SPROUT: A program for structure generation

Summary SPROUT is a new computer program for constrained structure generation that is designed to generate molecules for a range of applications in molecular recognition. It uses artificial intelligence techniques to moderate the combinatorial explosion that is inherent in structure generation. The program is presented here for the design of enzyme inhibitors. Structure generation is divided into two phases: (i) primary structure generation to produce molecular graphs to fit the steric constraints; and (ii) secondary structure generation which is the process of introducing appropriate functionality to the graphs to produce molecules that satisfy the secondary constraints, e.g., electrostatics and hydrophobicity. Primary structure generation has been tested on two enzyme receptor sites; the p-amidino-phenyl-pyruvate binding site of trypsin and the acetyl pepstatin binding site of HIV-1 protease. The program successfully generates structures that resemble known substrates and, more importantly, the predictive power of the program has been demonstrated by its ability to suggest novel structures.     

Combinatorial chemistry in food research

This mini-review is concerned with emerging applications of combinatorial chemistry relevant to the needs of the food industry. More specifically, recent advances in the use of combinatorial methods for the identification and analysis of flavours, "food chemical" libraries as a potential source of enzyme inhibitors for the food industry and the utility of biocatalysis for the generation of molecular diversity are discussed.     

Structure-based design and combinatorial chemistry yield low nanomolar inhibitors of cathepsin D

Background: The identification of potent small molecule ligands to receptors and enzymes is one of the major goals of chemical and biological research. Two powerful new tools that can be used in these efforts are combinatorial chemistry and structure-based design. Here we address how to join these methods in a design protocol that produces libraries of compounds that are directed against specific macromolecular targets. The aspartyl class of proteases, which is involved in numerous biological processes, was chosen to demonstrate this effective procedure.Results: Using cathepsin D, a prototypical aspartyl protease, a number of low nanomolar inhibitors were rapidly identified. Although cathepsin D is implicated in a number of therapeutically relevant processes, potent nonpeptide inhibitors have not been reported previously. The libraries, synthesized on solid support, displayed nonpeptide functionality about the (hydroxyethyl)amine isostere. The (hydroxyethyl)amine isostere, which targets the aspartyl protease class, is a stable mimetic of the tetrahedral intermediate of amide hydrolysis. Structure-based design, using the crystal structure of cathepsin D complexed with the peptide-based natural product pepstatin, was used to select the building blocks for the library synthesis. The library yielded a ‘hit rate’ of 6–7% at 1 μM inhibitor concentrations, with the most potent compound having a K_i value of 73 nM. More potent, nonpeptide inhibitors (K_i = 9–15 nM) of cathepsin D were rapidly identified by synthesizing and screening a small second generation library.Conclusions: The success of these studies clearly demonstrates the power of coupling the complementary methods of combinatorial chemistry and structure-based design. We anticipate that the general approaches described here will be successful for other members of the aspartyl protease class and for many other enzyme classes.     

Solid-phase combinatorial library of norstatine-type isosters by the nitroaldol reaction

A combinatorial library of norstatine-type peptide isosters as putative inhibitors of aspartic proteases is presented. The library was synthesized using a split-and-mix strategy designed to afford a one-bead-two-compounds library with the isosteric elements positioned centrally in peptide chains. Application of ladder synthesis during library generation enabled structure identification by MALDI-TOF mass spectroscopy. The library was screened against aspartic protease renin, and two types of inhibitors were identified, that is, XXX-psi[CHRCHOH)-XXX and an aldehyde arising from unreacted starting material. Selected hits were resynthesized and assayed in solution, revealing inhibitors of nanomolar potency.     

A probabilistic approach to high throughput drug discovery

A methodology is presented in which high throughput screening experimental data are used to construct a probabilistic QSAR model which is subsequently used to select building blocks for a virtual combinatorial library. The methodology is based upon statistical probability estimation and not regression. The methodology is applied to the construction of two focused virtual combinatorial libraries: one for cyclic GMP phosphodiesterase type V inhibitors and one for acyl-CoA:cholesterol O-acyltransferase inhibitors. The results suggest that the methodology is capable of selecting combinatorial substituents that lead to active compounds starting with binary (pass/fail) activity measurements.     

