terminal ends of the peptide, do not seem to be a requirement. These are important considerations in terms of drug design, since incorporation into peptidomimetics of groups capable of multiple Coulombic interactions—so important in protein-protein interactions—does not usually permit the development of permeable and drug-like small molecules.
The solvent-accessible surface on PCNA that is buried when a complex is formed with p21(139-160) has an area of 960 Å2. This compares with a buried surface area of 680 Å2 on complex formation with CM-peptide. The X-ray structures show that the p21(139-160) complex forms some 15 direct H-bonds with PCNA. This compares with only 8 direct H-bonds between the CM peptide and PCNA. Despite these differences the CM peptide has comparable affinity for PCNA (Kd=100 nM) compared with that of a closely related p21 peptide (residues 141-160) (Kd=88 nM)28. Isothermal calorimetry measurements28 show that the enthalpy of binding is only 2% more favourable for p21(141-160) (−9758 kcal/mol) compared with CM-peptide (−9518 kcal/mol) while the entropic contribution is significantly less favourable (0.09 cal mol−1 ° C.−1 compared with 0.61 cal mol−1 ° C.−1 for the CM-peptide). This suggests that the additional RRLIFS C-terminal residues (p21(155-160)) that bind to the linker strand do not contribute significantly to the net enthalpy of binding. Furthermore the less favourable entropic contribution to binding for p21(141-160) suggests that an entropic penalty must be paid when this longer, flexible tail binds to the linker strand. It is reasonable to argue that the unfavourable entropic term can be reduced if the peptide is pre-organised in the appropriate binding conformation. There is good evidence that this occurs in the helical region of the peptide from isothermal calorimetry experiments for two peptides: p21(141-160) and the site point mutant p21(141-160)D149A28. The structures of both CM-peptide and p21(139-160) (
Inspection of the CM-peptide binding pocket shows that the major interactions with PCNA involve the side chains Q5, I8, Y11 and F12 (
Comparison of the lipophilic pocket (formed in part by the linker strand) of the CM peptide structure with that of p21-bound PCNA reveals some interesting differences. As can be seen (
In normal, untransformed cells, quaternary complexes are formed between cyclins (A, B, D, and E), CDKs, p21, and PCNA. Furthermore, subunit rearrangement of these CDK complexes is associated with cellular transformation15. On cell transformation the expression of p21 is frequently depressed and CDKs dissociate from PCNA. This suggests that p21 may participate in the coordination of cellular DNA replication and cell-cycle progression and that upon transformation these processes become uncoupled, permitting escape from the G1 DNA-damage checkpoint. Quaternary complexes of CDK/cyclin pairs with p21 and PCNA exist in multiple cell cycle phases, including G1, S, and even G2/M, where CDK1/cyclin B is implicated35. The quaternary complex (
Phosphorylation provides an important and general control mechanism in cell-cycle events. The structures of the PCNA complexes (
Another aspect of the invention relates to a method of screening for a ligand capable of binding to a ligand binding domain, wherein said method comprises the use of a crystal as described hereinbefore or the structure co-ordinates of Table 3, Table 4 and/or Table 5.
Another aspect of the invention relates to a method of screening for a ligand capable of binding to a ligand binding domain, wherein the ligand binding domain is as defined hereinabove, the method comprising contacting the ligand binding domain with a test compound and determining if said test compound binds to said ligand binding domain.
Yet another aspect of the invention relates to a method of screening for a modulator of PCNA, wherein the method comprises using a crystal as defined hereinabove, or the structure coordinates of Table 3, Table 4 and/or Table 5.
In one preferred embodiment, the method comprises the steps of:
In a preferred embodiment, at least a portion of the structure co-ordinates of Tables 3, 4 and/or 5 and/or the putative modulator of PCNA and/or the substrate are provided on a machine-readable data storage medium comprising a data storage material encoded with machine readable data.
In a preferred embodiment, the putative modulator of PCNA is selected from a library of compounds. Preferably, the library is an in silico library. Suitable in silico libraries will be familiar to those skilled in the art, and include the Available Chemical Directory WL Inc), the Derwent World Drug Index (WDI), BioByteMasterFile, the National Cancer Institute database (NCI), and the Maybridge catalogue.
In another preferred embodiment, the putative modulator of PCNA is selected from a database.
In another preferred embodiment, the putative modulator of PCNA is designed de novo.
In yet another preferred embodiment, the putative modulator of PCNA is designed from a known PCNA modulator.
Preferably, the design or selection of the putative modulator of PCNA is performed in conjunction with computer modelling.
In one particularly preferred embodiment, the putative modulator of PCNA inhibits PCNA activity.
In a further preferred embodiment, the putative modulator of PCNA is useful in the prevention and/or treatment of a PCNA related disorder.
Even more preferably, the PCNA related disorder is a proliferative disorder.
More preferably still, the proliferative disorder is selected from cancer, leukemia, glomerulonephritis, rheumatoid arthritis, psoriasis and chronic obstructive pulmonary disorder.
A further aspect of the invention relates to an assay for a candidate compound capable of modulating PCNA, said assay comprising the steps of:
In one preferred embodiment, said candidate compound is selected by performing rational drug design with a 3-dimensional model of PCNA in conjunction with computer modelling.
Preferably, the assay is a competitive binding assay using a known modulator of PCNA.
Another aspect of the invention relates to a computer for producing a three-dimensional representation of PCNA wherein said computer comprises:
Another aspect of the invention relates to a machine-readable data storage medium comprising a data storage material encoded with machine readable data, wherein the data is defined by at least a portion of the structure co-ordinates of Table 3, Table 4 and/or Table 5.
A further aspect of the invention relates to the use of the above-described computer or machine readable data storage medium to predict the structure and/or function of potential modulators of PCNA.
Another aspect of the invention relates to the use of at least a portion of the structure co-ordinates of Table 3, Table 4 and/or Table 5 to screen for modulators of PCNA.
A further aspect of the invention relates to the use of at least a portion of the structure co-ordinates of Table 3, Table 4 and/or Table 5 to solve the structure of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of PCNA.
Preferably, the structure of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of PCNA is solved using molecular replacement.
Yet another aspect of the invention relates to the use of at least a portion of the structure co-ordinates of Table 3, Table 4 and/or Table 5 in molecular design techniques to design, select and synthesise modulators of PCNA.
Another aspect of the invention relates to the use of at least a portion of the structure co-ordinates of Table 3, Table 4 and/or Table 5 to screen small molecule databases for chemical entities or compounds that modulate PCNA.
Preferably, the modulator of PCNA, chemical entity, substrate or compound selectively modulates PCNA.
