Patent Application
20050090651
This invention relates to assembly of polymeric compounds using native chemical ligation as a method step. In particular, it relates to polypeptide assemblies, their use in therapy, and methods and intermediates for their production.
Combination therapy is a widely used approach to many diseases in the clinic It is found frequently that the effects of administering two or more drugs for the same indication of only modest efficacy may result in a clinical effect significantly greater than the sum of the parts. Such therapies are used widely in diseases such as cancer and AIDS.
Ligand directed therapy is another recent development that has received wide attention. The therapy relies on a pharmacophore (drug) being covalently or non-covalently attached to a ligand specific for a certain cell surface marker or tissue. The ligand directs the drug to the surface of the cell where it may exhibit its action at an increased local concentration. This may result in a lower dose being required, may reduce side-effects associated with systemic drug delivery, and thus may increase the therapeutic ratio. The drug-ligand conjugate may alternatively be taken up into the cell where it may exhibit its action, either in its conjugate form, or as free drug if a linker labile to intracellular conditions is incorporated into the conjugate. For instance, the linker may be labile to the acid conditions present in. lysosomal compartments. The directing ligand may be a small molecule, peptide, binding protein, antibody, or antibody fragment, identified by one of a number of combinatorial binding screens, for example.
A few examples in the prior art provide for bifunctional drug molecules that combine both elements of a combination therapy regime covalently linked in the same molecule (see for example U.S. Pat. No. 5,780,653).
Linker molecules are commercially available with three homofunctional groups arrayed around a central core (e.g. TMEA—Pierce Chemical Co—Rockford, Ill.) that theoretically allow for such an approach. In practice, however, it is difficult to direct the absolute order of addition of the three components, and complex and lengthy purification steps may be required between the addition of each ligand or pharmacophore.
Another example of the same general principle is the starburst dendrimer system whereby successive rounds of addition to a central homopolyfunctional core results in a large number of reactive groups on the surface of a spherical macromolecule that is available for ligand/drug attachment (see EP0271180). Again, it is difficult to specifically control the ratios of ligand/drug loading with so many possible sites of attachment. In addition, the resultant derivatised dendrimer is extremely large and difficult to quantify exactly.
EP6832096 and Dawson, P. E. et al. (1994; Science 266: 776-799) disclose the ligation of two peptidic compounds to form proteins by a process known as native chemical ligation. The basic requirements for this coupling are the presence on participant A of a thioester moiety such as a benzyl or ethyl thioester, and the presence on participant B of an N-terminal cysteine or functional equivalent containing the minimal grouping of a 1-amino-2 thiol. The coupling reaction commences when the thio-nucleophile initially attacks the thioester in a reversible fashion causing the thioalkyl group to leave. The thioester so formed then undergoes an intramolecular S→N acyl shift to irreversibly produce the ligated amide product A-B with a native cysteine (ie. as found naturally) with a free thiol group at the junction (
This process can be repeated if it is possible to unmask a further N-terminal cysteine at the N-terminal of A, in the product A-B, by enzymatic means or by classical protecting group chemistry. A further native chemical ligation coupling with a thioester-containing moiety E would therefore yield the product E-A-B. These methods have been used to generate a variety of proteins.
It is also possible to produce cyclic peptides/proteins by incorporating both the thioester and amino terminal cysteine moieties within the same molecule.
It is possible to perform the ligations described above without removing all of the reaction participants from a solid phase.
There is also a prior art method for incorporating a thioester moiety into a participant where one does not exist. This method relies on the reaction of a primary amine with thiolane-2,5-dione and subsequent alkylation of the thioacid formed with an appropriate alkyl bromide. This method does require a single primary amine to be present otherwise multiple thioesters may be coupled.
After a native chemical ligation (NCL) reaction has taken place, there exists at the junction site between A and B (
There are reports in which considerable effort has been made to remove the cysteine free thiol subsequent to native chemical ligation (WO 98/28434; Yan, L. Z. et al., Journal of the American Chemical Society 123/4: 526-533; Botti, P. et al., 2001, Tetrahedron Letters 42: 1831-1833; Low, D. W. et al., 2001, Proceedings of the National Academy of Sciences of the USA 98[12]:6554-6559).
