Transformation of low-affinity lead compounds into high-affinity protein capture agents

A simple and potentially general approach to the isolation of high-affinity and -specificity protein binding synthetic molecules is presented. A modest affinity lead compound is appended to the end of each molecule in a combinatorial library of oligomeric compounds, such as peptides or peptoids. The library is then screened under conditions too demanding for the lead to support robust binding to the protein target. It was anticipated that this procedure would select for bivalent ligands in which the oligomer library provides both a second binding element as well as an appropriate linker between this element and the lead compound. We report here synthetic ligands for the Mdm2 protein and ubiquitin able to capture their target proteins from dilute solutions in the presence of a large excess of other proteins.     

Bivalent inhibition of human beta-tryptase

BACKGROUND: Human beta-tryptase is a mast cell specific trypsin-like serine protease that is thought to play a key role in the pathogenesis of diverse allergic and inflammatory disorders like asthma and psoriasis. The recently resolved crystal structure revealed that the enzymatically active tetramer consists of four quasi-identical monomers. The spatial display of the four identical active sites represents an ideal basis for the rational design of bivalent inhibitors. RESULTS: Based on modeling experiments homobivalent inhibitors were constructed using (i) 6A,6D-dideoxy-6A,6D-diamino-beta-cyclodextrin as a rigid template to bridge the space between the two pairs of identical active sites and (ii) 3-(aminomethyl)benzene as a headgroup to occupy the arginine/lysine specific S1 subsites. A comparative analysis of the inhibitory potencies of synthetic constructs that differ in size and type of the spacer between headgroup and template revealed that the construct contained two 3-(aminomethyl)benzenesulfonyl-glycine groups linked to the 6A,6D-diamino groups of beta-cyclodextrin as an almost ideal bivalent inhibitor with a cooperativity factor of 1.9 vs. the ideal value of 2. The bivalent binding mode is supported by the inhibitor/tetramer ratio of 2:1 required for inactivation of tryptase and by X-ray analysis of the inhibitor/tryptase complex. CONCLUSION: The results obtained with the rigid cyclodextrin template underlined the importance of a minimal loss of conformational entropy in bivalent binding, but also showed the limitations imposed by such rigid core molecules in terms of optimal occupancy of binding sites and thus of enthalpic strains in bidentate binding modes. The main advantage of bivalent inhibitors is their high selectivity for the target enzyme that can be achieved utilizing the principle of multivalency.     

Exploration of structure--activity relationships among foldamer ligands for a specific protein binding site via parallel and split-and-mix library synthesis

We describe the use of parallel and split-and-mix library synthesis strategies for exploration of structure-activity relationships among peptidic foldamer ligands for the BH3-recognition cleft of the anti-apoptotic protein Bcl-xL. This effort began with a chimeric (alpha/beta+alpha)-peptide oligomer (composed of an alpha/beta-peptide segment and an alpha-peptide segment) that we previously identified to bind tightly to the target cleft on Bcl-xL. The side chains that interact with Bcl-xL were varied in a 1000-member one-bead-one-compound library. Fluorescence polarization (FP) screening identified four new analogues with binding affinities similar to that of the lead compound but no analogues with enhanced affinity. These results suggested that significant improvements in affinity were unlikely in this series. We then used library synthesis to examine backbone variations in the C-terminal alpha-peptide segment of the lead compound. These studies provided an opportunity for direct comparison of parallel and split-and-mix synthesis formats for foldamer libraries with respect to synthetic variability and assay sensitivity. We found that compounds from both the parallel and one-bead-one-compound libraries could be reliably screened in a competition FP assay without purification of library members. Our findings should facilitate the use of combinatorial library synthesis for exploration of foldamers as inhibitors of protein-protein interactions.     

One-armed artificial receptors for the binding of polar tetrapeptides in water: probing the substrate selectivity of a combinatorial receptor library

We have recently developed a new class of one-armed artificial receptors 1 for the binding of the polar tetrapeptide N-Ac-D-Glu-L-Lys-D-Ala-D-Ala-OH (EKAA) 2 in water using a combined combinatorial and statistical approach. We have now further probed the substrate selectivity of this receptor library 1 by screening a second tetrapeptide substrate (3) with the inverse sequence N-Ac-D-Ala-D-Ala-L-Lys-D-Glu-OH (AAKE). This "inverse" substrate is also efficiently bound by our receptors, with K(ass) approximately 6000 M(-1) for the best receptors, as determined both by a quantitative on-bead binding assay and by UV and fluorescence titration studies in free solution. Hence, the inverse tetrapeptide 3 is in general bound two to three times less efficiently than the "normal" peptide 2 (K(ass) approximately 17,000 M(-1)), even though the complexation mainly involves long-range electrostatic interactions and both the receptor and substrate are rather flexible. Molecular modeling and ab initio calculations have been used to rationalize the observed substrate selectivity and to analyze the various binding interactions within the complex.     