As used throughout, the term “selectively” refers to modulators, ligands or candidate compounds that are selective for PCNA. Preferably, the modulators, ligands or candidate compounds act independently of cyclin groove inhibitors. Preferably the modulators are selective for PCNA over the cyclin binding groove and do not substantially bind to the cyclin binding groove. Preferably the modulators of the invention have a selectivity ratio for PCNA over the cyclin binding groove of greater than 2, more preferably greater than 5, more preferably still greater than 10. Even more preferably, the selectivity ratio for PCNA over the cyclin binding groove is greater than 25, or more preferably still greater than 50 or 100. Selectivity ratios may readily be determined by the skilled person.
In one preferred embodiment, the PCNA modulator, ligand or candidate compound modulates PCNA activity but does not substantially bind to the cyclin binding groove.
In one particularly preferred embodiment, the PCNA modulator, ligand or candidate compound binds substantially exclusively to PCNA.
A further aspect of the invention relates to a PCNA modulator or ligand identified by the above-described methods, or a candidate compound identified by the above-described assay.
Preferably, the PCNA modulator, ligand or candidate compound of the invention inhibits PCNA activity.
More preferably, the PCNA modulator, ligand or candidate compound of the invention selectively inhibits PCNA.
In one particularly preferred embodiment, the PCNA modulator or candidate compound of the invention is capable of forming associations with one or more amino acid residues corresponding to I255, P253, A252, Y250, P234, P129, G127, L126, Q125, L47, V45, H44, W227 and W364.
Another aspect of the invention relates to a human PCNA ligand binding domain agonist, wherein said ligand binding domain comprises amino acid residues selected from one or more of the following: I255, P253, A252, Y250, P234, P129, G127, L126, Q125, L47, V45, H44, W227 and W364.
Yet another aspect of the invention relates to a human PCNA binding domain antagonist, wherein said ligand binding domain comprises amino acid residues selected from one or more of the following: I255, P253, A252, Y250, P234, P129, G127, L126, Q125, L47, V45, H44, W227 and W364.
The present invention permits the use of molecular design techniques to design, select and synthesise chemical entities and compounds, including PCNA modulating compounds, capable of binding to PCNA, in whole or in part.
By way of example, the structure co-ordinates of Table 3, Table 4 and/or Table 5 may be used to design compounds that bind to PCNA and may alter the physical properties of the compounds (eg. solubility) or PCNA itself. This invention may be used to design compounds that act as modulators, such as competitive inhibitors—of PCNA by binding to all or a portion of the active site of PCNA. Compounds may also be designed that act as non-competitive inhibitors of PCNA. These non-competitive inhibitors may bind to all or a portion of PCNA already bound to its substrate and may be more potent and specific than known PCNA inhibitors that compete only for the PCNA active site. Similarly, non-competitive inhibitors that bind to and inhibit PCNA whether or not it is bound to another chemical entity may be designed using the structure co-ordinates of PCNA described herein.
The present invention may also allow the development of compounds that can isomerise to reaction intermediates in the chemical reaction of a substrate or other compound that binds to PCNA. Thus, the time-dependent analysis of structural changes in PCNA during its interaction with other molecules may be performed. The reaction intermediates of PCNA may also be deduced from the reaction product in co-complex with PCNA. Such information is especially useful to design improved analogues of known PCNA modulators or to design new PCNA modulators based on the reaction intermediates of the PCNA enzyme and PCNA-modulator complex. This may provide a new route for designing PCNA modulators with high specificity and stability. Preferably, this provides a new route for designing PCNA modulators with high specificity, high stability and low toxicity.
Small molecule databases or candidate compounds may be screened for chemical entities or compounds that can bind in whole, or in part, to PCNA. Thus, in a preferred embodiment, the putative PCNA modulator is from a library of compounds or a database. In this screening, the quality of fit of such entities or compounds to the binding site may be judged by various methods—such as shape complementarity or estimated interaction energy (Meng, E. C. et al., J Comp. Chem., 13, pp. 505-524 (1992)).
The structure co-ordinates of Table 3, Table 4 and/or Table 5, or portions thereof, may also be useful in solving the structure of other crystal forms of PCNA. They may also be used to solve the structure of PCNA mutants, PCNA variants, PCNA homologues, PCNA derivatives, PCNA fragments and PCNA complexes.
Preferably, the structure co-ordinates of Table 3, Table 4 and/or Table 5 may be used to solve the structure of the crystalline form of proteins having significant amino acid sequence homology to any functional domain of PCNA. By way of example, molecular replacement may be used. In this method, the unknown crystal structure, whether it is another crystal form of PCNA, a PCNA mutant, a PCNA variant, a PCNA homologue (eg. another protein with significant amino acid sequence homology to any functional domain of PCNA), a PCNA derivative, a PCNA fragment or a PCNA co-complex may be determined using the PCNA structure co-ordinates of the present invention. This method will provide a more accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
In a preferred embodiment of the present invention, the PCNA crystal of unknown structure further comprises an entity bound to the PCNA protein or a portion thereof, for example, an entity that is an inhibitor of PCNA.
The crystal structures of such complexes may be solved by molecular replacement or in combination with MAD (Multiwavelength Anomalous Dispersion) and/or MIRAS (Multiple Isomorphous Replacement with Anomalous Scattering) procedures—and compared with that of wild-type PCNA. Potential sites for modification within the binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between PCNA and a chemical entity or compound.
The structures and complexes of PCNA may be refined using computer software—such as X-PLOR (Meth. Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)), MLPHARE (Collaborative computational project Number 4. The CCP4 Suite: Programs for Protein Crystallography (1994) Acta Crystallogr. D 50, 760-763) and SHARP [De La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameters refinement in the MIR and MAD methods (1997) Methods Enzymol. 276, 472-494). Preferably, the complexes are refined using the program CNS (Brünger et al. (1998) Acta Crystallogr. D 54, 905-921). During the final stages of refinement water molecules, ions and inhibitor molecules may be inserted in the structure. This information may thus be used to optimise known classes of PCNA modulators, eg. PCNA inhibitors, and more importantly, to design and synthesise novel classes of PCNA modulators.
The overall figure of merit may be improved by iterative solvent flattening, phase combination and phase extension with the program SOLOMON [Abrahams, J. P. & Leslie, A. G. W. Methods used in structure determination of bovine mitochondrial F1 ATPase. (1996) Acta Crystallogr. D 52, 110-119].
The structure co-ordinates of Table 3, Table 4 and/or Table 5 may also facilitate the identification of related proteins or enzymes analogous to PCNA in function, structure or both, thereby further leading to novel therapeutic modes for treating or preventing PCNA related diseases.
The design of compounds that bind to or modulate PCNA according to the present invention generally involves consideration of two factors. Firstly, the compound must be capable of physically and structurally associating with PCNA. Non-covalent molecular interactions important in the association of PCNA with its substrate may include hydrogen bonding, van der Waals and hydrophobic interactions. Secondly, the compound must be able to assume a conformation that allows it to associate with PCNA. Although certain portions of the compound may not directly participate in the association with PCNA, those portions may still influence the overall conformation of the molecule. This may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of a binding site of PCNA, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with PCNA.