In the present invention we show that the free thiol formed by native chemical ligation at the junction of A and B described earlier can in fact be derivatised with suitable reagents to form a branching point. A participant C may form a covalently-linked ‘trimeric’ entity comprising A, B, and C (for example:
The invention relates to the use of such assemblies in combinatorial ligand discovery. The invention teaches methods of assembling, for example, homo- and heterotrimers and tetramers by native chemical ligation/orthogonal grafting techniques. The invention is exemplified by a number of entities with therapeutic potential, where three or more functionalities are required to be arrayed in a predictable spatial fashion with defined molecular architecture at their junctions. Also described are a number of new classes of molecule that facilitate the assembly of molecular entities that would otherwise be unable to participate.
According to the present invention there is provided a process for forming a compound having the formula (I):
from chemical entities native [A], native [B] and native [C], where native [A] has a thioester group, native [B] has a 1-amino-2-thiol group with an unoxidised sulfhydryl side chain, and native [C] has a thiol reactive function (TRF) group,
The terms “native A”, “native B” and “native C” refer to the participant chemical entities in their form before reaction with each other. The “native” entities may thus be regarded as the pre-teaction entities.
Also provided is a process for the synthesis of a compound having the formula (I):
from chemical entities native [A], native [B] and native [C], where native [A] has a thioester group, native [B] has a 1-amino-2-thiol group with an unoxidised sulfhydryl side chain, and native [C] has a thiol reactive function (TRF) group,
Yet further provided is a process for the synthesis of a compound having the formula (I):
from chemical entities native [A], native [B] and native [C], where native [A] comprises a functional group having a chemical structure, excluding [A], shown in the formula (III):
where R1 is H or a sidechain,
In the above-mentioned process of the invention, native [C] has a thiol reactive function (TRF) group for reacting with the thiol at the junction site between [A] and [B]. An alternative is to provide a thiol reactive group (TRF) linker or spacer linker which reacts with the thiol at the junction site between [A] and [B], then reacting the resultant molecule with native [C]. Thus further provided according to the present invention is a process for forming a compound having the formula (I):
from chemical entities native [A], native [B] and native [C], where native [A] has a thioester group, native [B] has a 1-amino-2-thiol group with an unoxidised sulfhydryl side chain, and native [C] has a thiol group,
Also provided is a process for the synthesis of a compound having the formula (I)
from chemical entities native [A], native [B] and native [C], where native [A] has a thioester group, native [B] has a 1-amino-2-thiol group with an unoxidised sulfhydryl side chain, and native [C] has a thiol,
Further provided is a process for the synthesis of a compound having the formula (I):
from chemical entities native [A], native [B] and native [C], where native [A] comprises a functional group having a chemical structure, excluding [A], shown in the formula (III):
where R1 is H or a sidechain,
Alternatively, there is provided a process for forming a compound having the formula (I):
from chemical entities native [A], native [B] and native [C], where native [A] has a thioester group, native [B] has a 1-amino-2-thiol group with an unoxidised sulfhydryl side chain, and native [C] has a thiol reactive function (TRF) group,
The thiol reactive function (TRF) group linker may comprise, or the Spacer may be, a polyalkyloxy, alkyl, aryl, arylalkyl or peptidyl group.
Each of R1 and R2 shown in formula (III) and formula (IV) may be a substituted or unsubstituted aryl group or a substituted or unsubstituted alkyl group.
The functional group of native [B] may be a 1-amino-2-thiol group having an unoxidised sulfhydryl side chain.
The unoxidised sulfhydryl side chain of the 1-amino-2-thiol group of native [B] may be the only unoxidised sulfhydryl side chain present in native [B] . Alternatively, native [B] may comprise additional free thiols which are removed by mutation such that the unoxidised sulfhydryl side chain of the 1-amino-2-thiol group of native [B] becomes the only unoxidised sulfhydryl side chain present.
Native [B] may have a terminal cysteine moiety comprising the terminal 1-amino-2-thiol group.
