A combinatorial scaffold approach toward kinase-directed heterocycle libraries

A novel strategy for efficient synthesis of various substituted heterocycles as kinase-directed combinatorial libraries is described. The general scheme involves capture of various dichloroheterocycles onto solid support and further elaborations by aromatic substitution with amines at elevated temperature or by anilines, boronic acids, and phenols via palladium-catalyzed cross-coupling reactions, thus the scaffold itself is transformed into a diversity element within the combinatorial scheme. Libraries consisting of discrete and highly diverse heterocyclic small molecules constructed with these chemistries are currently being evaluated in a variety of cell and protein-based assays.     

Design of dimerization inhibitors of HIV-1 aspartic proteinase: A computer-based combinatorial approach

Inhibition of dimerization to the active form of the HIV-1 aspartic proteinase (HIV-1 PR) may be a way to decrease the probability of escape mutations for this viral protein. The Multiple Copy Simultaneous Search (MCSS) methodology was used to generate functionality maps for the dimerization interface of HIV-1 PR. The positions of the MCSS minima of 19 organic fragments, once postprocessed to take into account solvation effects, are in good agreement with experimental data on peptides that bind to the interface. The MCSS minima combined with an approach for computational combinatorial ligand design yielded a set of modified HIV-1 PR C-terminal peptides that are similar to known nanomolar inhibitors of HIV-1 PR dimerization. A number of N-substituted 2,5-diketopiperazines are predicted to be potential dimerization inhibitors of HIV-1 PR.     

A combinatorial approach toward the discovery of non-peptide, subtype-selective somatostatin receptor ligands

The tetradecapeptide somatostatin is widely distributed throughout the body and is thought to be involved with a variety of regulatory functions. Recently, five human somatostatin receptors (hSSTR1-5) have been cloned and characterized. Several selective peptidal agonists of the hSSTR receptors are known, and we sought to apply this information to the design of novel non-peptide small molecule ligands for each receptor. Initial computational methods identified a 200 nM murine SSTR2 active compound via a database search of our sample collection. A combinatorial library was designed around the structural class of the compound with the goal of rapidly developing this initial lead into the desired subtype-selective small molecules in order to characterize the pharmacology of each of the receptor subtypes. The library was synthesized using the resin-archive, iterative deconvolution format. The total number of unique compounds in the library was expected to be 131,670, present in 79 mixtures of 1330 or 2660 compounds per mixture. Through sequences of screening and mixture deconvolution, the components of selective and highly active (Ki = 50 pM to 200 nM) non-peptide small molecule ligands for somatostatin subtypes 1, 2, 4, and 5 were identified. In addition to discovering compounds with the desired activity and selectivity, useful structure/activity information was generated which can be used in the design of new compounds and second-generation combinatorial libraries.     

A consecutive Diels-Alder approach toward a Tet repressor directed combinatorial library

A combinatorial library of 180 tetracycline analogs was generated by solution phase parallel synthesis applying a consecutive Diels-Alder strategy. Chemical methodology suitable for three-dimensional solution phase parallel synthesis was developed that enabled us to generate a collection of potential TetR inducers. The synthesis was built on cross-conjugated trienes as central building blocks facilitating two consecutive cycloaddition processes with different dienophiles. Upon sequential exposure to naphthoquinone and maleimide derivatives, the generation of a carbocyclic skeleton of type 2 incorporating the diversity elements R¹-R⁵ was envisaged.     

A multilayer screening approach toward the discovery of novel Pf-DHFR inhibitors

A small yet diverse xanthone library was build and computationally docked against wild type Pf-DHFR by Molegro Virtual Docker (MolDock). For analysis of results an integrated approach based on re-ranking, scaling (based on heavy atom counts), pose clustering and visual inspection was implemented. Standard methods such as self-docking (for docking), EF analysis, average rank determinations (for size normalization), and cluster quality indices (for pose clustering) were used for validation of results. Three compounds X5, X113A and X164B displayed contact footprints similar to the known inhibitors with good scores. Finally, 16 compounds were extracted from ZINC data base by similarity based screening, docking score and drug/lead likeness. Out of these 16 compounds, 11 displayed very close contact footprints to experimentally known inhibitors, indicating there potential utility in further drug discovery efforts.     