The potential modulating or binding effect of a chemical compound on PCNA may be analysed prior to its actual synthesis and testing by the use of computer modelling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association with PCNA, then synthesis and testing of the compound may be obviated. However, if computer modelling indicates a strong interaction, the molecule may be synthesised and tested for its ability to bind to PCNA and modulate (eg. inhibit) using the fluorescent substrate assay of Thornberry et al. (2000) Methods Enzymol. 322, pp 100-110. In this manner, synthesis of inactive compounds may be avoided.
A modulating or other binding compound of PCNA may be computationally evaluated and designed by means of a series of steps in which chemical entities or candidate compounds are screened and selected for their ability to associate with PCNA.
A person skilled in the art may use one of several methods to screen chemical entities or candidate compounds for their ability to associate with PCNA and more particularly with the individual binding sites of PCNA. This process may begin by visual inspection of, for example, the active site on the computer screen based on the PCNA co-ordinates of the present invention. Selected chemical entities or candidate compounds may then be positioned in a variety of orientations, or docked, with PCNA. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimisation and molecular dynamics with standard molecular mechanics force fields—such as CHARMM and AMBER.
Specialised computer programs may also assist in the process of selecting chemical entities or candidate compounds. These include but are not limited to MCSS (Miranker and Karplus (1991) Proteins: Structure, Function and Genetics, 11, pp. 29-34); GRID (Goodford (1985) J. Med. Chem., 28, pp. 849-857) and AUTODOCK (Goodsell and Olsen (1990), Proteins: Structure. Function, and Genetics, 8, pp. 195-202.
Once suitable chemical entities or candidate compounds have been selected, they may be assembled into a single compound, such as a PCNA modulator. Assembly may proceed by visual inspection of the relationship of the chemical entities or candidate compounds in relation to the structure co-ordinates of PCNA. This may be followed by manual model building using software—such as Quanta, Sybyl, O, HOOK or CAVEAT [Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models (1991) Acta Crystallogr. A 47, 110-119].
Refinement of the model may be carried out using the program CNS [Brünger, A. T. et al. Crystallography & NMR System: A new software suite for macromolecular structure determination. (1998) Acta Crystallogr. D 54, 905-921].
Various programs may be used by a skilled person to connect the individual chemical entities or candidate compounds, such as 3D Database systems (Martin (1992) J. Med. Chem., 35, pp. 2145-2154) and CAVEAT (Bartlett et al. (1989) Royal Chem. Soc. 78, pp. 182-196).
Rather than build a PCNA inhibitor one chemical entity at a time, modulating or other PCNA binding compounds may be designed as a whole or de novo using either an empty binding site or optionally including some portion(s) of a known inhibitor(s). Such compounds may be designed using programs that may include but are not limited to LEGEND (Nishibata and Itai (1991) Tetrahedron, 47, p. 8985) and LUDI (Bohm (1992) J. Comp. Aid. Molec. Design, 6, pp. 61-78).
Other molecular modelling techniques may also be employed in accordance with this invention—such as those described by Cohen et al., J. Med. Chem., 33, pp. 883-894 (1990); Navia and Mureko (1992) Current Opinions in Structural Biology, 2, pp. 202-210 (1992).
Once a compound has been designed or selected by the above methods, the efficiency with which that compound may bind to PCNA may be computationally evaluated. Specific computer software may be used to evaluate the efficiency of binding (eg. to evaluate compound deformation energy and electrostatic interaction), such as QUANTA/CHARMM (Accelrys Inc., USA) and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif., USA). These programs may be implemented, for instance, using a suitable workstation. Other hardware systems and software packages will be known to those persons skilled in the art.
Once a PCNA-modulating compound has been selected or designed, as described above, substitutions may be made (eg. in atoms or side groups) to improve or modify the binding properties. The substitutions may be conservative ie. the replacement group may have approximately the same size, shape, hydrophobicity and charge as the original group. Such substituted chemical compounds may then be analysed for efficiency of binding to PCNA by the same computer methods described above.
Candidate compounds, ligands and modulators of PCNA etc. which are identified using the methods of the present invention may be screened in assays. Screening can be, for example in vitro, in cell culture, and/or in vivo. Biological screening assays preferably centre on activity-based response models, binding assays (which measure how well a compound binds), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity.
Current screening technologies are described in Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes. New York, N.Y., Marcel Dekker, (2001).
As herein, the term “modulating” or “modulates” refers to preventing, suppressing, inhibiting, alleviating, restorating, elevating, increasing or otherwise affecting PCNA.
The terms “PCNA modulator” or “modulator of PCNA” are used interchangeably and refer to a single entity or a combination of entities.
The PCNA modulator may be an antagonist or an agonist of PCNA.
As used herein, the term “agonist” means any entity, which is capable of interacting (eg. binding) with PCNA and which is capable of increasing a proportion of the PCNA that is in an active form, resulting in an increased biological response.
As used herein, the term “antagonist” means any entity, which is capable of interacting (eg. binding) with PCNA and which is capable of decreasing (eg. inhibiting) a proportion of the PCNA that is in an active form, resulting in a decreased biological response.
Preferably, the PCNA modulators of the present invention are antagonists of PCNA.
The modulator of PCNA may be an organic compound or other chemical. The modulator of PCNA may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial. The modulator of PCNA may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The modulator of PCNA may even be a polynucleotide molecule, which may be a sense or an anti-sense molecule. The modulator of PCNA may even be an antibody.
The modulator of PCNA may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.
By way of example, the modulator of PCNA may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised agent, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesiser or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof).
Typically, the modulator of PCNA will be an organic compound. Typically, the organic compounds will comprise two or more hydrocarbyl groups. Here, the term “hydrocarbyl group” means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. For some applications, preferably the modulator of PCNA comprises at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. For some applications, the modulator of PCNA comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
The modulator of PCNA may contain halo groups, for example, fluoro, chloro, bromo or iodo groups, or one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups, each of which may be branched or unbranched.
The modulator of PCNA may be a structurally novel modulator of PCNA, or may be an analogue of a known modulator of PCNA.
Preferably, the PCNA modulators have improved properties over those previously available, for example, fewer side effects.
The modulator of PCNA may be a mimetic, or may be chemically modified.
The modulator of PCNA may be capable of displaying other therapeutic properties.
The modulator of PCNA may be used in combination with one or more other pharmaceutically active agents. If combinations of active agents are administered, then they may be administered simultaneously, separately or sequentially.
As used herein, the term “candidate compound” includes, but is not limited to, a compound which may be obtainable from or produced by any suitable source, whether natural or not.
