WO2014150173A1

WO2014150173A1 - Method for identifying inhibitors of protein-protein interaction

Info

Publication number: WO2014150173A1
Application number: PCT/US2014/022489
Authority: WO
Inventors: Gerhard Wagner; Patrick Ryan HAGNER; Haribabu Arthanari; Vladimir M. GELEV; Ricard A. RODRIGUEZ; Amr FAHMY
Original assignee: President And Fellows Of Harvard College
Priority date: 2013-03-15
Filing date: 2014-03-10
Publication date: 2014-09-25

Abstract

A method of using NMR and combinatorial synthesis to identify compounds that bind to a target protein is provided. Embodiments of the present disclosure are directed to methods of making compounds including one or more fragments that bind to a target protein. The target protein may be one which is known to have a binding partner. The compound including one or more fragments that bind to the target protein may be an inhibitor of the binding or other interaction between the target protein and binding partner. According to one aspect, binding fragments are attached to a polypeptide backbone in a conformation which allows binding of the fragments to the target protein.

Description

METHOD FOR IDENTIFYING INHIBITORS OF PROTEIN-PROTEIN INTERACTION

RELATED APPLICATION DATA

This application claims priority to U.S. Provisional Patent Application No. 61/787,220 filed on March 15, 2013 and is hereby incorporated herein by reference in its entirety for all purposes. STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under NIH U54 GM094608, Grant Number CA068262, GM047467 and AI037581. The Government has certain rights in the invention.

FIELD

The present invention relates to methods of identifying compounds that inhibit protein-protein interactions using NMR fragment screening and high throughput synthesis to create peptide backbone structures having fragments bound thereto.

BACKGROUND

Fragment based drug discovery (FBDD) methods chemically link or extend known small molecule binders to construct high-affinity inhibitors of protein interactions. The initial binding fragments are readily identified by various biophysical methods, including Nuclear Magnetic Resonance (NMR) spectroscopy. Common NMR methods include Saturation Transfer Difference (STD) or water Logsy in combination with Inter-Ligand nuclear Overhauser Enhancement (iLOE) experiments. However, challenges exists to create compounds including fragments that are effective to inhibit protein-protein interactions. See Biochemistry 2012; 51(25):4990. SUMMARY

Embodiments of the present disclosure are directed to methods of making compounds including one or more fragments that bind to a target protein. The target protein may be one which is known to have a binding partner. The compound including one or more fragments that bind to the target protein may be an inhibitor of the binding or other interaction between the target protein and binding partner. According to one aspect, binding fragments are attached to a polypeptide backbone in a conformation which allows binding of the fragments to the target protein. According to an additional aspect, methods are provided for high throughput synthesis and screening methods of compounds including a polypeptide backbone and binding fragments to identify compounds optimized for binding to a target protein and inhibiting protein-protein interactions. Accordingly, a set of compounds having different polypeptide backbone structures and different binding fragments are prepared by high throughput synthesis and the set of compounds is then screened for binding to a target protein. Compounds according to the present disclosure include a polypeptide backbone, such as a cyclic polypeptide backbone including fragments that are linked to the backbone or otherwise extended from the backbone or integrated into the backbone itself and exhibit a 3 -dimensional conformation suitable for binding to a target protein. The present invention enables generation of a large number of rigid compounds that incorporate one or more protein binding fragments in a large number of three dimensional orientations, while presenting ample additional functionalities and geometries for interaction with the target protein.

According to certain aspects and with reference to Fig. 1A, NMR (or SPR) experiments are carried out on a target protein, such as MCT-1 or Calcineurin to identify small molecules, referred to herein as "fragments" that bind to the target protein. Such binding is exemplified by a range of binding strengths from weak binding to strong binding. According to one aspect, binding fragments can be identified from among a collection or library of candidate fragments using screening methods known to those of skill in the art. According to one aspect, once a set of fragments has been identified, pairs of fragments are then identified from within the set that bind non-competitively to adjacent sites on the protein, for example by competition experiments or by looking for nuclear Overhauser (NOE) contacts between the fragments such as by an interligand NOE (iLOE) experiment. Representative screening methods includes those described in Pellecchia et al., Chemistry and Biology 2005, 12, 961 and Huth et al., Methods in Enzymology 2005, 394 p. 549 each of which are hereby incorporated by reference herein in their entireties.

As illustrated in Fig. IB, a library of cyclic peptide backbones is generated in silico, containing amino acids. The term "amino acid" includes L- and D-amino acids, non-proteinogenic amino acids, N-alkyl and N-acyl amino acids. Examples of amino acids include those commonly understood by those of skill in the art and including alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, selenocysteine and pyrrolysine. The amino acid can be an L-amino acid or a D-amino acid. Amino acids also include compounds including an amino moiety and an acid moiety such as a carboxylic acid moiety or acyl moiety. These can be compounds having a main structure onto which an amino moiety and an acid moiety have been attached. Amino acids also include their reacted form, such as when attached to a peptide either as a side moiety or in series with other peptides. Non-proteinogenic amino acids are those commonly understood by one of skill in the art as not incorporated into proteins such as GABA, isonipecotic acid, beta alanine, hydroxyproline, L-DOPA, triiodothyronine and the fragment- derived molecules shown in Figure 2. Non-proteinogenic amino acids are those commonly understood by one of skill in the art as not produced directly and in isolation by standard cellular machinery, such as hydroxyproline and selenomethionine and the like. N-alkyl amino acids are those commonly understood by those of skill in the art to include an amino acid having an alkyl group attached to a nitrogen of the amino group. "Alkyl" includes straight or branched chain alkyl groups being substituted or unsubstituted and having between about 1 carbon and about 5 carbons, such as methyl, ethyl, propyl, butyl, and pentyl. Exemplary substitutions include hydroxyl, halide, ethyl, propyl, isopropyl, amino, sulfonamido, sulfonyl, nitro, etc. (limited only by the commercial availability of corresponding precursors). Additionally, all-carbon or heterocyclic aromatic groups such as furans, thiophenes, benzenes, indoles, pyrazoles, imidazoles, thiazoles, or other portions of a larger molecule can be substituted, such as a molecular fragment identified in a protein binding assay. "Acyl" refers to the same types of alkyl groups listed above, but instead of linked directly to the nitrogen atom of the peptide backbone, said groups are linked via the carbon of a carbonyl (C=0) group or the sulfur atom of a sulfonyl (SO₂) group (in other words, "alkyl" denotes "peptideN-R" and "acyl" denotes "peptideN-CO-R" or "peptideN-S02-R"). The method of utilizing N-alkylation and N-acylation to attach fragments to the peptide backbone is illustrated in Figure 2E.