Combinatorial synthesis of novel and potent inhibitors of NADH:ubiquinone oxidoreductase

BACKGROUND: NADH:ubiquinone oxidoreductase (complex I) is the first of three large enzyme complexes located in the cell's inner mitochondrial membrane which form the electron transport chain that carries electrons from NADH to molecular oxygen during oxidative phosphorylation. There is significant interest in developing small molecule inhibitors of this enzyme for use as biological probes, insecticides and potential chemopreventive/chemotherapeutic agents. Herein we describe the application of novel natural product-like libraries to the discovery of a family of potent benzopyran-based inhibitors. RESULTS: Initially a combinatorial library of benzopyrans, modeled after natural products, was synthesized using a solid phase cycloloading strategy. Screening of this diversity oriented library for inhibitory potency against NADH:ubiquinone oxidoreductase activity in vitro using bovine heart electron transport particles provided several lead compounds which were further refined through a series of focused libraries. CONCLUSIONS: Using this combinatorial library approach, a family of potent 2,2-dimethylbenzopyran-based inhibitors was developed with IC(50) values in the range of 18-55 nM. Cell-based assays revealed that these inhibitors were rather non-cytotoxic in the MCF-7 cell line; however, they were quite cytostatic in a panel of cancer cell lines suggesting their potential as chemotherapeutic/chemopreventive candidates.     

High-speed optimization of inhibitors of the malarial proteases plasmepsin I and II

Four focused libraries targeted for inhibition of the malarial proteases plasmepsin I and II were designed, synthesized, purified, and screened. Selected carboxylic acids and organometallic reactants with diverse physical properties were attached to the hydroxylethylamine scaffold in the P3 and P1' positions to furnish inhibitors with highly improved activity. The concept of controlled and sequential microwave heating was employed for rapid library generation. This combinatorial optimization protocol afforded plasmepsin inhibitors not only with K(i) values in the low nanomolar range, but also with high selectivity versus the human protease cathepsin D. With this class of inhibitory agents, modifications of the P1' substituents resulted in the largest impact on the plasmepsin/cathepsin D selectivity.     

Computational combinatorial ligand design: Application to human α-thrombin

Summary A new method is presented for computer-aided ligand design by combinatorial selection of fragments that bind favorably to a macromolecular target of known three-dimensional structure. Firstly, the multiple-copy simultaneous-search procedure (MCSS) is used to exhaustively search for optimal positions and orientations of functional groups on the surface of the macromolecule (enzyme or receptor fragment). The MCSS minima are then sorted according to an approximated binding free energy, whose solvation component is expressed as a sum of separate electrostatic and nonpolar contributions. The electrostatic solvation energy is calculated by the numerical solution of the linearized Poisson-Boltzmann equation, while the nonpolar contribution to the binding free energy is assumed to be proportional to the loss in solvent-accessible surface area. The program developed for computational combinatorial ligand design (CCLD) allows the fast and automatic generation of a multitude of highly diverse compounds, by connecting in a combinatorial fashion the functional groups in their minimized positions. The fragments are linked as two atoms may be either fused, or connected by a covalent bond or a small linker unit. To avoid the combinatorial explosion problem, pruning of the growing ligand is performed according to the average value of the approximated binding free energy of its fragments. The method is illustrated here by constructing candidate ligands for the active site of human -thrombin. The MCSS minima with favorable binding free energy reproduce the interaction patterns of known inhibitors. Starting from these fragments, CCLD generates a set of compounds that are closely related to high-affinity thrombin inhibitors. In addition, putative ligands with novel binding motifs are suggested. Probable implications of the MCSS-CCLD approach for the evolving scenario of drug discovery are discussed.     

Optimization of bivalent glutathione S-transferase inhibitors by combinatorial linker design

Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases, including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTP1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120-fold selectivity) and with stoichiometry of one inhibitor: one GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure (Asp-Gly-AMAB-Gly-Asp) and K(D) = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange. A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complementarity between the protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. The results demonstrate the utility of combinatorial, or "irrational", linker design for optimizing bivalent inhibitors.     