The candidate compound may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules and particularly new lead compounds. By way of example, the candidate compound may be a natural substance, a biological macromolecule, or an extract made from biological materials—such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic candidate compound, a semi-synthetic candidate compound, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised candidate compound, a peptide cleaved from a whole protein, or a peptide synthesised synthetically, for example, either using a peptide synthesiser or by recombinant techniques or combinations thereof, a recombinant candidate compound, a natural or a non-natural candidate compound, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof. The candidate compound may even be a compound that is a modulator of PCNA, such as a known inhibitor of PCNA, that has been modified in some way eg. by recombinant DNA techniques or chemical synthesis techniques.
Typically, the candidate compound will be prepared by recombinant DNA techniques and/or chemical synthesis techniques.
Once a candidate compound capable of interacting PCNA has been identified, further steps may be carried out to select and/or to modify the candidate compounds and/or to modify existing compounds, such that they are able to modulate PCNA.
In one aspect, the modulator of PCNA may act as a model (for example, a template) for the development of other compounds.
A further aspect relates to the use of candidate compounds or PCNA modulators identified by the assays and methods of the invention in one or more model systems, for example, in a biological model, a disease model, or a model for PCNA inhibition. Such models may be used for research purposes and for elucidating further details of the biological, physicochemical, pharmacological and/or pharmacokinetic activity of a particular candidate compound. By way of example, the candidate compounds or PCNA modulators of the present invention may be used in biological models or systems in which the cell cycle is known to be of particular significance, e.g. in models relating to cell fertilization, especially in animals.
As used herein, the term “mimetic” relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a known compound. That is, the mimetic is a functional equivalent of a known compound.
Preferably, the modulator of PCNA of the present invention may be prepared by chemical synthesis techniques.
It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound of the invention. This may be achieved by conventional techniques, for example as described in “Protective Groups in Organic Synthesis” by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P. J. Kocienski, in “Protecting Groups”, Georg Thieme Verlag (1994).
It is possible during some of the reactions that any stereocentres present could, under certain conditions, be racemised, for example if a base is used in a reaction with a substrate having an having an optical centre comprising a base-sensitive group. This is possible during e.g. a guanylation step. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc. as is well-known in the art. The compounds and salts may be separated and purified by conventional methods.
Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallisation, chromatography or H.P.L.C. of a stereoisomeric mixture of a compounds or suitable salts or derivatives thereof. An individual enantiomer of a compound may also be prepared from a corresponding optically pure intermediate or by resolution, such as by H.P.L.C. of the corresponding racemate using a suitable chiral support or by fractional crystallisation of the diastereomeric salts formed by reaction of the corresponding racemate with a suitably optically active acid or base.
PCNA, modulators of PCNA or variants, homologues, derivatives, fragments or mimetics thereof may be produced using chemical methods to synthesise the PCNA or the modulator of PCNA in whole or in part. For example, a PCNA peptide or a modulator of PCNA that is a peptide can be synthesised by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
Synthesis of peptides (or variants, homologues, derivatives, fragments or mimetics thereof) may be performed using various solid-phase techniques (Roberge J Y et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising the modulator of PCNA, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant modulator of PCNA.
In one embodiment, the modulator of PCNA may be a chemically modified modulator of PCNA. The chemical modification of a modulator of PCNA may either enhance or reduce interactions between the modulator of PCNA and the target, such as hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der Waals interactions or dipole interactions.
Another aspect of the invention relates to a process comprising the steps of:
A further aspect of the invention relates to a process comprising the steps of:
A further aspect relates to a process comprising the steps of:
Another aspect of the invention relates to a pharmaceutical composition comprising a PCNA modulator, ligand or candidate compound of the invention and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant or any combination thereof. Even though the PCNA modulators, ligands or candidate compounds (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid: Antioxidants and suspending agents may be also used.
The PCNA modulators, ligands or candidate compounds of the present invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
Pharmaceutically acceptable salts of the PCNA modulators, ligands or candidate compounds of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers and tautomers of the PCNA modulators, ligands or candidate compounds of the invention. The man skilled in the art will recognise compounds that possess an optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
Some of the PCNA modulators, ligands or candidate compounds of the invention may exist as stereoisomers and/or geometric isomers, e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the PCNA modulators. ligands or candidate compounds, or pharmaceutically acceptable salts thereof. An isotopic variation of a PCNA modulator, ligand or candidate compound of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 17O, 18O, 31P, 32P, 35S, 18F and 36Cl, respectively. Certain isotopic variations of the agents and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the PCNA modulators, ligands or candidate compounds of the present invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
The present invention also includes solvate forms of the PCNA modulators, ligands or candidate compounds. The terms used in the claims encompass these forms.
The invention furthermore relates to PCNA modulators, ligands or candidate compounds of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
The invention further includes PCNA modulators, ligands or candidate compounds of the present invention in prodrug form. Such prodrugs are generally compounds of the invention wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
Certain PCNA modulators, ligands or candidate compounds of the present invention have been found to possess anti-proliferative activity and are therefore believed to be of use in the treatment of proliferative disorders, such as cancers, leukaemias or other disorders associated with uncontrolled cellular proliferation such as psoriasis and restenosis.
One aspect of the invention therefore relates to a method of preventing and/or treating a PCNA related disorder comprising administering a PCNA modulator, ligand or candidate compound of the invention and/or a pharmaceutical composition according to the invention, wherein said PCNA modulator, said ligand, said candidate compound or said pharmaceutical, is capable of causing a beneficial preventative and/or therapeutic effect.
A further aspect of the invention relates to the use of a PCNA modulator, ligand or candidate compound according to the invention in the preparation of a medicament for treating a PCNA related disorder. Preferably, the PCNA related disorder is a proliferative disorder, more preferably cancer.
As used herein the phrase “preparation of a medicament” includes the use of the compound directly as the medicament in addition to its use in a screening programme for further therapeutic agents or in any stage of the manufacture of such a medicament.
Preferably, the PCNA dependent disorder is a disorder associated with increased PCNA activity. Even more preferably, the disorder is cancer.
The term “proliferative disorder” is used herein in a broad sense to include any disorder that requires control of the cell cycle, for example cardiovascular disorders such as restenosis and cardiomyopathy, auto-immune disorders such as glomerulonephritis and rheumatoid arthritis, dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema and alopecia. In these disorders, the compounds of the present invention may induce apoptosis or maintain stasis within the desired cells as required.
Preferably, the proliferative disorder is a cancer or leukaemia.
In another preferred embodiment, the proliferative disorder is psoriasis.
The compounds of the invention may inhibit any of the steps or stages in the cell cycle, for example, formation of the nuclear envelope, exit from the quiescent phase of the cell cycle (G0), G1 progression, chromosome decondensation, nuclear envelope breakdown, START, initiation of DNA replication, progression of DNA replication, termination of DNA replication, centrosome duplication, G2 progression, activation of mitotic or meiotic functions, chromosome condensation, centrosome separation, microtubule nucleation, spindle formation and function, interactions with microtubule motor proteins, chromatid separation and segregation, inactivation of mitotic functions, formation of contractile ring, and cytokinesis functions. In particular, the compounds of the invention may influence certain gene functions such as chromatin binding, formation of replication complexes, replication licensing, phosphorylation or other secondary modification activity, proteolytic degradation, microtubule binding, actin binding, septin binding, microtubule organising centre nucleation activity and binding to components of cell cycle signalling pathways.