N-acyl amino acids are those commonly understood by those of skill in the art to include amino acids having an acyl group attached to a nitrogen and include hippuric acid, phenaceturic acid and the like. "Acyl" includes a functional group derived by the removal of one or more hydroxyl groups from an oxoacid including inorganic acids. According to one aspect, the acyl group may be derived from a carboxylic acid and having the formula RCO-, where R may represent an alkyl group or other group that is attached to the CO group with a single bond. Acyl groups can be derived from other types of acids such as sulfonic acids and phosphonic acids. According to one aspect, an acyl groups is attached to a larger molecular fragment, in which case the carbon and oxygen atoms are linked by a double bond and the carbon atom is linked to Ri and R₂ which may be portions of a larger molecule. An exemplary acyl group is acetyl.

According to one aspect, embodiments include synthetic amino acids, such as non-naturally occurring amino acids, modified amino acids and the like. These may include specifically designed amino acids or reactive amino acids capable of binding to the fragments that have been specifically modified to possess a complementary functionality, as shown in Figure 2D. Conceptually, such reactive amino acids can be referred to as chemical 'handles' for attachment of the fragments insofar as they provide reactive sites for covalent attachment of one or more fragments. Fragments are modified in such a way as to be incorporated in various orientations into cyclic peptide scaffolds. Accordingly, the present disclosure contemplates amino acids having reactive sites or other sites to which a fragment can be bound using chemistries known to those of skill in the art and disclosed herein. Computer simulations are used to identify a subset of scaffolds that position the fragments in plausible orientations thereby limiting the number of compounds that need to be synthesized and tested. Accordingly, in one aspect, in silico methods are used to identify scaffold possessing a conformation that presents the attached fragments in a manner consistent with the manner in which the individual fragments are believed to bind on the surface of the protein. This identifies candidate scaffolds for synthesis and testing. Accordingly, methods provided herein screen scaffolds based on their likelihood of binding to a target protein and to identify candidate scaffolds for synthesis and testing. In one optional aspect, the simulations first identify and discard peptide backbone scaffolds that are likely to ne non-cell-permeable. In another aspect, scaffolds unlikely to present fragments in a manner consistent with individual fragment binding are excluded. Further, scaffolds that sterically clash with the protein when presenting the fragments are excluded. Further, for a given scaffold that presents fragments in a plausible prientation, the computer simulation and related computations identify candidate side chains potentially complementary to the target protein surface. As illustrated in Fig. 1C, the set of peptides identified by the simulations as candidate peptides is synthesized by solid phase peptide synthesis methods known to those of skill in the art or by in vitro peptide synthesis known to those of skill in the art. Each candidate peptide scaffold is diversified by combinatorial synthesis to find the optimal fragment / peptide combination. Methods for combinatorial synthesis of a plurality or set of peptide scaffolds having one or more, or two or more binding fragments are known to those of skill in the art. The peptides having the fragments attached thereto, i.e., candidate inhibitor compounds, are screened for inhibitory activity by one of several assays familiar to those skilled in the art including FRET such as described in Camarero et al., Chemistry Today 2007, 25, 20, reverse two-hybrid such as described in Tavassoli et al., ACS Chem Biol 2008, 3, 757, fluorescence polarization such as described in Roehrl et al., Biochemistry 2004, 43, 16056, or pulldown as described in Grossman et al., PNAS, 2012 Oct 30: 109(44): 17942-7, doi: 10.1073/pnas, 1208396109 each of which are hereby incorporated by reference in their entireties. The assays can be conducted with immobilized protein incubated with mixtures of peptides, or with immobilized peptides incubated with the target protein(s), or with cells containing a reporter system incubated with mixtures of peptides. Inhibitors of protein- protein interaction are identified by a combination of deconvolution synthesis and mass spectrometry methods, e.g. using positionally encoded mixtures, stable isotope labeling, and tandem MS-MS. An example of using a useful approach for decoding a peptide library is described in Pastor et al., JACS 2007 vol. 129(48) pp. 14922- 14932. BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which: FIG. 1 is a schematic showing methods according to the present disclosure.