Combinatorial library of five-membered iminocyclitol and the inhibitory activities against glyco-enzymes.

BACKGROUND: Oligosaccharide processing enzymes are important classes of catalysts involved in synthesizing specific oligosaccharide structures on proteins and sphingolipids. Development of specific inhibitors of such enzymes is of current interest as these inhibitors may be used to control cellular functions. Five-membered iminocyclitols have been shown to be potent inhibitors of such enzymes. Since a rational design and synthesis of inhibitors is often extremely difficult due to the limited information regarding the structure of the active site, we carried out a combinatorial library approach. RESULTS: To create diversity, we decided to use an aldehyde group of a protected iminocyclitol for reductive amination and the Strecker reaction. After transformation of the nitrile group introduced by the Strecker reaction into an amine and amide and complete deprotection, a small library of five-membered iminocyclitols consisting of 27 compounds was synthesized. A series of compounds obtained by reductive amination was first screened as potential inhibitors of glycosidases and glycosyltransferases. Among them, compounds carrying a C(10)-alkyl group showed marked enhancement of inhibitory activity against alpha-mannosidase at 10 microM concentration when compared with its parent compound and deoxymannojirimycin. Furthermore, compounds having the phenylethyl group showed an extremely strong inhibitory effect against alpha-galactosaminidase at a K(i) value of 29.4 nM. Compounds with an aminomethyl and amide group at the C-1' position of these two molecules showed a decrease in inhibitory activities. CONCLUSIONS: A combinatorial approach based on five-membered iminocyclitols with a galacto-configuration was exploited. The potential usefulness of the library as a source of inhibitors of glycoenzymes is clearly shown in this study.     

Simulated molecular evolution in a full combinatorial library

BACKGROUND: The Darwinian concept of 'survival of the fittest' has inspired the development of evolutionary optimization methods to find molecules with desired properties in iterative feedback cycles of synthesis and testing. These methods have recently been applied to the computer-guided heuristic selection of molecules that bind with high affinity to a given biological target. We describe the optimization behavior and performance of genetic algorithms (GAs) that select molecules from a combinatorial library of potential thrombin inhibitors in 'artificial molecular evolution' experiments, on the basis of biological screening results. RESULTS: A full combinatorial library of 15,360 members structurally biased towards the serine protease thrombin was synthesized, and all were tested for their ability to inhibit the protease activity of thrombin. Using the resulting large structure-activity landscape, we simulated the evolutionary selection of potent thrombin inhibitors from this library using GAs. Optimal parameter sets were found (encoding strategy, population size, mutation and cross-over rate) for this artificial molecular evolution. CONCLUSIONS: A GA-based evolutionary selection is a valuable combinatorial optimization strategy to discover compounds with desired properties without needing to synthesize and test all possible combinations (i.e. all molecules). GAs are especially powerful when dealing with very large combinatorial libraries for which synthesis and screening of all members is not possible and/or when only a small number of compounds compared with the library size can be synthesized or tested. The optimization gradient or 'learning' per individual increases when using smaller population sizes and decreases for higher mutation rates.     

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.     

Dynamic combinatorial chemistry: on the road to fulfilling the promise

Dynamic combinatorial chemistry makes use of reversible reactions between functionalised monomeric building blocks to generate a mixture of products (dimers or oligomers) under thermodynamic equilibrium. This system reorganises upon addition of a target so that species that bind to, and are therefore stabilised by the target, are favourably formed and are thus amplified. Since the mid-1990's, dynamic combinatorial chemistry has been successfully applied to the identification/selection of ion receptors, enzyme inhibitors, catalysts, materials and nucleic acid ligands. Although it is now established as a powerful tool with broad applications some intrinsic limitations appeared when working on systems of increasing complexity. We present here the most recent advances in the field of dynamic combinatorial chemistry that have been developed to overcome these limitations and explore new areas of application.     