As defined herein, an anti-proliferative effect within the scope of the present invention may be demonstrated by the ability to inhibit cell proliferation in an in vitro whole cell assay, for example using any of the cell lines A549, HeLa, HT-29, MCF7, Saos-2, CCRF-CEM, HL-60 and K-562, or by showing kinase inhibition in an appropriate assay. These assays, including methods for their performance, will be familiar to the skilled artisan. Using such assays it may be determined whether a compound is anti-proliferative in the context of the present invention.
In one preferred embodiment, the compound of the invention is administered orally. Another aspect of the invention relates to a method of modulating PCNA activity in a cell, said method comprising contacting the cell with a modulator of PCNA as defined above and/or a pharmaceutical composition as defined above.
Preferably, the cell is a cancer cell.
The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Depending upon the need, the agent may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
In an exemplary embodiment, one or more doses of 10 to 150 mg/day will be administered to the patient for the treatment of malignancy.
Another aspect of the invention relates to a fragment of PCNA, or a homologue, mutant, or derivative thereof, comprising a ligand binding domain, said ligand binding domain being defined by amino acid residue structural coordinates selected from one or more of the following: I255, P253, A252, Y250, P234, P129, G127, L126, Q125, L47, V45, H44, W227 and W364. As used herein, the term “ligand binding domain (LBD)” means the ligand binding region of PCNA which is responsible for ligand binding. The term “ligand binding domain” also includes a homologue of the ligand binding domain, or a portion thereof.
As used herein, the term “portion thereof” means the structural co-ordinates corresponding to a sufficient number of amino acid residues of the PCNA sequence (or homologue thereof) that are capable of interacting with a candidate compound capable of binding to the LBD. This term includes ligand binding domain amino acid residues having amino acid residues from about 4 Å to about 5 Å of a bound compound or fragment thereof. Thus, for example, the structural co-ordinates provided in the homology model may contain a subset of the amino acid residues in the LBD which may be useful in the modelling and design of compounds that bind to the LBD.
In one preferred embodiment, the fragment of PCNA, or a homologue, mutant or derivative thereof, corresponds to a portion of the structure co-ordinates of Table 3, Table 4 and/or Table 5.
Another aspect of the invention relates to the use of the above-described fragment of PCNA, or a homologue, mutant, or derivative thereof, in an assay or method for identifying candidate compounds capable of modulating PCNA. Suitable assays/methods are identical to those described hereinabove.
As used herein, the term “nucleotide sequence” refers to nucleotide sequences, oligonucleotide sequences, polynucleotide sequences and variants, homologues, fragments and derivatives thereof (such as portions thereof) which comprise the nucleotide sequences encoding PCNA.
The nucleotide sequence may be DNA or RNA of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or antisense strand or combinations thereof.
Preferably, the term nucleotide sequence is prepared by use of recombinant DNA techniques (e.g. recombinant DNA). The nucleotide sequences may include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3′ and/or 5′ ends of the molecule. For the purposes of the present invention, it is to be understood that the nucleotide sequences described herein may be modified by any method available in the art.
It will be understood by a skilled person that numerous different nucleotide sequences can encode the same protein as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not substantially affect the activity encoded by the nucleotide sequence of the present invention to reflect the codon usage of any particular host organism in which the target is to be expressed. Thus, the terms “variant”, “homologue” or “derivative” in relation to nucleotide sequences include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acids from or to the sequence providing the resultant nucleotide sequence encodes a functional protein according to the present invention (or even a modulator of PCNA according to the present invention if said modulator comprises a nucleotide sequence or an amino acid sequence).
As used herein, the term “amino acid sequence” is synonymous with the term “polypeptide” and/or the term “protein”. In some instances, the term “amino acid sequence” is synonymous with the term “peptide”.
The amino acid sequence may be isolated from a suitable source, or it may be made synthetically or it may be prepared by use of recombinant DNA techniques.
The PCNA described herein is intended to include any polypeptide, which has the activity of the naturally occurring PCNA and includes all vertebrate and mammalian forms. Such terms also include polypeptides that differ from naturally occurring forms of PCNA by having amino acid deletions, substitutions, and additions, but which retain the activity of PCNA.
The term “variant” is used to mean a naturally occurring polypeptide or nucleotide sequences which differs from a wild-type or a native sequence.
The term “fragment” indicates that a polypeptide or nucleotide sequence comprises a fraction of a wild-type or a native sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections. The sequence may also comprise other elements of sequence, for example, it may be a fusion protein with another protein. Preferably the sequence comprises at least 50%, more preferably at least 65%, more preferably at least 80%, most preferably at least 90% of the wild-type sequence.
The present invention also encompasses the use of variants, homologues and derivatives of nucleotide and amino acid sequences. Here, the term “homologue” means an entity having a certain homology with amino acid sequences or nucleotide sequences. Here, the term “homology” can be equated with “identity”.
In the present context, an homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), it is preferred to express homology in terms of sequence identity.
An homologous sequence is taken to include a nucleotide sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the subject sequence.
Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences.
% homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion will cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local homology.
However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible—reflecting higher relatedness between the two compared sequences—will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is −12 for a gap and 4 for each extension.
Calculation of maximum % homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter 18), PASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2): 247-50; FEMS Microbiol Lett 1999 177(1): 187-8)
Although the final % homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
The sequences may also have deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
Conservative substitutions may be made, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
Homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
Replacements may also be made by unnatural amino acids include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p-Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, β-alanine*, L-α-amino butyric acid*, L-γ-amino butyric acid*, L-α-amino isobutyric acid*, L-ε-amino caproic acid#, 7-amino heptanoic acid*, L-methionine sulfone#*, L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline#, L-thioproline*, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe*, pentamethyl-Phe*, L-Phe (4-amino)#, L-Tyr (methyl)*, L-Phe (4-isopropyl)*, L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid)*, L-diaminopropionic acid# and L-Phe (4-benzyl)*. The notation * has been utilised for the purpose of the discussion above (relating to homologous or non-homologous substitution), to indicate the hydrophobic nature of the derivative whereas # has been utilised to indicate the hydrophilic nature of the derivative, #* indicates amphipathic characteristics.
The term “derivative” or “derivatised” as used herein includes chemical modification of an entity, such as candidate compound or a PCNAmodulator. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or β-alanine residues. A further form of variation, involves the presence of one or more amino acid residues in peptoid form, will be well understood by those skilled in the art. For the avoidance of doubt, “the peptoid form” is used to refer to variant amino acid residues wherein the cc-carbon substituent group is on the residue's nitrogen atom rather than the α-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, Trends Biotechnol. (1995) 13(4), 132-134.