Strategy for discovering chemical inhibitors of human cyclophilin a: focused library design, virtual screening, chemical synthesis and bioassay

The discovery of cyclophilin A (CypA) inhibitor is now of special interest in the treatment of immunological disorders. In this work, using a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay, a series of novel small molecular CypA inhibitors have been discovered. First, using the fragments taken from our previously discovered CypA inhibitors (Bioorg. Med. Chem. 2006, 14, 2209-2224) as building blocks, we designed a focused combinatorial library containing 255 molecules employing the LD1.0 program (J. Comb. Chem. 2005, 7, 398-406) developed by us. Sixteen compounds (1a-e, 2a-b, 3a-b, and 4a-g) were selected by using virtual screening against the X-ray crystal structure of CypA as well as druglike analysis for further synthesis and bioassay. All these sixteen molecules are CypA binders with binding affinities (K(D) values) ranging from 0.076 to 41.0 microM, and five of them (4a, 4c, and 4e-g) are potent CypA inhibitors with PPIase inhibitory activities (IC(50) values) of 0.25-6.43 microM. The hit rates for binders and inhibitors are as high as 100% and 31.25%, respectively. Remarkably, both the binding affinity and inhibitory activity of the most potent compound increase approximately 10 times than that of the most active compound discovered previously. The high hit rate and the high potency of the new CypA inhibitors demonstrated the efficiency of the strategy for focused library design and screening. In addition, the novel chemical entities reported in this study could be leads for discovering new therapies against the CypA pathway.     

Microarrays of tagged combinatorial triazine libraries in the discovery of small-molecule ligands of human IgG

A novel and highly diverse tagged triazine library incorporating a triethylene glycol-based linker was synthesized using an orthogonal combinatorial approach on the solid phase and covalently immobilized on a glass substrate as a small molecule microarray (SMM). The SMM was screened with a fluorophore-conjugated human IgG, and 4 novel binders from a library of 2688 compounds were identified from the fully spatially addressable array without the need for compound decoding. Using surface plasmon resonance (SPR) analysis, binding seen on the array was confirmed, and a binding constant as low as Kd = 2.02 x10(-6) M was measured.     

DNA‐Encoded Dynamic Combinatorial Chemical Libraries

Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA‐encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal‐to‐noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA‐encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high‐affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA‐templated chemical reactions.     

High-affinity mu opioid receptor ligands discovered by the screening of an exhaustively stereodiversified library of 1,5-enediols

In this communication, we report the synthesis of an exhaustively stereodiversified library of 16 1,5-enediols (2) and the screening of these compounds for mu opioid receptor (MOR) binding. The stereochemical configuration of 2 strongly impacted the binding affinity, and (S,S,S,R)-2 exhibited a Ki of 8.8 nM for MOR, comparable to that of endomorphin-2 (Ki = 1.2 nM). Moreover, compounds 2 exhibited 5-86-fold selectivity for MOR over delta opioid receptor (DOR) and 16-150-fold selectivity for MOR over kappa opioid receptor (KOR). Additionally, analogues of 2 were synthesized which showed the importance of the trans olefin for receptor binding but that modifications of the C-terminal amino acid were well tolerated. Ligand 11 is noteworthy because it retains only one of the amide bonds present in 1, but binds MOR with an affinity of 10 nM and 110- and 600-fold selectivity for MOR over DOR and KOR. These results demonstrate the utility of stereochemical diversity in the discovery of bioactive small molecules.     

