Combinatorial chemistry of β-hairpins

Combinatorial chemistry is expanding rapidly both in terms of chemistry development and application to the synthesis of compound libraries for lead discovery and optimization. Combinatorial technologies continue evolving and developing, in fact they are being used as basic research tools in different fields that include peptide/protein folding. This review examines the use of combinatorial chemistry in the design of peptides and protein domains that adopt beta-sheet conformations. In particular, the use of conformationally restricted peptide libraries has allowed the identification of linear peptides that are folded in a beta-hairpin structure in plain aqueous solutions.     

Structural aspects of antibody-antigen interaction revealed through small random peptide libraries

Summary Two small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 10⁷ hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.Abbreviations ABTS 2,2-azino-di-3-ethylbenzthiazoline sulfonate - EGF epidermal growth factor - Mab monoclonal antibody - OD_ccd optical density obtained using CCD camera - RAMPO rabbit-antimouse peroxidase - SDS sodium dodecylsulfate - TGEV transmissible gastroenteritis virus     

Combining 2D and 3D in silico methods for rapid selection of potential PDE5 inhibitors from multimillion compounds’ repositories: biological evaluation

Rapid in silico selection of target focused libraries from commercial repositories is an attractive and cost-effective approach when starting new drug discovery projects. If structures of active compounds are available rapid 2D similarity search can be performed on multimillion compounds’ databases. This in silico approach can be combined with physico-chemical parameter filtering based on the property space of the active compounds and 3D virtual screening if the structure of the target protein is available. A multi-step virtual screening procedure was developed and applied to select potential phosphodiesterase 5 (PDE5) inhibitors in real time. The combined 2D/3D in silico method resulted in the identification of 14 novel PDE5 inhibitors with <1 μMIC₅₀ values and the hit rate in the second in silico selection and in vitro screening round exceeded the 20%.     

Rational selection of structurally diverse natural product scaffolds with favorable ADME properties for drug discovery

Natural product analogs are significant sources for therapeutic agents. To capitalize efficiently on the effective features of naturally occurring substances, a natural product-based library production platform has been devised at Aurigene for drug lead discovery. This approach combines the attractive biological and physicochemical properties of natural product scaffolds, provided by eons of natural selection, with the chemical diversity available from parallel synthetic methods. Virtual property analysis, using computational methods described here, guides the selection of a set of natural product scaffolds that are both structurally diverse and likely to have favorable pharmacokinetic properties. The experimental characterization of several in vitro ADME properties of twenty of these scaffolds, and of a small set of designed congeners based upon one scaffold, is also described. These data confirm that most of the scaffolds and the designed library members have properties favorable to their utilization for creating libraries of lead-like molecules.These authors have contributed equally to this work.     

Application of computer assisted combinatorial chemistry in antivirial,antimalarial and anticancer agents design

Combinatorial chemistry and technologies have been developed to a stage where synthetic schemes are available for generation of a large variety of organic molecules. The innovative concept of combinatorial design assumes that screening of a large and diverse library of compounds will increase the probability of finding an active analogue among the compounds tested. Since the rate at which libraries are screened for activity currently constitutes a limitation to the use of combinatorial technologies, it is important to be selective about the number of compounds to be synthesized. Early experience with combinatorial chemistry indicated that chemical diversity alone did not result in a significant increase in the number of generated lead compounds. Emphasis has therefore been increasingly put on the use of computer assisted combinatorial chemical techniques. Computational methods are valuable in the design of virtual libraries of molecular models. Selection strategies based on computed physicochemical properties of the models or of a target compound are introduced to reduce the time and costs of library synthesis and screening. In addition, computational structure-based library focusing methods can be used to perform in silico screening of the activity of Compounds against a target receptor by docking the ligands into the receptor model. Three case studies are discussed dealing with the design of targeted combinatorial libraries of inhibitors of HIV-1 protease, P. falciparum plasmepsin and human urokinase as potential antivirial, antimalarial and anti-cancer drugs. These illustrate library focusing strategies.     

Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: Deconvolution strategy

Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X₁–X₂–hinge–X₃–X₄–NH₂ (capped) and X₁–hinge–X₂–X₃ (uncapped), where X₁ through X₄ are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a central hinge residue in small peptides increases the opportunity for side chain interactions, providing a distinct advantage for hinge peptide libraries over conventional peptide libraries. Hinge peptide libraries are a rich source of novel ligands for modulation of biomechanisms. The library subsets uncovered in this study may possess peptides that will lead to effective therapies to neurotoxin poisoning.     

Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries

Summary Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.     

An integrated drug-likeness study for bicyclic privileged structures: from physicochemical properties to in vitro ADME properties

The concept of drug-likeness has been widely applied in combinatorial chemistry as an approach to reduce attrition in drug discovery and development. Meanwhile, bicyclic privileged structures with versatile binding properties have emerged as ideal source of core scaffolds for the design and synthesis of combinatorial libraries. For the purpose of better assisting the design of bicyclic privileged structure-based combinatorial libraries, we conducted an integrated drug-likeness study on compounds of these scaffolds. Distributions of physicochemical properties (PCPs) were analyzed and in silico prediction models were built. Our results showed that there exist much difference between the drug-like ranges (DLRs) of bicyclic privileged structures and that of others, which have significant impact on compound selection. The DLRs for bicyclic privileged structures were defined as 260 ≤ MW ≤ 524; 0.9 ≤ ALogP ≤ 5.4; 2 ≤ Hacc ≤ 8; Hdon ≤ 3; 21.0 ≤ PSA ≤ 128.6; 6.3 ≤ FPSA ≤ 34.2; 1 ≤ RotB ≤ 10; 2 ≤ Nr ≤ 5; 1 ≤ Nc ≤ 7; SA ≤ 4. Two accurate and easy to understand in silico prediction models, Caco-2 permeability model and metabolic stability classification model, had been built to guide drug candidate optimization. In these models, hydrogen-bond donor and rotatable bond showed major impact on the permeability of compounds, while lipophilicity, flexibility, degree of branching and the existence of some functional groups determined the fate of a drug in metabolic process. Suggestions on structural modification toward higher permeability and metabolic stability were given according to the in silico models.     

Exploring privileged structures: The combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classesof receptor with high affinity. They may therefore be considered to beprivileged structures. This review outlines the strategies by which bothmacrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines havebeen synthesised in combinatorial libraries. It also briefly outlines someof the biological applications of these molecules, thereby justifying theirinclusion as privileged structures.     

Utilization of multiple phage display libraries for the identification of dissimilar peptide motifs that bind to a B7-1 monoclonal antibody

Summary Seven random peptide libraries (two displaying linear peptides and five displaying cysteine-constrained peptides) were constructed as gene III fusion proteins of the bacteriophage fd-tet. These libraries were used to screen a blocking monoclonal antibody raised against B7-1 (CD80), a human cell surface antigen that binds two T cell receptors, CD28 and CTLA-4. After three rounds of screening against the immobilized antibody, 1000-fold enrichment was observed in libraries displaying both linear and cysteineconstrained peptides. DNA sequencing of the enriched phage revealed two distinct consensus sequences: HXG(A/Y)XH and DVCXXGGPGC. Phage expressing these consensus sequences bound to L307.4 but not to an isotype matched antibody, indicating that binding was antibody specific. Synthetic peptides corresponding to both motifs inhibited phage binding to L307.4, indicating that the gene III protein is not required for peptide binding. In addition, the cyclized forms of synthetic peptides containing the DVCXXGGPGC motif were capable of inhibiting L307.4 binding to soluble B7-1/Fc fusion. Moreover, phage expressing only the HXG(A/Y)XH consensus sequence were inhibited from binding to L307.4 by the presence of chelating agents. These results indicate that the framework within which the peptide is presented on the surface of the phage may allow the identification of unique peptide motifs with distinct binding characteristics. These peptide motifs could be used for the design of peptidomimetics with therapeutic applications if they inhibit the binding of B7-1 to its T cell receptors.     

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.     