As used herein, the term “mutant” refers to PCNA comprising one or more changes in the wild-type PCNA sequence.
The term “mutant” is not limited to amino acid substitutions of the amino acid residues in PCNA, but also includes deletions or insertions of nucleotides which may result in changes in the amino acid residues in the amino acid sequence of PCNA.
The present invention also enables the solving of the crystal structure of PCNA mutants. More particularly, by virtue of the present invention, the location of the active site of PCNA based on the structural coordinates of Tables 3, 4 and/or 5 permits the identification of desirable sites for mutation. For example, one or more mutations may be directed to a particular site—such as the active site—or combination of sites of PCNA. Similarly, only a location on, at or near the enzyme surface may be replaced, resulting in an altered surface charge of one or more charge units, as compared to the wild-type enzyme. Alternatively, an amino acid residue in PCNA may be chosen for replacement based on its hydrophilic or hydrophobic characteristics.
Such mutants may be characterised by any one of several different properties as compared with wild-type PCNA. For example, such mutants may have altered surface charge of one or more charge units, or have an increased stability to subunit dissociation, or an altered substrate specificity in comparison with, or a higher specific activity than, wild-type PCNA.
The mutants may be prepared in a number of ways that are known by a person skilled in the art. For example, mutations may be introduced by means of oligonucleotide-directed mutagenesis or other conventional methods. Alternatively, mutants of PCNA may be generated by site specific replacement of a particular amino acid with an unnaturally occurring amino acid. This may be achieved by growing a host organism capable of expressing either the wild-type or mutant polypeptide on a growth medium depleted of one or more natural amino acids but enriched in one or more corresponding unnaturally occurring amino acids.
As used herein, the term “host cell” refers to any cell that comprises nucleotide sequences that are of use in the present invention, for example, nucleotide sequences encoding PCNA.
Host cells may be transformed or transfected with a nucleotide sequence contained in a vector e.g. a cloning vector. Preferably, said nucleotide sequence is carried in a vector for the replication and/or expression of the nucleotide sequence. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
The gram-negative bacterium E. coli is widely used as a host for cloning nucleotide sequences. This organism is also widely used for heterologous nucleotide sequence expression. However, large amounts of heterologous protein tend to accumulate inside the cell. Subsequent purification of the desired protein from the bulk of E. coli intracellular proteins can sometimes be difficult.
In contrast to E. coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.
Depending on the nature of the polynucleotide and/or the desirability for further processing of the expressed protein, eukaryotic hosts including yeasts or other fungi may be preferred. In general, yeast cells are preferred over fungal cells because yeast cells are easier to manipulate. However, some proteins are either poorly secreted from the yeast cell, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a different fungal host organism should be selected.
Examples of expression hosts are fungi—such as Aspergillus species (such as those described in EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria—such as Bacillus species (such as those described in EP-A-0134048 and EP-A-0253455), Streptomyces species and Pseudomonas species; yeasts—such as Kluyveromyces species (such as those described in EP-A-0096430 and EP-A-0301670) and Saccharomyces species; and mammalian cells—such as CHO-K1 cells.
The PCNA proteins produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the nucleotide sequence and/or the vector used.
The use of host cells may provide for post-translational modifications as may be needed to confer optimal biological activity on recombinant expression products of the present invention.
Aspects of the present invention also relate to host cells comprising the PCNA constructs of the present invention. The PCNA constructs may comprise a nucleotide sequence for replication and expression of the sequence. The cells will be chosen to be compatible with the vector and may for example be prokaryotic (for example bacterial), fungal, yeast or plant cells.
In a preferred embodiment, the host cells are mammalian cells, such as CHO-K1 cells.
Aspects of the present invention relate to a vector comprising a nucleotide sequence, such as a nucleotide sequence encoding PCNA or a modulator of PCNA, administered to a subject.
Preferably, PCNA or the modulator of PCNA is prepared and/or delivered using a genetic vector.
As it is well known in the art, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant DNA techniques allow entities, such as a segment of DNA (such as a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a host and/or a target cell for the purpose of replicating the vectors comprising nucleotide sequences and/or expressing the proteins encoded by the nucleotide sequences. Examples of vectors used in recombinant DNA techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes or viruses.
The term “vector” includes expression vectors and/or transformation vectors.
The term “expression vector” means a construct capable of in vivo or in vitrolex vivo expression.
The term “transformation vector” means a construct capable of being transferred from one species to another.
In some applications, nucleotide sequences are operably linked to a regulatory sequence which is capable of providing for the expression of the nucleotide sequence, such as by a chosen host cell. By way of example, a vector comprising the PCNA nucleotide sequence is operably linked to such a regulatory sequence i.e. the vector is an expression vector.
The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
The term “regulatory sequences” includes promoters and enhancers and other expression regulation signals.
The term “promoter” is used in the normal sense of the art, e.g. an RNA polymerase binding site.
Enhanced expression of a nucleotide sequence, for example, a nucleotide sequence encoding PCNA, may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, secretion leader and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of PCNA. In eukaryotes, polyadenylation sequences may be operably connected to the PCNA nucleotide sequence.
Preferably, the PCNA nucleotide sequence is operably linked to at least a promoter.
Aside from the promoter native to the gene encoding the PCNA nucleotide sequence, other promoters may be used to direct expression of the PCNA polypeptide. The promoter may be selected for its efficiency in directing the expression of the PCNA nucleotide sequence in the desired expression host.
In another embodiment, a constitutive promoter may be selected to direct the expression of the PCNA nucleotide sequence. Such an expression construct may provide additional advantages since it circumvents the need to culture the expression hosts on a medium containing an inducing substrate.
Hybrid promoters may also be used to improve inducible regulation of the expression construct.
The promoter can additionally include features to ensure or to increase expression in a suitable host. For example, the features can be conserved regions such as a Pribnow Box or a TATA box. The promoter may even contain other sequences to affect (such as to maintain, enhance, decrease) the levels of expression of the PCNAnucleotide sequence. For example, suitable other sequences include the Sh1-intron or an ADH intron. Other sequences include inducible elements—such as temperature, chemical, light or stress inducible elements. Also, suitable elements to enhance transcription or translation may be present.
The PCNA encoding sequence may be fused (eg. ligated) to nucleotide sequences encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53). Preferably, the polypeptide domain which facilitates purification of soluble proteins is fused in frame with the PCNA encoding sequence. Such purification facilitating domains include, but are not limited to, metal chelating peptides—such as histidine-tryptophan modules that allow purification on immobilised metals (Porath J (1992) Protein Expr Purif 3, 263-281), protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and PCNA is useful to facilitate purification.
Preferably, nucleotide sequences, such as nucleotide sequences encoding PCNA or modulators of PCNA, are inserted into a vector that is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell.