Synthesis and characterization of potent bivalent amyloidosis inhibitors that bind prior to transthyretin tetramerization

The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Monovalent small molecules that bind to one or both of the unoccupied thyroid hormone binding sites at the TTR quaternary structure interface stabilize the native state, raising the kinetic barrier for tetramer dissociation sufficiently that the rate of dissociation, and therefore amyloidosis, becomes slow. Bivalent amyloid inhibitors that bind to both binding sites simultaneously are reported herein. The candidate bivalent inhibitors are generally unable to bind to the native TTR tetramer and typically do not engage in monovalent binding owing to a strong inhibitor orientation preference. However, the TTR quaternary structure can assemble around several of the bivalent inhibitors if the inhibitor intercepts the protein before assembly occurs. Some of the wild-type TTR.bivalent inhibitor complexes prepared in this fashion retain a tetrameric structure when subjected to substantial denaturation stresses (8 M urea, 120 h). The best bivalent inhibitor reduced acid-mediated TTR (3.6 microM) amyloid fibril formation to 6% of that exhibited by TTR in the absence of inhibitor, a significant improvement over the approximately 30% observed for the best monovalent inhibitors (3.6 microM, 72 h). The apparent dissociation rate of the best bivalent inhibitor is effectively zero, consistent with the idea that TTR tetramer dissociation and inhibitor dissociation are linked-as a result of the inhibitor-templating tetramer assembly. X-ray cocrystal structures of two of the complexes demonstrate that the bivalent inhibitors simultaneously occupy both sites in TTR, consistent with the 1:1 binding stoichiometry derived from HPLC analysis. The purpose of this study was to demonstrate that bivalent inhibitors could be useful; what resulted are the best inhibitors produced to date. In this context, molecules capable of intercepting TTR during folding and assembly in the lumen of the endoplasmic reticulum would be of obvious interest.     

Systems-based design of bi-ligand inhibitors of oxidoreductases: filling the chemical proteomic toolbox

Genomics-driven growth in the number of enzymes of unknown function has created a need for better strategies to characterize them. Since enzyme inhibitors have traditionally served this purpose, we present here an efficient systems-based inhibitor design strategy, enabled by bioinformatic and NMR structural developments. First, we parse the oxidoreductase gene family into structural subfamilies termed pharmacofamilies, which share pharmacophore features in their cofactor binding sites. Then we identify a ligand for this site and use NMR-based binding site mapping (NMR SOLVE) to determine where to extend a combinatorial library, such that diversity elements are directed into the adjacent substrate site. The cofactor mimic is reused in the library in a manner that parallels the reuse of cofactor domains in the oxidoreductase gene family. A library designed in this manner yielded specific inhibitors for multiple oxidoreductases.     

Studies of the mechanism of selectivity of protein tyrosine phosphatase 1B (PTP1B) bidentate inhibitors using molecular dynamics simulations and free energy calculations

Bidentate inhibitors of protein tyrosine phosphatase 1B (PTP1B) are considered as a group of ideal inhibitors with high binding potential and high selectivity in treating type II diabetes. In this paper, the binding models of five bidentate inhibitors to PTP1B, TCPTP, and SHP-2 were investigated and compared by using molecular dynamics (MD) simulations and free energy calculations. The binding free energies were computed using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) methodology. The calculation results show that the predicted free energies of the complexes are well consistent with the experimental data. The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy decomposition analysis indicates that the residues ARG24, ARG254, and GLN262 in the second binding site of PTP1B are essential for the high selectivity of inhibitors. Furthermore, the residue PHE182 close to the active site is also important for the selectivity and the binding affinity of the inhibitors. According to our analysis, it can be concluded that in most cases the polarity of the portion of the inhibitor that binds to the second binding site of the protein is positive to the affinity of the inhibitors while negative to the selectivity of the inhibitors. We expect that the information we obtained here can help to develop potential PTP1B inhibitors with more promising specificity.     

Molecular design and structural optimization of potent peptide hydroxamate inhibitors to selectively target human ADAM metallopeptidase domain 17