The thiol reactive function (TRF) group may be a thiol, an alkyl halide, a 2-pyridyl disulfide, a 4-pyridyl disulfide or a Michael acceptor (eg. a vinyl sulfone or a maleimide).
An example of a suitable thiol reactive function (TRF) is an alkyl halide and the reaction is an SN2 displacement. The reaction product is a thioether. Incorporation of alkyl halides into various substrates is a straightforward process well known to those skilled in the art.
Another example is an activated disulfide such as a 2- or 4-pyridyl disulfide and the reaction is a disulfide exchange. The reaction product is a disulfide. Pyridyl disulfides may be incorporated synthetically into many substrates. Additionally, a range of reagents is available to install pyridyl disulfides on the surface of proteins and other molecules.
A further example is a Michael acceptor such as a vinyl sulfone or a maleimide and the reaction is a Michael-type addition. The reaction product is a thioether. Maleimides may be incorporated synthetically into many substrates. Additionally, a range of reagents is available to install maleimides on the surface of proteins and other molecules.
Examples of the three resultant reaction products are shown (
The thioester group or functional group of native [A] may be a benzyl, ethyl or 2-aminoethyl group.
Native [A] and/or native [B] and/or native [C] may comprise any one of the following entities or derivatives thereof: pharmacophore, ligand, small molecule, purification handle/immunochemical tag (eg biotin, a dinitrophenyl compound), fluorescent moiety (eg. Alexafluor dye, Texas Red dye), solubilising agent, chelating ligand, chelating ligand plus radioimaging agent, therapeutic protein, antibody or fragment thereof, peptide, peptidomimetic or macroscopic particle (eg. a bead).
Prior art (for example U.S. Pat. No. 5,789,653) does not suggest or teach a covalently linked combination therapeutic covalently linked to a directing ligand or antibody or antibody fragment.
Native [A] and/or native [B] and/or native [C] may comprise an entity from a combinatorial array of chemicals. The combinatorial array may be a peptide library.
Also provided according to the present invention is compound obtainable or obtained by the above-mentioned processes.
Further provided is a compound having the general formula (I):
wherein [A], [B] and [C] are derived from chemical entities native [A], native [B] and native [C], respectively, and are covalently linked by linker group L, the compound of formula (I) having a chemical structure shown in formula (VI):
or formula (XI):
where TRFD is a thiol reactive function derivative and the Spacer is a linking group,
Yet further provided is the use of the compound according to the present invention in a binding screen for detecting binding to a target entity. The binding screen may select compounds which have increased or decreased binding to a target entity.
Also provided is the use of a compound formed by native chemical ligation in a process for the production of the compound of the invention.
In one embodiment, native [A] and/or native [B] and/or native [C] may comprise a myristoyl/electrostatic switch peptide (MSWP) or derivative thereof, for example MSWP2278 (SEQ. ID NO: 2).
In another embodiment, native [A] comprises EGFP, native [B] comprises MSWP2278 (SEQ. ID NO: 2) and native [C] comprises Texas Red Dye.
In yet another embodiment, native [A] comprises EGFP, native [B] comprises MSWP2278 (SEQ. ID NO: 2), and native [C] comprises an antibiotic (for example vancomycin).
In an alternative embodiment, native [A] comprises Hirulog anticoagulant peptide, native [B] comprises MSWP2278 (SEQ. ID NO: 2), and native [C] comprises SCR1-3.
WO 98/02454 relates to the derivatisation of soluble complement regulators by disulfide exchange reaction with pyridyl disulfide activated myristoyl/electrostatic switch peptides (MSWPs). These derivatives demonstrate improved complement inhibitory activity owing to their membrane localisation by the MSWP. The myristoyl group was designed to be minimally membrane insertive, while the lysine rich peptidic portion of MSWP-1 (Seq. ID No 1) was designed to interact with the negatively charged phospholipid headgroups present on the lipid bilayer. The concept is exemplified in WO 98/02454 by SCR1-3 of LHR-A of CR1, altered by a single C-terminal cysteine residue. After treatment with an appropriate reducing agent to ensure the presence of a free thiol on the terminal cysteine sidechain, the protein is reacted with MSWP-1 (N-(Myristoyl)-Gly-Ser-Ser-Lys-Ser-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-Pro-Gly-Asp-(S-2-Thiopyridyl) CyS-NH2) to yield a membrane binding derivative of SCR1-3 (
This chemistry relies on there existing within the protein participant of the reaction a single free thiol resulting in derivatisation at a single point only. There are numerous other cysteines in SCR1-3, but all save the C-terminal cysteine are participants in intramolecular disulfide bridges. Some proteins, however, may have free thiols other than that specific free thiol on which derivatisation is desired to occur. In instances such as this, it may be possible to mutate using molecular biological techniques these cysteine residues to other amino acids, while retaining the desired biological function. In other cases this mutation process may result in the loss of the desired biological function.