FIG. 2A identifies fragments that bind Calcineurin at adjacent sites. Experimentally observed STYD and iLOE (red dotted lines) between hydrogen atoms in the fragments suggest that when the two fragments are bound to the Calcineurin protein, their relative orientation is similar to what is shown in the figure. Based on this relative orientation, the fragments will be incorporated in a library of macrocycles and in a manner consistent with the observed STD and iLOEs. FIG. 2B is a schematic showing various strategies for incorporation of fragments in a library of macrocycles. FIG. 2C depicts various ways to incorporate the fragments into the macrocycles by first converting the fragments into amino acids such as by attaching an amino and a carboxy functionality, and subsequently using the resulting amino acids in peptide synthesis. Accordingly, aspects of the present disclosure are directed to a scaffold structure as shown that is modified with an amino functionality and an acid functionality. The resulting amino acid can be incorporated into a cyclic peptide by attachment to a cyclic peptide as a side moiety or forming part of the ring structure of the cyclic polypeptide. FIG. 2D depicts an alternate approach to attachment of fragments to cyclic polypeptides. In this embodiment, cyclic polypeptides which contain one of the shown "anchor points" are synthesized first, followed by attaching one of the fragments iunctionalized as shown using one of the chemistries shown. Anchor points are reactive groups or moieties on the cyclic polypeptide and are shown, for example in schematic with the anchor point included into a cyclic polypeptide with the line indicating amino acids ("AA") in series in the desired number, such as between about 4 and about 9 amino acids. The cyclic polypeptides with the reactive groups can be used as a desirable reactant in making cyclic polypeptide compounds as inhibitors. The cyclic polypeptides with the reactive groups are reacted with desirable compound to result in the inhibitor cyclic polypeptide. Accordingly, aspects of the present disclosure are directed to reacting a cyclic polypeptide having an anchor point to a FIG. 2E depicts an alternate method for attaching the fragments to the macrocycle, by alkylation or acylation of the amide nitrogens of the peptide backbone, to attach fragments thereto.

FIG. 3 depicts a schematic of the solid phase synthesis of diversified cyclic peptides containing fragments as side chains or in the backbone, and containing a PEG solubility tag. DETAILED DESCRIPTION

Embodiments of the present invention are directed to a method of identifying a binding ligand to a target protein comprising identifying one or more fragment compounds that bind to the target protein, incorporating the one or more fragment compounds into a plurality of candidate polypeptide scaffolds, identifying selected polypeptide scaffolds from among the candidate polypeptide scaffolds that position the one or more fragment compounds near their respective binding sites on the target protein, modifying the selected polypeptide scaffolds by combinatorial synthesis to produce diversified polypeptide scaffolds, and identifying one or more optimized polypeptide scaffolds from among the diversified polypeptide scaffolds that have binding affinity to the target protein. Methods according to the present disclosure and described above can also be carried out by identifying two or more fragment compounds and then incorporating them into a plurality of polypeptide scaffolds, and then identifying the polypeptide scaffolds that position the two or more fragment compounds near their respective binding sites on the target protein, modifying the selected polypeptide scaffolds and identifying one or more optimized polypeptide scaffolds. Methods according to the present disclosure also include identifying inhibitors of protein-protein interactions by identifying a binding ligand to a target protein having a known binding protein partner.

According to one aspect, the step of identifying the one or more fragment compounds includes screening candidate fragment compounds for binding to the target protein. According to an additional aspect, the screening is performed in vitro or in vivo. According to an additional aspect, two or more fragments bind to the target protein non-competitively at adjacent sites on the target protein. According to an additional aspect, the first polypeptide scaffolds are cyclic polypeptides.

According to an additional aspect, the step of incorporating the one or more fragment compounds into a plurality of candidate polypeptide scaffolds is performed in silico. According to an additional aspect, the step of identifying selected polypeptide scaffolds from among the candidate polypeptide scaffolds that position the two or more fragment compounds near their respective binding sites on the target protein is performed in silico. According to an additional aspect, the step of identifying one or more optimized polypeptide scaffolds from among the diversified polypeptide scaffolds that have binding affinity to the target protein includes screening for binding of the diversified polypeptide scaffolds to the target protein. According to an additional aspect, the screening is in vitro or in silico. According to an additional aspect, the step of identifying the two or more fragment compounds includes screening candidate fragment compounds for binding to the target protein using NMR. According to an additional aspect, the target protein is multiple copies in T-cell lymphoma- 1 (MTC-1). According to an additional aspect, the target protein is multiple copies in T-cell lymphoma- 1 and wherein the one or more optimized polypeptide scaffolds inhibit binding between MTC- 1 and DENR. According to an additional aspect, the target protein is calcineurin. According to an additional aspect, the target protein is calcineurin and wherein the one or more optimized polypeptide scaffolds inhibit binding between calcineurin and NFAT.

One of ordinary skill in the art will readily understand that the disclosure is not limited to MCT- 1 or calcineurin, but that other target proteins will become evident in view of the present disclosure. According to an additional aspect, the polypeptide scaffolds include between about 5 and about 10 amino acids. According to an additional aspect, the polypeptide scaffolds include between about 6 and about 8 amino acids. According to an additional aspect, the polypeptide scaffolds are cyclic. According to an additional aspect, the polypeptide scaffolds are cyclic and include between about 6 and about 8 amino acids. According to an additional aspect, the polypeptide scaffolds are cyclic and two or more fragment compounds are attached to a respective cyclic polypeptide scaffold. Fragment compounds can be attached directly to the scaffold, such as by covalent bond, or by any suitable chemical linking group. According to an additional aspect, the polypeptide scaffolds are cyclic and at least one of the one or more, or two or more, fragment compounds is internal to a respective cyclic polypeptide scaffold to the extent that the fragment compound is bound at a first position and a second position such that it forms part of the cyclic scaffold. According to an additional aspect, the polypeptide scaffolds are cyclic and at least two of the one or more, or two or more, fragment compounds are internal to a respective cyclic polypeptide scaffold.