Human ADAMs (a disintegrin and metalloproteinases) have been established as an attractive therapeutic target of inflammatory disorders such as inflammatory bowel disease (IBD). The ADAM metallopeptidase domain 17 (ADAM17 or TACE) and its close relative ADAM10 are two of the most important ADAM members that share high conservation in sequence, structure and function, but exhibit subtle difference in regulation of downstream cell signaling events. Here, we described a systematic protocol that combined computational modeling and experimental assay to discover novel peptide hydroxamate derivatives as potent and selective inhibitors for ADAM17 over ADAM10. In the procedure, a virtual combinatorial library of peptide hydroxamate compounds was generated by exploiting intermolecular interactions involved in crystal and modeled structures. The library was examined in detail to identify few promising candidates with both high affinity to ADAM17 and low affinity to ADAM10, which were then tested in vitro with enzyme inhibition assay. Consequently, two peptide hydroxamates Hxm-Phe-Ser-Asn and Hxm-Phe-Arg-Gln were found to exhibit potent inhibition against ADAM17 (K_i = 92 and 47 nM, respectively) and strong selectivity for ADAM17 over ADAM10 (∼7-fold and ∼5-fold, S = 0.86 and 0.71, respectively). The structural basis and energetic property of ADAM17 and ADAM10 interactions with the designed inhibitors were also investigated systematically. It is found that the exquisite network of nonbonded interactions involving the side chains of peptide hydroxamates is primarily responsible for inhibitor selectivity, while the coordination interactions and hydrogen bonds formed by the hydroxamate moiety and backbone of peptide hydroxamates confer high affinity to inhibitor binding.     

Combining combinatorial chemistry and affinity chromatography: highly selective inhibitors of human betaine: homocysteine S-methyltransferase

A new method to find novel protein targets for ligands of interest is proposed. The principle of this approach is based on affinity chromatography and combinatorial chemistry. The proteins within a crude rat liver homogenate were allowed to interact with a combinatorial library of phosphinic pseudopeptides immobilized on affinity columns. Betaine: homocysteine S-methyltransferase (BHMT) was one of the proteins that was retained and subsequently eluted from these supports. The phosphinic pseudopeptides, which served as immobilized ligands for the isolation of rat BHMT, were then tested for their ability to inhibit human recombinant BHMT in solution. The most potent inhibitor also behaved as a selective ligand for the affinity purification of BHMT from a complex media. Further optimization uncovered Val-Phe-psi[PO(2-)-CH(2)]-Leu-His-NH(2) as a potent BHMT inhibitor that has an IC(50) of about 1 microM.     

Parallel synthesis of H-pin polyamides by alkene metathesis on solid phase

A small library of H-pin polyamides with variable aliphatic bridge lengths (CH(2))(n)(), where n = 4-8, connecting a central Py/Py pair was prepared via parallel synthesis with Ru-catalyzed alkene metathesis on solid phase as a complexity-generating cross-linking reaction. DNA binding affinities and sequence specificities were analyzed for each member of the library to determine the optimum linker length. An H-pin polyamide with a six-methylene bridge was found to have the highest affinity to its match site with high selectivity over a 1-bp mismatch site. The relationship between the number of methylenes in the linker (CH(2))(n)() and affinity is n = 6 > 4 > 7 > 5 > 8. These results indicate that 6 followed by 4 methylene-bridged polyamides represent the optimum spacer length for the H-pin motif in the DNA minor groove. Importantly, the H-pin is competitive with hairpin polyamides with respect to affinity and specificity. The metathesis-based convergent synthetic route to H-pin polyamides expands the scope of readily available DNA recognition motifs for small molecule-based gene regulation studies.     

Multivalency effects in protein--carbohydrate interaction: the binding of the Shiga-like toxin 1 binding subunit to multivalent C-linked glycopeptides

A series of monovalent and bivalent glycopeptides displaying a C-linked analogue of the Pk trisaccharide, the in vivo ligand for the pentavalent Shiga-like toxin binding subunit (SLT-1B), were prepared and evaluated as ligands for SLT-1B by isothermal titration microcalorimetry and competitive enzyme-linked immunosorbent assay (ELISA). Although none of the monovalent ligands showed any enhancement in affinity compared to O-methyl glycoside, two bivalent ligands show significant enhancements in affinity in assays. This observation represents the first calorimetric observation of an enhancement in affinity for this system. In contrast, only one of the two ligands shows an enhancement in the competitive ELISA. Together, these data signal a difference in the means by which the two ligands achieve affinity, apparently triggered by a change in the nature of the linker domain. These results provide a rationalization for apparently contradictory reports from the recent literature and again emphasize the importance of investigating complex binding phenomena by multiple techniques.     

Triple-helical transition state analogues: a new class of selective matrix metalloproteinase inhibitors

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).     