Nucleotide sequences produced by a host recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing a PCNA encoding nucleotide sequence or a mutant, variant, homologue, derivative or fragment thereof can be designed with signal sequences, which direct secretion of the nucleotide sequence through a particular prokaryotic or eukaryotic cell membrane.
Preferably, the expression vectors are stably expressed in CHO cells as described previously (Ehlers et al. (1996) Biochemistry 35, 9549-9559). More preferably, the expression vectors are pLEN-tACEΔ36g(1, 2, 3, 4) and pLEN-tACEΔ36g(1,3).
PCNA or a modulator of PCNA may be expressed as a fusion protein to aid extraction and purification and/or delivery of the modulator of PCNA or the PCNA protein to an individual and/or to facilitate the development of a screen for modulators of PCNA.
Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase.
It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably, the fusion protein will not hinder the activity of the protein of interest.
The fusion protein may comprise an antigen or an antigenic determinant fused to the substance of the present invention. In this embodiment, the fusion protein may be a non-naturally occurring fusion protein comprising a substance, which may act as an adjuvant in the sense of providing a generalised stimulation of the immune system. The antigen or antigenic determinant may be attached to either the amino or carboxy terminus of the substance.
The term “organism” in relation to the present invention includes any organism that could comprise PCNA and/or modulators of PCNA. Examples of organisms may include mammals, fungi, yeast or plants.
Preferably, the organism is a mammal. More preferably, the organism is a human.
As indicated earlier, the host organism can be a prokaryotic or a eukaryotic organism. Examples of suitable prokaryotic hosts include E. coli and Bacillus subtilis. Teachings on the transformation of prokaryotic hosts are well documented in the art, for example see Sambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley & Sons, Inc. Examples of suitable eukaryotic hosts include mammalian cells.
If a prokaryotic host is used then the nucleotide sequence, such as the PCNA nucleotide sequence, may need to be suitably modified before transformation—such as by removal of introns.
Thus, the present invention also relates to the transformation of a host cell with a nucleotide sequence, such as PCNA or a modulator of PCNA. Host cells transformed with the nucleotide sequence may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell may be secreted or may be contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing coding sequences can be designed with signal sequences which direct secretion of the coding sequences through a particular prokaryotic or eukaryotic cell membrane. Other recombinant constructions may join the coding sequence to nucleotide sequence encoding a polypeptide domain, which will facilitate purification of soluble proteins (Kroll D J et al (1993) DNA Cell Biol 12:441-53) e.g. 6-His or Glutathione-S-transferase.
Vectors comprising for example, the PCNA nucleotide sequence, may be introduced into host cells, for example, mammalian cells, using a variety of methods.
Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotech. (1996) 14, 556), multivalent cations such as spermine, cationic lipids or polylysine, 1,2,-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.
Uptake of nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Example of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition.
Such methods are described in many standard laboratory manuals—such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
By way of summary, the present invention provides an X-ray crystal structure of PCNA complexed with a 16mer peptide related to p21, which binds with a Kd of 100 nM. Two additional crystal structures of native PCNA provide the first structures of free human PCNA and show that the only significant changes on ligand binding involve rigidification of a number of flexible regions on the surface of PCNA. The competitive binding experiments described herein show that a 20mer sequence from p21 can associate simultaneously with PCNA and CDK/cyclin complexes. A structural model for such quaternary complexes is presented, in which the C-terminal sequence of p21 acts as a double-sided tape in that it docks to both the PCNA and cyclin molecules. The quaternary complex shows little direct interaction between PCNA and cyclin, assigning to p21 the role of an adaptor. Taken together, the biochemical and structural results delineate a compact inhibitor site on the surface of PCNA that may be exploited in the design of peptidomimetics, which will act independently of cyclin-groove inhibitors. Blocking this site with drug-like small molecules may be both chemically feasible and therapeutically relevant in proliferative diseases.
The identification of a well-defined ‘druggable’ site on PCNA adjacent to (and potentially independent of) the cyclin binding site identified in the model structure opens up the possibility of using these sites to probe the biological function of PCNA. In particular it will be interesting to study the effects of small and specific ‘cyclin-groove’ inhibitors which are now available27, along with smaller CM-related peptides to examine a possible synergy of action. Such complementary effects could have important consequences in developing inhibitor cocktails to manipulate cell-cycle events.
The present invention is further described by way of example and with reference to the following figures wherein:
The methods described here may employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press; Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by Edward Harlow, David Lane, Ed Harlow (1999, Cold Spring Harbor Laboratory Press, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y., Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Edited Jane Roskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN 0-87969-630-3. Each of these general texts is herein incorporated by reference.
PCNA: Recombinant human PCNA was expressed in Escherichia coli BL21(DE3) from a pT7-PCNA expression vector. The protein was purified from the soluble fraction using a four-step chromatographic procedure, including anion exchange (Q-Sepharose, Pharmacia), cation exchange (SP-Sepharose, Pharmacia), hydroxyapatite (BioRad), and size-exclusion (Superose-12, Pharmacia) modes, as described28.
CDK4: The N-terminally His6-tagged human recombinant protein was expressed in Sf9 insect cells using a baculovirus construct. Sf9 culture (1.6×106 cells/mL) was infected (MOI of 3) for two days. The cells were harvested by low speed centrifugation and the protein was purified from the insect cell pellet by metal affinity chromatography. In brief: the insect cell pellet was lysed in buffer A (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% Nonidet P40, 5 MM β-mercaptoethanol, 20 mM NaF, 1 mM Na3VO4 and Sigma Protease Inhibitor Cocktail) by sonication. The soluble fraction was obtained by centrifugation and was loaded onto Ni-NTA-Agarose (Qiagen). Non-bound protein was removed with 300 mM NaCl, 5-15 mM imidazole in buffer A, and bound protein was eluted with buffer A, supplemented with 250 mM imidazole. The purified protein was dialyzed extensively against storage buffer (20 mM HEPES pH 7.4, 50 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.02% Nonidet P40, 10% v/v glycerol) and stored at −70° C.
Cyclin D1: Recombinant human cyclin D1 was expressed in E. coli BL21 (DE3) using a PET expression vector. BL21 (DE3) was grown at 37° C. with shaking (200 rpm) to mid-log phase (OD600=0.6 AU). Expression was induced by addition of 1 mM IPTG and the culture was incubated for a further 3 h. The bacteria were then harvested by centrifugation, and the cell pellet was re-suspended in 50 mM Tris-HCl pH 7.5, 10% sucrose. Cyclin D1 was purified from the inclusion bodies. The bacterial cells were lysed by treatment with lysozyme and sonication. The insoluble fraction was pelleted by centrifugation. The inclusion bodies were purified by repetitive washing of the insoluble fraction with 50 mM Tris-HCl pH 8.0, 2 mM EDTA, 100 mM NaCl, and 0.5% Triton. Purified inclusion bodies were solubilized with the same buffer, containing 6 M urea. The protein was refolded by slow dilution with 25 mM Tris HCl pH 8.0, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, 0.2% Nonidet P40. After concentration by ultrafiltration (Amicon concentration unit) the protein was further purified by size-exclusion HPLC (Superose 12, Pharmacia).