Identification of alpha-galactosyl epitope mimetics through rapid generation and screening of C-linked glycopeptide library

A general methodology has been established for rapid generation and screening of combinatorial glycopeptide library and subsequent mass spectrometric sequencing to identify the mimetics of Galalpha(1,3)Gal epitopes. Using this approach, several active glycopeptide sequences were recognized and found to inhibit the binding of human natural anti-Gal antibodies with comparable IC(50)s to synthetic Galalpha(1,3)Gal oligosaccharides. The most active glycopeptides detected from the library included Gal-Tyr-Trp-Arg-Tyr, Gal-Thr-Trp-Arg-Tyr, and Gal-Arg-Trp-Arg-Tyr. These glycopeptides showed higher affinities to anti-Gal antibodies than known Galalpha(1,3)Gal peptide mimetics, such as DAHWESWL and SSLRGF. Our results suggest that, by combining a peptide sequence (the "functional" mimic part) with a terminal alpha-linked galactose moiety (the "structural" mimic part), the resulting glycopeptide could be a very good Galalpha(1,3)Gal mimetic. Analysis of these active glycopeptides provided first-hand information regarding the binding site of anti-Gal antibodies to facilitate the structurally based design of more potent and stable inhibitors.     

The SHAPES strategy: an NMR-based approach for lead generation in drug discovery.

BACKGROUND: Recently, it has been shown that nuclear magnetic resonance (NMR) may be used to identify ligands that bind to low molecular weight protein drug targets. Recognizing the utility of NMR as a very sensitive method for detecting binding, we have focused on developing alternative approaches that are applicable to larger molecular weight drug targets and do not require isotopic labeling. RESULTS: A new method for lead generation (SHAPES) is described that uses NMR to detect binding of a limited but diverse library of small molecules to a potential drug target. The compound scaffolds are derived from shapes most commonly found in known therapeutic agents. NMR detection of low (microM-mM) affinity binding is achieved using either differential line broadening or transferred NOE (nuclear Overhauser effect) NMR techniques. CONCLUSIONS: The SHAPES method for lead generation by NMR is useful for identifying potential lead classes of drugs early in a drug design program, and is easily integrated with other discovery tools such as virtual screening, high-throughput screening and combinatorial chemistry.     

Successful screening of large encoded combinatorial libraries leading to the discovery of novel p38 MAP kinase inhibitors

Screening of more than 2 million compounds comprising 41 distinct encoded combinatorial libraries revealed a novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors. The methodology used for screening large encoded combinatorial libraries combined with the statistical interpretation of screening results is described. A strong preference for a particular triaminotriazine aniline amide was discovered based on biological activity observed in the screening campaign. Additional screening of a focused follow-up combinatorial library yielded data expanding the unique combinatorial SAR and emphasizing an extraordinary preference for this particular building block and structural class. The preference is further highlighted when the p38 inhibitor data set is compared to data obtained for a panel of other kinases.     

DBU-assisted cyclorelease elimination: combinatorial synthesis and gamma-glutamyl cysteine synthetase and glutathione-S-transeferase modulatory effect of C-nucleoside analogs

A combinatorial library of 60C- nucleoside analogs was synthesized by sequential coupling of building blocks followed by cyclative cleavage with DBU in an efficient manner. Only DMSO soluble compounds were tested for their modulatory effect against filarial gamma-glutamyl cysteine synthetase (gamma-GCase) and glutathione-S-transeferases (GSTs). Several compounds were found to be weak inhibitors of filarial gamma-GCase, whereas, most of them stimulated filarial GSTs.     

Potential protease inhibitors based on a functionalized cyclic sulfamide scaffold

Exploratory studies related to the design and synthesis of functionalized cyclic sulfamides (I) as potential inhibitors of proteolytic enzymes were carried out. The structural motif and three diversity sites embodied in the scaffold render it amenable to combinatorial parallel synthesis and the facile generation of lead discovery prospecting libraries. The scaffold was readily assembled starting with (DL) serine methyl ester, and a series of compounds was generated and screened against human leukocyte elastase. Modification of the P(1) recognition element, believed to be accommodated at the primary specificity site (S(1) subsite) of the enzyme, yielded compounds that inhibited the enzyme by an apparent hyperbolic partial mixed-type inhibition.