Chemical synthesis of TASP arrays and their application in protein design

A combinatorial synthesis of de novo proteins is described. The concept of template-assembled synthetic proteins (TASP) has been adapted to an orthogonal assembly of small libraries of purified peptide building blocks. It is combined with the spot synthesis of peptides which is exploited to array cyclic decapeptide templates on cellulose membranes. A cleavable linker on the cellulose allows control of the synthesis. The hydrophilic proteins are constructed by successive cleavage of orthogonal protecting groups on the template, followed by coupling of amphipathic helices in a predefined orientation and finally by incorporation of a cofactor. Libraries of peptides with variation of the amino acids expected to be close to the cofactor were coupled to the cellulose-bound template in all combinations, yielding up to 500 variants of a protein. Cofactors have been inserted either at non-covalent binding sites as heme and Cu2+ or by covalent modification of amino acids as Ru-bipyridine or flavin. The proteins were screened by recording their UV-vis spectra directly on the solid support. The properties screened include the redox potential of heme proteins, charge transfer bands indicating the ligation of Cu-centers, enzymatic activity, and folding stability. Synthesis of the best hits as soluble variants was used for detailed characterization. Iterative improvement in a second screening cycle was efficient in finding novel copper proteins. We discuss the prospects of synthesizing proteins by extending the concept to beta-sandwich proteins and construction of efficient peptide libraries with computer-supported design, as well as the possible usage of improved solid phase materials.     

Molecular diversification in spider venoms: A web of combinatorial peptide libraries

Summary Spider venoms are a rich source of novel pharmacologically and agrochemically interesting compounds that have received increased attention from pharmacologists and biochemists in recent years. The application of technologies derived from genomics and proteomics have led to the discovery of the enormous molecular diversity of those venoms, which consist mainly of peptides and proteins. The molecular diversity of spider peptides has been revealed by mass spectrometry and appears to be based on a limited set of structural scaffolds. Genetic analysis has led to a further understanding of the molecular evolution mechanisms presiding over the generation of these combinatorial peptide libraries. Gene duplication and focal hypermutation, which has been described in cone snails, appear to be common mechanisms to venomous mollusks and spiders. Post-translational modifications, fine structural variations and new molecular scaffolds are other potential mechanisms of toxin diversification, leading to the pharmacologically complex cocktails used for predation and defense.     

Leadlikeness and structural diversity of synthetic screening libraries

Summary High program failure rates in the pharmaceutical industry have prompted the development of predictive software that can profile compound libraries as being ‘druglike’ (resembling existing drugs) and/or ‘leadlike’ (possessing the structural and physicochemical profile of a quality lead). In recent years, these two notions prompted pharmaceutical companies to clean up their corporate libraries of screening compounds. In order to maintain and expand the size and diversity of these corporate libraries, pharmaceutical companies still continue to add compounds to these, mainly by the acquisition of screening libraries. In this paper, we have analyzed 45 commercially available libraries, offered by suppliers of screening chemistry, for leadlikeness and diversity of the offered structures. To this end we have used a set of structural and physicochemical filters for leadlikeness that was developed in-house. These 45 supplier libraries contained a total of 5.3 million structures, of which 49% (2,592,778 structures) turned out to be unique, and only 12% (677,328 structures) were found to be both unique and leadlike. A diversity analysis revealed that big differences exist between the various offered libraries.     

Novel α-glucosidase inhibitors identified using multiple cyclic peptide combinatorial libraries

Summary Twenty-six cyclic synthetic peptide combinatorial libraries (disulfides and lactams) of varying size and composition, representing 6.8 × 10³ to 4.7 × 10⁷ individual peptides, were synthesized along with their respective linear analogs. One of the hexapeptide lactam libraries (cyclo[xXxXxN]) was found to have significant -glucosidase inhibitory activity. This library was carried through an iterative process of synthesis and screening, during which all of the five mixture positions (x and X) were successively defined. As the result of this process, potent and selective a-glucosidase inhibitors were identified.     