WO 98/02454 describes polypeptide derivatives in terms of a combinatorial array of membrane binding elements corresponding to the general structure:
[P]-{L-[W]}n-X 1
In the context of the current invention where such an assembly is constructed by native chemical ligation, and where n=1, structure 1 can be rewritten whereby entity A represents [P], entity B represents [W] and X, and thus L is comprised of the nascent cysteine residue at the junction between A and B. We will for simplicity continue our discussion of the scope of the invention using A, B, and C, but with an additional linker function L which is comprised of the nascent cysteine residue at the junction between A and B and any further atoms that may make up a flexible linker to moiety C.
Where [A] is a protein, one method of formation of the [A]-L-[B] portion of the invention is provided by the IMPACT™ system (New England Biolabs). Conjugates are afforded by the native chemical ligation of peptides containing an N-terminal cysteine, to proteins expressed in frame with a controllable intervening peptide sequence (CIVPS) and an affinity purification handle. The desired protein is cleaved from its affinity purification matrix by elution with a free thiol such as MESNA to yield a C-terminal thioester. The MESNA thioester is displaced by the thiol of the N-terminal cysteine before an S→N acyl shift installs the peptide bond in an irreversible fashion. Alternatively, the affinity matrix is incubated with the free peptide, displacing the desired protein from the matrix and ligating in a single step (
The most favoured MSWP in WO 98/02454 comprised an N-terminal myristoyl function, a C-terminal 2-thiopyridylated cysteine primary amide, and a series of intervening residues including the hexalysine region (
In the specific case where [C]=[A]-L-[B] it is possible to form a symmetrical homodimer. The alkyl halide, activated disulfide, and maleimide methods described above are also applicable in this case to yield symmetrical thioethers, symmetrical disulfides, and symmetrical maleimides respectively. However a further method also applies. The invention allows for the spontaneous air oxidation of two identical [A]-L-[B] functions to the homodimer in aqueous solution.
Many other linking chemistry are possible. Some are explained in WO 98/02454 and are incorporated herein by reference.
Clearly if one or more of the following:
A≠A′
B≠B′
is true, then the product of such a coupling reaction will be pseudoheterotetra- or heterotetrameric i.e.
In both of these cases, a simple air oxidation may result in mixtures of products formed, therefore the activated disulfide and Michael type addition methods are preferred. For instance, in the case of the air oxidation of [A]-L-[B]+[A′]-L-[B], the following three products would be observed in the reaction mixture:
The incorporation of C orthogonally to the existing linear molecular backbone is a fundamental utility described within the invention. The entity C is, as a result of the inventive step, not constrained within the confines of a linear sequence but is attached to a point between the entities A and B.
In the case where one of A, B, or C is a probe such as biotin, or a fluorophore such as an Alexafluor dye, an immunochemical marker such as a dinitrophenyl compound, or a radiochemical label, the trimers or tetramers described above gain utility in that their fate is able to be monitored. These monitoring techniques include but are not limited to an immunohistochemical screen, a fluorescence localisation assay, or radiochemical screen. The scale of monitoring may vary from a whole body screen, to intra-organ differential localisation, to the identification of sub-cellular fate.