The term "binding site", "active binding site," "binding pocket," or epitope refers to a region of a target protein that binds or interacts or has an affinity for a particular compound. The terms "associates with" or "interacts with" refers to conditions of proximity between a chemical entity, compound or portion thereof with another chemical entity, compound or portion thereof. The association or interaction may be non-covalent or covalent. Non-covalent interactions include hydrogen bonding, van der Waals or electrostatic interactions, and hybrophobic effect. A fragment may have a binding affinity for a binding site. Such binding affinities may vary depending on the fragment chemical structure. According to certain aspects, structure coordinates identified from NMR experiments may be used to design ligands with enhanced affinities. This can be achieved by intuition inspecting the co- crystal structures of protein-protein binding pairs, or by computer modeling techniques and binding energy calculations to develop tighter binding ligands. This may include optimizing interactions by matching van der Waals contacts, salt bridges, hydrogen bonds and other electrostatic interactions. Computer simulation techniques are then used to map interaction positions for functional groups including protons, hydroxyl groups, amine groups, divalent cations, aromatic and aliphatic functional groups, amide groups, alcohol groups, etc. that are designed to interact with the model site. These groups may be designed into a candidate compound with the expectation that the candidate compound will specifically bind to the site. Candidate compound design thus involves a consideration of the ability of the candidate compound to interact with a site through any or all of the available types of chemical interactions, including hydrogen bonding, van der Waals, electrostatic, and covalent interactions.

The ability of a candidate compound to bind to an active binding site or epitope of a target protein can be analyzed prior to actual synthesis using computer modeling techniques. Only those candidates that are indicated by computer modeling to bind the target as described herein may be synthesized and tested for their ability to bind to the target protein or to prevent binding of the target protein to its protein binding partner using assays known to those of skill in the art. Such assays are known to those of skill in the art and are described in part in USSN 1 1/795,078. A candidate compound may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with individual binding target sites on the target protein. One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with the target protein, and more particularly with active binding sites or epitopes on the target protein. The process may begin by visual inspection of, for example, an active binding site or epitope on a computer screen, based on the protein-protein complex structure coordinates, or a subset of those structure coordinates. Such structure coordinates may be readily available to one of skill in the art, such as by being in the published literature. Alternatively, structure coordinates may be obtained using NMR methods readily available to those of skill in the art of a crystal of the protein-protein binding pair. Once the crystal structure coordinates have been determined and entered into a suitable software program, selected fragments or chemical entities may then be positioned in a variety of orientations or "docked" within an active binding site or epitope of the target protein as defined from analysis of the crystal structure data. Docking may be accomplished using software such as Quanta (Molecular Simulations, Inc., San Diego, Calif.) and Sybyl (Tripos, Inc. St. Louis, Mo.) followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields such as CHARMM (Molecular Simulations, Inc., San Diego, Calif.) and AMBER (University of California at San Francisco).

Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include but are not limited to: GRID (Goodford, P. J., "A Computational Procedure for Determining Energetically Favorable Binding Sites on Biologically Important Macromolecules," J. Med. Chem., 28, pp. 849 857 (1985)); GRID is available from Oxford University, Oxford, UK; MCSS (Miranker, A. and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method," Proteins: Structure, Function and Genetics, 1 1, pp. 29 34 (1991)); MCSS is available from Molecular Simulations, Inc., San Diego, Calif; AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing," Proteins: Structure, Function, and Genetics, 8, pp. 195 202 (1990)); AUTODOCK is available from Scripps Research Institute, La Jolla, Calif; DOCK (Kunts, I. D., et al. "A Geometric Approach to Macromolecule-Ligand Interactions," J. Mol. Biol., 161, pp. 269 288 (1982)); DOCK is available from University of California, San Francisco, Calif; CERIUS II (available from Molecular Simulations, Inc., San Diego, Calif); Flexx (Raret, et al. J. Mol. Biol. 261, pp. 470 489 (1996)); TreeDock (Fahmy and Wagner, J. Am. Chem. Soc, 124, 1241-1250 (2002); and Octopus (Fahmy and Wagner, Biophysical J., 101, 1690-1698 (201 1)).

After selecting suitable chemical entities or fragments, one or more, such as two or more fragments can be assembled into a single compound. Assembly may proceed by visual inspection of the relationship of the fragments to each other on a three-dimensional image of the fragments in relation to the target protein structure or portion thereof displayed on a computer screen. Visual inspection may be followed by manual model building using software such as the Quanta or Sybyl programs described above.

Software programs also may be used to aid one skilled in the art in connecting the individual chemical entities or fragments. These include, but are not limited to CAVEAT (Bartlett, P. A., et al. "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules" In "Molecular Recognition in Chemical and Biological Problems," Special Publ, Royal Chem. Soc, 78, pp. 182 196 (1989)); CAVEAT is available from the University of California, Berkeley, Calif; 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif); this area is reviewed in Martin, Y. C, "3D Database Searching in Drug Design," J. Med. Chem., 35:2145 2154 (1992)); and HOOK (available from Molecular Simulations Inc., San Diego, Calif). As an alternative to building candidate compounds up from individual fragments or chemical entities, they may be designed de novo using the structure of an active binding site or epitope of a target protein, optionally, including information from known inhibitor(s) that bind to the target site. De novo design may be included by programs including, but not limited to LUDI (Bohm, H. J., "The Computer Program LUDI: A New Method for the De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61 78 (1992)); LUDI is available from Molecular Simulations, Inc., San Diego, Calif; LEGEND (Nishibata, Y., and Itai, A., Tetrahedron 47, p. 8985 (1991); LEGEND is available from Molecular Simulations, San Diego, Calif; and LeapFrog (available from Tripos Associates, St. Louis, Mo.). Additional molecular modeling techniques also may be employed in accordance with the invention. See, e.g., Cohen, N. C, et al. "Molecular Modeling Software and Methods for Medicinal Chemistry," J. Med. Chem., 33, pp. 883 894 (1990); Hubbard, Roderick E., "Can drugs be designed?" Curr. Opin. Biotechnol. 8, pp. 696 700 (1997); and Afshar, et al. "Structure-Based and Combinatorial Search for New RNA-Binding Drugs," Curr. Opin. Biotechnol. 10, pp. 59 63 (1999).