GST-pRb(773-928): The hyperphosphorylation domain of pRb (residues 773-982) with an N-terminal GST tag was expressed in E. coli BL21(DE3) and was purified on a glutathione-Sepharose column (Pharmacia) according to the manufacturer's instructions. For the 96-well format in vitro kinase assay GST-pRb was used immobilized on glutathione-Sepharose beads.
CDK4/cyclin D1 kinase assay: The reaction mixture consisted of 1 μM of CDK4 and cyclin D1, 5 μg GST-pRb, 100 mM ATP and 0.2 μCi 32P-ATP. The kinase reaction was carried out for 10 min at 30° C., the reaction was stopped with the addition of sample buffer and after heating the sample was resolved on 10% SDS-PAGE. The aouthoradiography images were scanned and quantified using QuickScan software. The IC50 values were determined using GraFit software.
Peptides were assembled using standard solid-phase chemistry based on the Fmoc protecting group was employed38. Peptides were side-chain deprotected and cleaved from the synthesis support using an acidolysis method as described39. All peptides were purified by preparative reversed-phase HPLC, isolated by lyophilization, and analysed by analytical HPLC and mass spectrometry (Dynamo DE MALDI-TOF spectrometer, ThermoBioAnalysis).
PCNA monoclinic form: Crystals of PCNA were grown by the hanging drop vapour diffusion method. A 2-μL solution of PCNA (8˜10 mg/mL) in a buffer consisting of 25 mM Tris and 2 mM DTT was added to 2 μL well solution comprising 20% PEG-3,350 and 0.2 M magnesium acetate. Crystals grew after 7-10 days at 18° C. A crystal of about 0.05 mm in length was collected in a 0.05-0.1 mm cryo-loop (Hampton Research), dipped briefly in immersion oil (Type B, Cargille) and frozen by plunging into liquid nitrogen. The frozen crystal was then transferred to a magnetic goniometer head in a stream of liquid nitrogen at 100 K (Cryostream, Oxford Cryosystems). Diffraction data were collected on a CCD detector using the synchrotron source in Daresbusy station 9.6 (Table 1). X-ray data were processed by DENZO40. Molecular replacement was carried out using MOLREP41 based on the trimeric molecule of the published structure of PCNA complexed with a 22mer p21 peptide (PDB #1AXC) and was calculated within the resolution range of 35-3.1 Å. The rotation function gave three equivalent peaks with Rf/σ=10. The next highest peak height was 5.5. The translation function gave an unambiguous peak of Tf/σ=26 (next best solution 10), R factor 0.54 (next best solution 0.60) and correlation coefficient of 0.57 (next best solution 0.43). Following the translation function, rigid-body refinement of the optimum molecular replacement solution was performed by the program RIGID in CNS42 using data to 3.5 Å. 5% of the data was flagged as Rfree and omitted from refinement for cross-validation. After 40 cycles of rigid-body refinement, the R-factor reduced from 45.2% to 36.6% (Rfree from 48.7% to 39.5%).
Following rigid-body refinement, the model was subjected to the stimulated annealing, positional and B-factor refinements by program REFINE in CNS using all data. R-factor reduced from 39.1% to 25.3% (Rfree from 41.1% to 30.8%) after refinement. ARP/wAR43 was used for initial density interpretation and the addition of water molecules. Subsequent rounds of manual remodelling with the program Quanta (Accelrys, San Diego, USA) and restrained refinement using the program REFMAC44 gave an R value of 18.7 and an Rfree of 27.9.
PCNA trigonal form: The crystal of the trigonal form of PCNA was grown similarly. A 1 μL solution of PCNA (6 mg/mL in 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.01% Nonidet P40, 10% glycerol, 2 mM benzamidine, 1 mM PMSF, 1 mM DTT, and 25 mM NaCl) was added to 1 μL well solution containing 30-40% monomethylated PEG-2,000, 0.1 M sodium acetate buffer (pH 4.6) and 0.2 M ammonium sulfate. Crystals formed after 3-5 days growth at 18° C. A crystal of 0.5 mm in length was dipped into immersion oil and frozen in liquid nitrogen. Diffraction data were collected on a CCD detector using the synchrotron source at Daresbury station 14.1. The data were processed using DENZO40. A similar protocol to that used for the monoclinic form was applied for the structure determination; statistics are shown in Table 1.
PCNA-CM peptide complex: Crystals of this complex were grown by the hanging drop vapour diffusion method. A 6-μL solution of PCNA (6-8 mg/mL) and 0.4 mM CM in a buffer consisting of 5 mM HEPES (pH 7.5), 5 mM NaCl and 1.2% v/v DMSO was added to 2 μL well solution comprising 2.7 M ammonium sulfate, HEPES (pH 8.0). Crystals grew after 3˜4 days at 18° C. A crystal of about 0.2 mm in length was collected in a 0.1-0.2 mm cryo-loop (Hampton Research), dipped briefly in 2.7 M ammonium sulphate, HEPES (pH 8.0), 26% glycerol, and was frozen by plunging into liquid nitrogen. The frozen crystal was then transferred to a magnetic goniometer head in a stream of liquid nitrogen at 100 K (Cryostream, Oxford Cryosystems). Diffraction data were collected on MAR345 image plate using station BW7B at DESY, Hamburg. The data were processed using MOSFLM45 (Table 1). Molecular replacement was carried out using AMoRe46. The trimer molecule of the published structure of hPCNA complexed with the 22mer p21 peptide Waf1 (PDB 1AXC) was used as a starting model. The molecular replacement calculations were performed in the resolution range of 10 Å-3.5 Å. A clear solution was found for two trimers in the asymmetric unit with an R-factor of 53% and a correlation coefficient of 0.30. The rotation function gave three equivalent peaks with Rf/σ around 12 and three more with Rf/σ around 10. The next highest peak was 6. The three unique rotation solutions were used in translation search. The translation function gave an unambiguous peak of correlation intensity=35.1% (next best solution 26.6), R factor 47% (next best solution 50%). The first solution was fixed and translation search performed again. With the second trimer found, correlation intensity increased to 60% and the R factor dropped to 39%. Ten cycles of rigid body refinement using the program REFMAC gave R factor and Rfree values of 29% and 33%, respectively. ARP/wARP43 was used for initial density interpretation and the addition of water molecules. Subsequent rounds of manual remodelling with the program Quanta (Accelrys, San Diego, USA) and restrained refinement using the program REFMAC44 gave an R value of 17.9% and an Rfree of 25.8%.
Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.
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1The numbers in parenthesis are statistics for the highest resolution shell.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0410498.0 | May 2004 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB05/01764 | 5/10/2005 | WO | 00 | 8/21/2007 |