An example is:
This heterotrimer (
A further example is:
The antibiotic in can be vancomycin for example (
A further example is:
This heterotrimer contains all the elements of a ligand directed combination therapy as described earlier. The MSWP2278 peptide serves to localise the remainder of the molecule on the surface of mammalian cells. On these surfaces, the antiocoagulant properties and the complement inhibitory activities of both of the components A and C respectively are conserved. The synthesis was accomplished by the native chemical ligation of MSWP2278 with a hirulog type peptide thioester ((D)-PheProArgProGlyGlyGlyGlyAspGlyAspPheGluGluIleProGluGluTyrLeuGlySerGlySerGlySerGlySerGlySerGlySerGly-SEtCONH2) (Seq ID No 3). The product formed was reacted with a bismaleimide, and then with SCR1-3. The single exposed C-terminal cysteine reacts solely with the maleimide to provide the heterotrimer (
In a further example, C can be incorporated as described above not as a single chemical entity but as a combinatorial array of n chemical entities where C=C(1→n).
Alternatively, this combinatorial array of n chemicals was comprised of a peptide library.
Further, each of n chemical entities was comprised of a different peptide sequence, and was linked to L by disulfide exchange chemistry. This principle permitted the use of the combinatorial array of C(1→n) in a binding screen where the binding energy gained from C was additive and augments that gained from B. The process of identifying binding ligands identifies C sequences capable of increasing the overall binding to the cell type of choice, and thus confers selectivity over other cell types
i.e. for library member λ:
ΔG(λ)=Σ(ΔGA+ΔGB+ΔGC)
And in the case where B is a MSWP, ΔGB=ΔGW+ΔGX.
There are several possibilities to enable the reduction to practice of such a binding screen. One method involved a one-pot preparation of a multiplicity of library members which were incubated with a tissue for which selective targeting was desired. Each member of this library shall have incorporated into C an affinity handle such as biotin, and shall have a disulfide linkage between C and L. After a washing process to remove library members that either do not bind or bind weakly, the sequence identity of C ligands that bind can be determined by reduction of the disulfide, extraction of C by supported avidin, and identification of C by mass spectrometry.
A different method involves the combinatorial assembly of heterotrimers where the members of the library C(1→n) are used individually in the fashion described above to furnish a library of discrete heterotrimers in a high throughput fashion. The members of this protein library can then be individually screened for binding to a particular cell type in, for instance, a 96-well format. The extent of binding can be detected immunohistochemically (ELISA) or by direct fluorescence in the case where A is fluorescent such as GFP, or where A or B or C or any combination of the three is a fluorescently labeled entity. Both of these methods are exemplified (vide infra)
Any of the components brought together with this enabling technology may be selected from the group consisting of: a pharmacophore, a ligand, a small molecule, a purification handle/immunochemical tag (eg biotin), a fluorescent moiety, a solubilising agent, a chelating ligand, a chelating ligand plus radioimaging agent, a therapeutic protein, an antibody or fragment thereof, a peptide or a peptidomemtic.
This list is not exhaustive. Other moieties may be linked in the fashion to be described and will be apparent to those skilled in the art.
The peptide:
The peptide:
The crude peptide was desalted via gel filtration (Sephadex G10, 0.1% TFA in water) before purification by preparative C18 Vydac) high performance liquid chromatography (HPLC) using 0.1% trifluoroacetic acid and 0.1% trifluoroacetic acid/90% acetonitrile as gradient components. After lyophilisation, the peptide was a white amorphous powder, soluble to at least 10 mg/mL in both dimethylsulfoxide and water. Fast atom bombardment spectrometry gave a main peak at 2101 amu corresponding to the molecular ion of the peptide.
Standard molecular biological techniques were used to replace the VP1 gene in vector pET21-VP1Int [Schmidt U., Rudolph, R. & Böhm, G. J. Virol. 74, 1658-1662 (2000)] with the gene for enhanced green fluorescent protein (EGFP), using the vector pEGFP-N1 (Clontech) as template. The resulting vector, pET107-01, expresses the EGFP gene under the control of the T7lac promoter as a C-terminal fusion with the VMA1 gene for the intein from Saccharomyces cerevisiae and the gene for the chitin binding domain from Bacillus circulans. The construction of pET21-VP1Int from vectors pCYB2 and pET21d (New England Biolabs) has been described elsewhere (Schmidt et al., vide supra). Upon transformation into the bacterial strain Hams113, an overnight starter culture of 25 mL LB medium containing 100 μg/mL ampicillin was inoculated with a single colony and, on the next morning, diluted 1:100 into 2 L of LB medium containing 100 μg/mL ampicillin. The culture was fermented at 37° C. until an optical density at 600 nm of 0.5 was achieved, at which point the temperature was lowered to 20° C. and expression of the gene induced by the addition of 1 mM IPTG after 30 mins. Cells were harvested 21 h post induction by low-speed centrifugation and stored in aliquots at −40° C.