Following candidate compound design or selection according to any of the above methods or other methods known to one skilled in the art, the efficiency with which a candidate compound binds to an active binding site or epitope of the target protein may be tested and optimized using computational evaluation. A candidate compound may be optimized, e.g., so that in its bound state it would preferably lack repulsive electrostatic interaction with the target site. These repulsive electrostatic interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions. It is preferred that the sum of all electrostatic interactions between the candidate compound and the active binding site or epitope when the candidate compound is bound to the active binding site or epitope make a neutral or favorable contribution to the binding enthalpy. Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include, but are not limited to Gaussian 92, revision C (Frisch, M. J., Gaussian, Inc., Pittsburgh, Pa. (1992)); AMBER, version 4.0 (Kollman, P. A., University of California at San Francisco, (1994)); QUANT A/CHARMM (Molecular Simulations, Inc., San Diego, Calif. (1994)); and Insight II/Discover (Biosym Technologies Inc., San Diego, Calif. (1994)). These programs may be run, using, e.g., Unix workstations or clusters of workstations. Other hardware and software combinations may be used to carry out the functions described herein, and are known to those of skill in the art. Once a candidate compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups to improve or modify its binding properties. Generally, initial substitutions are conservative in that the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Components known in the art to alter conformation should be avoided in making substitutions. Substituted candidates may be analyzed for efficiency of fit to the active binding site or epitope of the target protein using the same methods described above.

Once a candidate compound has been identified using any of the methods described above, it can be screened for biological activity. Any one of a number of assays to determine binding to the target protein or inhibition of binding of the target protein to its binding partner known to those of skill in the art may be used.

Candidate compounds identified according to the methods of the invention may be provided from libraries of compounds available from a number of sources or may be derived by combinatorial chemistry approaches known in the art. Such libraries include but are not limited to the available Chemical Director, Maybridge, and natural product collections. These include libraries available through the Institute for Cell Biology and Chemical Biology, described at world wide website iccb.med.harvard.edu. In one embodiment of the invention, libraries of compounds with known or predicted structures may be docked to the active binding sites or epitopes of target proteins described herein. EXAMPLE I

Peptide Targets

The ultimate goal of therapeutic research is to identify molecular abnormalities present in cancer, which are absent in the normal matched tissues. The oncogene Multiple Copies in T-cell Lymphoma- 1 (MCT- 1) is located on chromosome Xq22-24 and was found to be highly over- expressed in the 85% of primary diffuse large B-cell lymphoma (DLBCL) samples compared with normal lymph nodes when examined by tissue microarray (TMA) analysis (Cancer Res. 2009;69(19):7835-43). MCT-1 belongs to a family of PseudoUridine synthase and Archaeosine transglycosylase (PUA) containing proteins, which have the ability to interact with RNA and be involved in translation initiation ( £¾SV 274(19):4972-4984, Mol Cell 34(4):427-439, Genes Dev 24(16): 1787- 1801). RNAi inhibition of MCT- 1 in DLBCL significantly reduced cell viability through an apoptotic mechanism (Cancer Res. 2009 Oct l ;69(19):7835-43), providing the first direct genetic evidence that interfering with MCT-1 function was able to induce apoptosis in lymphoma cells with high endogenous levels of MCT- 1 protein. The mechanism of how MCT- 1 binds the ribosomal complex is yet to be discovered. However, once bound, MCT- 1 recruits a partner protein, DENR, to the translation initiation complex (Cancer Res 66(18):8994-9001). DENR contains a SUIl domain which is also found in the translation initiation factor elFl (FEBS J 274(19):4972-4984). The recruitment of DENR results in an increase in the translation initiation rates of a subset of mRNAs, containing a long and highly structured 5' UTR, typically found in cancer related messages (Cancer Res 66(18):8994-9001). A partial mechanism underlying this selective increase in translation has been described and is attributed to the increased scanning capability of the 40S ribosome and increased recognition of the correct initiation codon within oncogenic mRNA conferred by the MCT- 1/DENR complex (FEBS J 274(19):4972-4984, Cancer Res 66(18):8994-9001, Genes Dev 20(10): 1294-1307). Therefore, the identification of inhibitors that disrupt the MCT- 1/DENR complex would be a significant advantage to therapeutic interventions concerning tumors in which MCT- 1 is overexpressed.

Calcineurin (Cn) is a cellular phosphatase that is the target of the widely used immunosuppressive drug cyclosporine A (CsA). A compound that can selectively inhibit the Cn dephosphorylation of NFAT, but not the dephosphorylation of other Cn substrates is expected to exhibit as potent immunosuppressive activity as CsA but fewer of the harsh side effects of CsA. In addition, development of a new class of Cn-inhibitors may open other avenues for fine-tuning of immunosuppressive activity vs. undesired side effects.