Diversity through semisynthesis: the chemistry and biological activity of semisynthetic epothilone derivatives

Epothilones are myxobacterial natural products that inhibit human cancer cell growth through the stabilization of cellular microtubules (i.e., a “taxol-like” mechanism of action). They have proven to be highly productive lead structures for anticancer drug discovery, with at least seven epothilone-type agents having entered clinical trials in humans over the last several years. SAR studies on epothilones have included a large number of fully synthetic analogs and semisynthetic derivatives. Previous reviews on the chemistry and biology of epothilones have mostly focused on analogs that were obtained by de novo chemical synthesis. In contrast, the current review provides a comprehensive overview on the chemical transformations that have been investigated for the major epothilones A and B as starting materials, and it discusses the biological activity of the resulting products. Many semisynthetic epothilone derivatives have been found to exhibit potent effects on human cancer cell growth and several of these have been advanced to the stage of clinical development. This includes the epothilone B lactam ixabepilone (Ixempra^®, which has been approved by the FDA for the treatment of advanced and metastatic breast cancer.     

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage

Summary We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 × 10⁹ members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of -endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v -ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.     

High-volume cellular screening for anticancer agents with combinatorial chemical libraries: A new methodology

Summary A single-step cancer cell cytotoxic assay system for anticancer drug discovery has been developed which facilitates rapid screening of large combinatorial chemical libraries synthesized using the one-bead-one-compound (OBOC) methodology. Each OBOC library bead incorporates two orthogonally cleavable linkers that release the bead-bound compound at a different pH. The assay utilizes high concentrations of tumor cells mixed directly with OBOC beads and plated in soft agarose containing tissue culture medium. One of the orthogonal linkers is cleaved at neutral pH in tissue culture releasing an aliquot of compound to diffuse at a relatively high local concentration into the soft agarose immediately surrounding the bead. Active compounds are identified visually from a clear ring of tumor cell lysis which forms within 48 h around just the rare bead releasing a cytotoxic compound. The bead releasing a cytotoxin is then plucked from the agar and the remaining compound still linked to the bead can be released for structural analysis, followed by compound resynthesis and confirmatory testing. This assay system has been successfully applied to identification of lead cytotoxic compounds from model peptidic and non-peptidic combinatorial chemical libraries. Use of this methodology may facilitate anticancer drug discovery.     

Mining combinatorial data in protein sequences and structures

Combinatorial searches of structural and physical chemical properties involving the components of libraries of dipeptide, tripeptide and tetra peptide fragments were carried out in the Protein Data Bank and the SwissProt databases. The properties investigated are structural propensities, co localization of peptide fragments in protein sequences, interactions between peptide fragments in close structural proximity and the participation of physical chemical profiles in the distribution of structural motifs among peptide fragments. The results obtained for each combinatorial search in the study are classified according to the structural motifs alpha-helix, beta-sheet, reverse turn I and reverse turn II. The application of combinatorial data mined in protein databases to the design of new peptide libraries is discussed. The present findings have implications for the study of protein structure which are also discussed.     

Emerging approaches for the syntheses of bicyclic imidazo[1,2-x]-heterocycles

Imidazo-[1,2-x]heterocycles are versatile building blocks for use in both a 'drug hunters' quest to discover new leads and a chemical biologists search for effective molecular tools in 'cell perturbation' studies. At the front end of the drug discovery flow chart, the last 5-10 years have witnessed the discovery of new high-throughput methodologies which very quickly have enabled access to virtual libraries of these chemo-types in the realm of 10(7) derivatives. Interestingly, these often neglected cores in patent cooperation treaty (PCT) applications appear in several highly effective marketed drugs, completing the medicinal chemists search for clinical success. Such rigid chemo-types, all containing a bridgehead nitrogen atom, are thus poised for an ever increasing impact on the discovery and development of new molecular therapeutics. The following mini-review will briefly cover therapeutic utility, chemical methodologies and automation developed to enable preparation of arrays of these chemo-types in a high-throughput manner. Synthetic emphasis is placed on a 3-component-3-center isocyanide based multi-component reaction (IMCR), which spans solution, solid phase, flourous and microwave assisted organic synthesis.