To lyse cells, an aliquot of bacterial paste corresponding to 333 mL of fermentation broth was resuspended in 40 mL of ice-cold Buffer 160 (20 mM HEPES pH 8.0, 100 mM NaCl, 1 mM EDTA) and homogenised by three passages through an Emulsiflex C5 homogeniser at a pressure of 12 kpsi. The soluble fraction was obtained by centrifugation (20000 g, 30 min, 4° C.) and immediately passed over a column containing 15 mL chitin beads (New England Biolabs). The column was washed with four column volumes of Buffer 100, eight column volumes of Buffer 2000 (20 mM HEPES pH 8.0, 2000 mM NaCl, 1 mM EDTA), four column volumes of Buffer 100, and finally with three column volumes of Cleavage Buffer 1 (Buffer 100 containing 50 mM cysteine). After incubation at 6° C. for 20 h, EGFP was eluted from the column using Buffer 100 as the elution buffer.
The procedure outlined here resulted in an EGFP labelled at the C-terminal with a single additional cysteine residue designated APT2269. The availability of this thiol was tested by derivatisation with the thiol-reactive MSWP1. 500 μL of a 26 μM solution of APT2269 was mixed with 100 μL 10 mM TCEP/20 mM HEPES over 1 h to ensure complete reduction of the terminal cysteine. The mixture was dialysed against 1 L PBS/EDTA (1 mM)/TCEP (1 mM), against 1 L PBS/EDTA (1 mM) and MSWP1 added to a final concentration of 34 SM. This reaction resulted in the derivatisation of APT2269 with between 1 and 3 tails (a process referred to as supertailing).
EGFP was purified as described in Example 4, but Cleavage Buffer 2 (Buffer 100 containing 50 mM MESNA and 50 μM APT2278) was used in the overnight incubation. The cleavage resulted in almost quantitative tailing of EGFP with no evidence of supertailing. Even upon addition of 5 mM APT2278 (equivalent to a 1000-fold molar excess) no further tailing was detected. The reaction product of EGFP with APT2278 was designated APT2308.
To demonstrate the accessibility of the newly introduced thiol in APT2308, we subjected it to further derivatisation with the thiol-reactive compound APT542.
APT2308 was treated with 1 mM TCEP overnight and the buffer exchanged by passing the solution over a Nap-5 column equilibrated with PBS. APT542 was added to a concentration of 13 μM and the reaction mixture analysed by SDS-PAGE after 1 h.
The addition of each tail was confirmed by a 2 kDa shift in molecular weight.
COS7 cells were incubated with either APT2308 or recombinant EGFP at a concentration of 6 μM in Dulbecco's modified eagle medium (Sigma). After 1 h, the cells were visualised by fluorescence microscopy and the images recorded with identical parameters. EGFP demonstrated no binding to COS7 cells (which had been demonstrated with other cell lines for concentrations up to 2.5 mg/mL). APT2308 bound to the cells with very high efficiency (
pET107-01 was used as a template for mutagenesis with the aim of replacing two cysteine residues in APT 2253 (C49 and C71), with two serine residues. Single base changes (TGC to AGC) were made to each codon at positions 5349 and 5415 of pET107-01.
Mutagenesis primers were designed to extend 15 bases in either direction of each mismatched base. A restriction site was also incorporated into each pair of primers to aid the identification of positive clones. In both cases, this necessitated the introduction of a silent mutation in the coding sequence.
Primers Designed for the Mutation of Cysteine Residue at Position 49 (C49S)
In addition to generating a thymine to adenine transition (at position 5349 in pET107-01), these 41mers incorporate an Age I site (ACCGGT) by silent mutation of a glycine codon (G52 in APT 2253), replacing cytosine with thymine (position 5360 in pET107-01).