EXAMPLE II

Identification of Cn Binding Fragments by NMR and Constructing a Library of Potential Inhibitors

STD NMR experiments on 5 μΜ natural abundance calcineurin (Cn) with 10 compound mixtures of fragments from a commercial Maybridge(TM) library (1000 fragments) identified thirteen binding fragments. An exemplary NMR method is described in Huth et al., Methods in Enzymology 2005, 394, 549. iLOE experiments of various pairs of those fragments in the presence of calcineurin identified two molecules (fragments) that bind non-competitive ly to adjacent sites as shown in Fig. 2A. The red dotted lines indicate hydrogen atoms on the fragments that are within iLOE distance, i.e. between about 2-10 angstroms. See Huth et al., Methods in Enzymology 2005,

394, 549

Fig. 2B is a schematic representation of the various ways in which the two fragments can be incorporated in a library of macrocycles. A macrocycle is a cyclic polypeptide having a plurality of amino acids. "X" and "Y" denote between about 0 to about 8 amino acids, with about 3 to about 6 amino acids being preferred, initially selected from the shown set of D- and L- , N-H, N-alkyl, and N-acyl amino acids to simulate various backbone conformations. For example, the cyclic polypeptides shown in schematic in Figure 2B include various amino acids as shown. According to one aspect, an amino acid may include group R which can be H or isopropyl or as otherwise defined herein. These two groups will be used for the permeability simulations described herein. Hydrogen is useful for the docking simulations described herein. R may also be any proteinogenic side chain. Proteinogenic side chains are useful for side-chain filling simulations described herein. Useful amino acids may include either NH or N-alkyl or N-acyl as described herein. As shown in Figure 2B, the two fragments are covalently bound to the cyclic polypeptide as shown using chemistries known to those of skill in the art and reactive sites on the fragments. After initial membrane permeability simulations and fragment-positioning simulations, X and Y within the cyclic polypeptide are diversified at the 'R' side chain of the amino acid. As indicated, the NH shown in the generic amino acid structures for X or Y and as incorporated into a cyclic polypeptide can be either NH or N-Me and either in the D- or L- configuration. Amino acid side chains are well known to those of skill in the art as are methods of diversification. The cyclic polypeptides shown in Figure 2B are exemplary. As shown in Fig. 2C, in practice, the fragments can be attached directly by converting them into the shown amino acids and using those amino acids in the peptide synthesis, such as by reacting them to a cyclic polypeptide as a side group or as part of the ring structure, as opposed to being attached to the ring structure. R as depicted in Figure 2C can be hydrogen, methyl, ethyl, isopropyl, buytl, t-butyl, hydroxyl, chloro, fluoro, bromo, ethoxy, methoxy, amino, acetyl, nitro, thiomethyl or -S-CH₃. As shown in Fig. 2D, alternatively, the fragments can be attached at the end of the synthesis employing one of the shown chemistries: First, one or two of the shown non-proteinogenic amino acids serving as attachment 'handles' or reactive groups or moieties are incorporated in the library of cyclic peptides which then allow attachment of a desired reactant, i.e. fragment as disclosed herein, to the cyclic polypeptide. Subsequent reaction with the appropriately functionalized fragments yields the corresponding fragment- derived side chain. In Figure 2D, Rl as indicated herein or can be R or as indicated. "AA" is an amino acid in the amount indicated and including any amino acids described herein and those identified in Figures 2C and 2D. In the figures, the wavy lines indicate a D- or L- amino acid configuration, following standard convention. As shown in Fig. 2E, the fragments can be attached by alkylation/acylation of amides of the peptide backbone. The methods of Fig. 2D or 2E are not limited to specific fragments but can be generally used with any fragments discovered to bind to any protein target. The alkylation/acylation in Fig. 2E as well as some of the reactions in Fig. 2D require a strong base and therefore require that all other amide positions be alkylated/acylated a priori. In addition, R can be a proteinogenic amino acid side chain. R can be straight or branched chain alkyl groups, substituted or unsubstituted and having between 1 carbon and 5 carbons, such as methyl, ethyl, propyl, butyl and pentyl. Substitutions include hydroxyl, halide, ethyl, propyl, isopropyl, amino, sulfonamido, sulfonyl, nitro, etc. (limited only by the commercial availability of corresponding precursors). Additionally, all-carbon or heterocyclic aromatic groups such as furans, thiophenes, benzenes, indoles, pyrazoles, imidazoles, thiazoles, or other portions of a larger molecule can be substituted, such as a molecular fragment identified in a protein binding assay. Also, R in some cases is meant as any future modification in a second generation library where R may be randomized to make a library. As shown in Figures 2B-E, fragment incorporation occurs in one of three ways: via pre- synthesized fragment-amino acids which can be reacted into or on cyclic polypeptides (Figure 2c), via amino acids designed as anchor points or reactive sites on a cyclic polypeptide (Figure 2d), or via alkylation/acylation at backbone amides. The attachment chemistry includes copper (I) and ruthenium-catalyzed click chemistry, amide and ester biond formation, and reactions of thiols with iodoacetyl and maleimide groups. In addition, haloalkyl and haloacyl fragments can be attached directly to the amide nitrogen of the peptide backbone by deprotonation followed by alkylation or acylation (Figure 2e). A person skilled in the art will recognize that other standard reactions can be employed in the same fashion.

It is to be understood that compounds identified herein can be synthesized using common organic chemistry synthesis materials and methods.