二价HIV-1融合抑制剂的设计、合成与活性评价

针对人免疫缺陷病毒跨膜糖蛋白(HIV-1 gp41)N末端重复序列靶标设计二价融合抑制剂, 以C肽为模板, 通过共价交联形成类似发夹结构的相互平行的2条肽链, 研究了二价C肽分子不同连接位点与不同连接臂对抗HIV融合活性的影响. 细胞-细胞融合活性测试表明, 与单价分子相比, 所设计的基于N末端交联的C34或T20的二价分子在前体共价交联后, 活性明显提升. 基于C34或T20的N末端与C末端均存在发生协同效应的可能性, 在C34的N末端设计中β-丙氨酸为最适连接臂, 而在C末端的设计中C34C的融合活性提高最大. 单价分子CβAC34经过氧化形成二硫键连接的二价分子BiCβAC34, 融合活性从43.7 nmol/L提高到6.4 nmol/L, 表明二价抑制剂中2条C肽链间具有良好的协同效应. 本文结果表明, 针对gp41靶标设计的二价融合抑制剂能够相互协同.     

Biologically-Inspired Peptide Reagents for Enhancing IMS-MS Analysis of Carbohydrates

The binding properties of a peptidoglycan recognition protein are translated via combinatorial chemistry into short peptides. Non-adjacent histidine, tyrosine, and arginine residues in the protein’s binding cleft that associate specifically with the glycan moiety of a peptidoglycan substrate are incorporated into linear sequences creating a library of 27 candidate tripeptide reagents (three possible residues permutated across three positions). Upon electrospraying the peptide library and carbohydrate mixtures, some noncovalent complexes are observed. The binding efficiencies of the peptides vary according to their amino acid composition as well as the disaccharide linkage and carbohydrate ring-type. In addition to providing a charge-carrier for the carbohydrate, peptide reagents can also be used to differentiate carbohydrate isomers by ion mobility spectrometry. The utility of these peptide reagents as a means of enhancing ion mobility analysis of carbohydrates is illustrated by examining four glucose-containing disaccharide isomers, including a pair that is not resolved by ion mobility alone. The specificity and stoichiometry of the peptide–carbohydrate complexes are also investigated. Trihistidine demonstrates both suitable binding efficiency and successful resolution of disaccharides isomers, suggesting it may be a useful reagent in IMS analyses of carbohydrates.     

Structural analysis of charge discrimination in the binding of inhibitors to human carbonic anhydrases I and II

Despite the similarity in the active site pockets of carbonic anhydrase (CA) isozymes I and II, the binding affinities of benzenesulfonamide inhibitors are invariably higher with CA II as compared to CA I. To explore the structural basis of this molecular recognition phenomenon, we have designed and synthesized simple benzenesulfonamide inhibitors substituted at the para position with positively charged, negatively charged, and neutral functional groups, and we have determined the affinities and X-ray crystal structures of their enzyme complexes. The para-substituents are designed to bind in the midsection of the 15 A deep active site cleft, where interactions with enzyme residues and solvent molecules are possible. We find that a para-substituted positively charged amino group is more poorly tolerated in the active site of CA I compared with CA II. In contrast, a para-substituted negatively charged carboxylate substituent is tolerated equally well in the active sites of both CA isozymes. Notably, enzyme-inhibitor affinity increases upon neutralization of inhibitor charged groups by amidation or esterification. These results inform the design of short molecular linkers connecting the benzenesulfonamide group and a para-substituted tail group in "two-prong" CA inhibitors: an optimal linker segment will be electronically neutral, yet capable of engaging in at least some hydrogen bond interactions with protein residues and/or solvent. Microcalorimetric data reveal that inhibitor binding to CA I is enthalpically less favorable and entropically more favorable than inhibitor binding to CA II. This contrasting behavior may arise in part from differences in active site desolvation and the conformational entropy of inhibitor binding to each isozyme active site.     

A potent and highly selective inhibitor of human alpha-1,3-fucosyltransferase via click chemistry

Potent inhibitors of fucosyltransferases, and glycosyltransferases in general, have been elusive due to the inherent barriers surrounding the family of glycosyltransfer reactions. The problems of weak substrate affinity and low catalytic proficiency of fucosyltransferase was offset by recruiting additional binding features, in this case hydrophobic interactions, to produce a high affinity inhibitor, 24, with Ki = 62 nM. The molecule was identified from a GDP-triazole library of 85 compounds, which was produced by the Cu(I)-catalyzed [2 + 3] cycloaddition reaction between azide and acetylene reactants, followed by in situ screening without product isolation.