Primers Designed for the Mutation of Cysteine Residue at Position 71 (C71S).
In addition to generating a thymine to adenine transition (at position 5415 in pET107-01), these 32 mers incorporate a Hind III site (AAGCTT) by silent mutation of a glutamine codon (Q70 in APT 2253), replacing guanine with adenine.
A two step process was used whereby the C49S variant was generated first via site-directed mutagenesis using the QuickChange mutagenising kit (Stratagene), according to the manufacturers instructions. pET107-01 was amplified using primers 1200951 and 1200952, followed by treatment with Dpn I and transformation into XL10 competent cells (Stratagene) to generate pET107-02. pET107-02 was then used as a template for C71S mutagenesis with primers 1200961 and 1200962, using the same process. The resultant plasmid, pET107-04, encodes APT 2307.
Doubly mutated EGFP was tailed using the appropriate peptide or amino acid at 50 μM as described in Example 5. Each reaction mixture was ultrafiltered exhaustively with PBS to remove excess peptide or amino acid.
APT2360 (40 μl of a 10 μM solution in PBS) was mixed with TCEP (2.5 μl of a 1 mM solution in 50 mM HEPES pH 4.5) and left at ambient temperature overnight. EZ-Link PEO-maleimide biotinylation reagent (Pierce, UK). 8 μL of a 400 uM solution in PBS) was added and the mixture left at ambient temperature over 1 h. Freshly made cysteine solution (5 μL of a 10 mM solution in PBS) was added to quench the reaction. Excess reagents were removed by microdialysis. A small gel shift was observed, the new product was immunoreactive with an anti-biotin antibody in a Western Blot, and the expected increase in molecular mass was observed by MALDI mass spec. The reaction proceeded in quantitative yield.
APT2360 was treated with TCEP as described in Example 11 before an activated disulfide derivative of vancomycin (APT2033), at 13.2 μL of a 610 μM solution in PBS, was added. After 2 h at ambient temperature, excess reagents were removed by -microdialysis. A significant gel shift was observed, and the new product was formed quantitatively.
APT2361 was treated with TCEP as described for APT2360 in Example 11 before a 3-fold molar equivalency of −OOC-MTAAPLRGSXGC(2-S-Py)-NH2 (X=biotinylated lysine) (APT2400) was added. The mixture was incubated at ambient temperature over 2 h, before excess TCEP and APT2400 were removed by dialysis. An appropriate gel shift was observed by SDS-PAGE corresponding to the addition of a single peptide to the native chemical ligation junction.
APT2360 was prepared as described in Example 9. Upon incubation overnight in the presence of 1 mM TCEP, the sample was dialysed against degassed PBS and Texas Red-maleimide (Molecular Probes) was added to a final concentration of 2 mM (corresponding to an approximate 200-fold excess over APT2360). The mixture was incubated for 2 h at room temperature and then the protein separated from free fluorophore via gel filtration with a PD-10 column. An analysis using SDS-PAGE, UV/VIS spectroscopy and cell binding studies showed that APT2455, the reaction product
The production of APT2434 takes place in a three-step synthesis. The first step is the Native Chemical Ligation of APT2383, the hirulog-thioester (including a Glycine-Serine linker segment) to APT2278, yielding APT2384. Typically, both reaction partners are used at mM concentrations and the reaction proceeds overnight at 22 degrees centigrade in the presence of 1 mM TCEP and in 40 mM HEPES buffer, pH 8.5. APT2384 was purified using RP-HPLC and derivatised with a tenfold surplus of BM(PEO)4 in PBS, a bismaleimide with an 18 Å long linker segment (Pierce). The reaction product, APT2433, was purified via RP-HPLC and reacted with APT154 to yield APT2434, the final heterotrimer.
APT2434 was assessed regarding its three proposed functions—membrane localisation, complement inhibition and anticoagulant activity. All three functions were found to be preserved in the final molecule.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0123262.8` | Sep 2001 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB02/04352 | 9/26/2002 | WO |