EXAMPLE III

In Silico Peptide Library

An initial collection of candidate cyclic peptide backbones is composed of all penta-, hexa-, hepta- and octa-peptides containing the fragment-derived and "X","Y" amino acids shown in Fig. 2B and Fig. 2C, including all D- and L-, and N-methylated versions thereof. The scaffolds also contain the PEG-functionalized D- or L- Ser shown in Fig. 3. The peptides are cyclized head to tail as shown in Fig. 3. This initial set of peptides is subjected to a simple computational procedure intended to remove compounds that are likely to be membrane impermeable. A minimum energy conformation in low dielectric medium is computed for each peptide backbone. If this conformation contains exposed amide hydrogens, the cyclic peptide is likely to be relatively impermeable. This is based on existing methods for estimating cell permeability (JACS 2006, 128: 14073) with three notable improvements. One improvement is the use of a Loop Closure algorithm on cyclic peptides linearized by a single cleavage, leading to more exhaustive enumeration of backbone conformations. The second improvement is in the side chain conformer enumeration, also resulting in more precise conformational analysis. Thirdly, the present method extends the calculations to include any arbitrary non-proteinogenic amino acid backbone, including beta-amino acids and amino acids formed from fragments. The output of this protocol is a reduced set of peptide scaffolds that are more likely to passively penetrate the cell membrane. The reduced set of scaffolds is further processed as follows. First, the individual fragments discovered in the NMR assay as illustrated in Fig. 2A are computationally docked to the calcinuerin surface proximal to the NFAT binding site, for example, when a precise location for the fragments is not available by experiment. For each predicted binding geometry of each fragment, each peptide backbone is rotated and translated attempting to find an orientation that can accommodate one or both of the fragments without any steric clashes with the protein surface. If this is not possible, the given backbone scaffold is deleted from the set. Second, if a proper orientation is found, the backbone is subjected to an algorithm suggesting amino acid side chains complementary to the protein surface. A subset of scaffolds with minimal contacts to the protein surface is removed at this stage. The final result from these computations is a set of 'plausible' peptide scaffolds serving as the basis for a diversified library.

Note that if a protein structure is not available, the docking and side chain filling steps are omitted. On the other hand, if a protein structure is available and fragment binding has been successfully mapped by NMR experiments, the fragment docking step is omitted. EXAMPLE IV

Synthesis of the Library

Synthesis of the compounds follows the scheme depicted in Figure 3. A solid phase support that is functionalized with PEG, such as Tentagel(TM) which swells in both organic and aqueous solutions is used if on-bead screening is desired. Otherwise, any polystyrene or similar resin would work. A PEG amino acid possessing between one and ten ethylene glycol units is attached to the resin, preferably via an acid labile linker such as a Wang linker. The PEG 'tail' in this way is preserved in the cleaved peptide, with dual purpose.

First it enhances water solubility, simplifying NMR analysis of lead candidates and increasing potential bioavailability of the final drug lead. Second, it increases specificity by reducing undesired interactions of the compounds' 'back face' with proteins other than the target protein. This PEG-linker also serves as a cyclization point, via an allyl-protected carboxylic acid side chain. Chain elongation proceeds by standard FMOC solid phase synthesis using the FMOC-protected versions of the amino acid monomers shown in Figure 2. As shown in the Figure, fragment incorporation occurs in one of three ways: via pre- synthesized fragment-amino acids (Figure 2c), via amino acids designed as anchor points (Figure 2d), or via alkylation/acylation at backbone amides. The attachment chemistry includes copper (I) and ruthenium-catalyzed click chemistry, amide and ester bond formation, and reactions of thiols with iodoacetyl and maleimide groups. In addition, haloalkyl and haloacyl fragments can be attached directly to the amide nitrogen of the peptide backbone by deprotonation followed by alkylation or acylation. A person skilled in the art will recognize that other standard reactions can be employed in the same fashion.

Alternative to solid phase synthesis, the library of peptides can be generated by in vitro (cell-free) translation using ribosomal extract from bacteria, wheat germ, or other cells. In this approach, non- natural amino acids are attached to tRNA using traditional chemical coupling methods or a promiscuous ribozyme-based amino acid RNA synthethase (Nature Methods 2001; 6: 779). The in vitro translation method is limited in the types of non-proteinogenic amino acids that can be used. Specifically, the non-alpha amino acids and some of the alpha amino acids carrying very bulky fragment side chains (Figure 2c) can not be incorporated successfully in the peptides. Thus, the in vitro translation approach is more suitable when fragment attachment is conducted post-synthesis on the library (Figure 2c,d). This method generates mixtures of peptides in solution making it more suitable for certain assays that require solution mixtures of compounds.

EXAMPLE V Screening and Hit Identification

Screening is conducted by pull-down assay with fluorescently labeled calcineurin protein, or by FRET assay in which the beads are incubated with a fluorescently labeled Cn/NFAT complex and screened for fluorescence enhancement due to complex dissociation. Other screening methods familiar to a person skilled in the art can be used. Hit identification is via cleavage of the compound followed by MS analysis and deconvolution. Numerous variations of this method will be familiar to persons skilled in the art. A suitable method for decoding a peptide library is provided in Pastor et al., JACS 2007 vol. 129 (48) pp. 14922-14932.

EXAMPLE VI Compounds

According to aspects of the present disclosure, compounds useful in the described methods include

MB

HO

20

22

o





Reactions within the scope of the present disclosure include reacting a cyclic polypeptide having a reactive functional group, such as shown in each reaction below, with a reactant as shown to produce a cyclic polypeptide with a fragment as described herein.

where

32

Claims

Claims:

1. A method of identifying a binding compound to a target protein comprising

identifying one or more fragment compounds that bind to the target protein,

incorporating the fragment compounds into a plurality of candidate polypeptide scaffolds, identifying selected polypeptide scaffolds from among the candidate polypeptide scaffolds that position two or more fragment compounds near their respective binding sites on the target protein,

modifying the selected polypeptide scaffolds by combinatorial synthesis to produce diversified polypeptide scaffolds, and

identifying one or more binding compounds that have binding affinity to the target protein.

2. The method of claim 1 wherein the step of identifying the one or more fragment compounds includes screening candidate fragment compounds for binding to the target protein.

3. The method of claim 2 wherein the screening is performed in vitro.

4. The method of claim 1 wherein the two or more fragments bind to the target protein non- competitively at adjacent sites on the target protein.

5. The method of claim 1 wherein the polypeptide scaffolds are cyclic polypeptides.

6. The method of claim 1 wherein the step of incorporating the one or more fragment compounds into a plurality of candidate polypeptide scaffolds is performed in silico.

7. The method of claim 1 wherein the step of identifying selected polypeptide scaffolds from among the candidate polypeptide scaffolds that position the two or more fragment compounds near their respective binding sites on the target protein is performed in silico. 8. The method of claim 1 wherein the step of identifying one or more optimized polypeptide scaffolds from among the diversified polypeptide scaffolds that have binding affinity to the target protein includes screening for binding of the diversified polypeptide scaffolds to the target protein.

9. The method of claim 7 wherein the screening is in vitro.

10. The method of claim 1 wherein the step of identifying the two or more fragment compounds includes screening candidate fragment compounds for binding to the target protein using NMR. 1 1. The method of claim 1 wherein the target protein is multiple copies in T-cell lymphoma- 1.

12. The method of claim 1 wherein the target protein is multiple copies in T-cell lymphoma- 1 and wherein the one or more optimized polypeptide scaffolds inhibit binding between multiple copies in T-cell lymphoma- 1 and DENR.

13. The method of claim 1 wherein the target protein is calcineurin.

14. The method of claim 1 wherein the target protein is calcineurin and wherein the one or more optimized polypeptide scaffolds inhibit binding between calcineurin and NFAT.

15. The method of claim 1 wherein the polypeptide scaffolds include between about 5 and about 10 amino acids.

16. The method of claim 1 wherein the polypeptide scaffolds are cyclic and include between about 5 and about 10 amino acids.

17. The method of claim 1 wherein the polypeptide scaffolds are cyclic and include between about 6 and about 8 amino acids. 18. The method of claim 1 wherein the polypeptide scaffolds are cyclic and one end of the one or more fragment compounds is attached to a respective cyclic polypeptide scaffold.

19. The method of claim 1 wherein the polypeptide scaffolds are cyclic and at least one of the one or more fragment compounds is internal to a respective cyclic polypeptide scaffold.

20. The method of claim 1 wherein the polypeptide scaffolds are cyclic and at least two of the one or more fragment compounds is internal to a respective cyclic polypeptide scaffold.

21. A polypeptide having bound thereto a member selected from the group consisting of

, and , and a member selected from the group consisting of

22. The polypeptide of claim 21 including between about 5 amino acids and about 10 amino acids.

23. A cyclic polypeptide having bound thereto a member selected from the group consisting of

An amino acid having bound thereto

A amino acid having bound thereto

wherein the amino acid is a D-amino acid, L-amino acid, N-alkyl amino acid or N-acyl amino acid.

28. A polypeptide including a member selected from the group consisting of

39

configuration and where R is H or a side chain having binding affinity to a target protein or a proteinogenic amino acid side chain; a straight or branched chain alkyl groups, substituted or unsubstituted and having between 1 carbon and 5 carbons, such as methyl, ethyl, propyl, butyl and pentyl; wherein substitutions include hydroxyl, halide, ethyl, propyl, isopropyl, amino, sulfonamido, sulfonyl, nitro, all-carbon or heterocyclic aromatic groups such as furans, thiophenes, benzenes, indoles, pyrazoles, imidazoles, thiazoles, or other portions of a larger molecule can be substituted, such as a molecular fragment identified in a protein binding assay.

29. The polypeptide of claim 28 including between about 5 amino acids and about 10 amino acids.

30. The polypeptide of claim 28 wherein the amino acids are one or more of a D-amino acid, L-amino acid, N-alkyl amino acid or N-acyl amino acid.

31. The polypeptide of claim 28 being a cyclic polypeptide.

32. The polypeptide of claim 28 being a linear polypeptide.

33. The polypeptide of claim 21 including polyethylene glycol bound thereto. 34. The cyclic polypeptide of claim 23 including polyethylene glycol bound thereto.

35. The polypeptide of claim 26 including polyethylene glycol bound ther eto.

36. A compound having a formula

45

47

o

51

53

wherein R is a proteinogenic amino acid side chain; a straight or branched chain alkyl groups, substituted or unsubstituted and having between 1 carbon and 5 carbons, such as methyl, ethyl, propyl, butyl and pentyl; wherein substitutions include hydroxyl, halide, ethyl, propyl, isopropyl, amino, sulfonamido, sulfonyl, nitro, all-carbon or heterocyclic aromatic groups such as furans, thiophenes, benzenes, indoles, pyrazoles, imidazoles, thiazoles, or other portions of a larger molecule can be substituted, such as a molecular fragment identified in a protein binding assay.

37. One or more of the following chemical reactions, reactants, products or compounds identified therein

where R is a proteinogenic amino acid side chain; a straight or branched chain alkyl groups, substituted or unsubstituted and having between 1 carbon and 5 carbons, such as methyl, ethyl, propyl, butyl and pentyl; wherein substitutions include hydroxyl, halide, ethyl, propyl, isopropyl, amino, sulfonamido, sulfonyl, nitro, all-carbon or heterocyclic aromatic groups such as furans, thiophenes, benzenes, indoles, pyrazoles, imidazoles, thiazoles, or other portions of a larger molecule can be substituted, such as a molecular fragment identified in a protein binding assay.