Solid-phase synthesis of a peptide-based P,S-ligand system designed for generation of combinatorial catalyst libraries

An efficient methodology for the solid-phase synthesis of diverse combinatorial peptide-based P,S-ligand libraries based on a modular approach was developed. Chiral thioethers were introduced into a series of peptide scaffolds using commercially available Fmoc-protected cysteine derivatives, and secondary amines were incorporated into the peptide backbones by reductive alkylation using readily available Fmoc-protected amino aldehydes. Phosphinylation of the secondary amines of the scaffolds, applying two different reagents, yielded two different types of ligands. Subsequent complexation with palladium afforded six- or seven-membered chelates, respectively. The selectivity, in an asymmetric allylic substitution reaction, of the two different types of chelates, derived from the same peptide scaffold, was complementary in all cases studied, affording the product as opposite stereoisomers with up to 60% ee. These results hold great promise for the identification of highly selective catalysts upon screening of larger P,S-based catalyst libraries.     

Greener approach toward the generation of dimedone derivatives

The use of dimedone in green chemistry has been described for the synthesis of selective heterocyclic motifs which are both pharmacologically and industrially important. The objective of this review is to summarize some of the selected recent advances of dimedone in the synthesis of organic compounds utilizing green chemistry procedures.     

A library approach to the generation of bisubstrate analogue sulfotransferase inhibitors

[reaction: see text]. A library of potential bisubstrate analogue inhibitors (1) targeting sulfotransferase enzymes was generated by the chemoselective ligation of the PAPS mimic 2 with a panel of 447 aldehydes. Preliminary screening has identified compounds that inhibit estrogen sulfotransferase (EST), an enzyme relevant to breast cancer.     

TAC-scaffolded tripeptides as artificial hydrolytic receptors: a combinatorial approach toward esterase mimics

In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.     

A peptide-based catalyst approach to regioselective functionalization of carbohydrates

Two small peptide libraries (150 members and 36 members) have been subjected to screening experiments to evaluate their potential for regioselective (i.e. site-selective) acylation of carbohydrate monomers. Two substrates, one diol derived from N-acetyl glucosamine and one tetraol derived from glucose, have served as the test cases. In each case, the inherent regioselection of catalyzed acylation was defined as that derived from the reaction where N-methylimidazole (NMI) is used as the catalyst. With both substrates, peptides were found to perturb the inherent selectivities from those observed with NMI. From the libraries, the catalysts that provide the largest deviation from NMI were subjected to optimization studies. The work sets the groundwork for studies of expanded peptide libraries and development of structure-selectivity relationships to obtain catalysts that can selectively derivatize the unique sites in stereochemically complex polyols.     

A combinatorial approach to the electron correlation problem

Starting from a path-integral formulation of quantum statistical mechanics expressed in a space of Slater determinants, we develop a method for the Monte Carlo evaluation of the energy of a correlated electronic system. The path-integral expression for the partition function is written as a contracted sum over graphs. A graph is a set of distinct connected determinants on which paths can be represented. The weight of a graph is given by the sum over exponentially large numbers of paths which visit the vertices of the graph. We show that these weights are analytically computable using combinatorial techniques, and they turn out to be sufficiently well behaved to allow stable Monte Carlo simulations in which graphs are stochastically sampled according to a Metropolis algorithm. In the present formulation, graphs of up to four vertices have been included. In a Hartree-Fock basis, this allows for paths which include up to sixfold excitations relative to the Hartree-Fock determinant. As an illustration, we have studied the dissociation curve of the N(2) molecule in a VDZ basis, which allows comparison with full configuration-interaction calculations.     

A combinatorial approach toward smart libraries of discontinuous epitopes of HIV gp120 on a TAC synthetic scaffold

We describe rapid and convenient access to smart libraries of protein surface discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a triazacyclophane scaffold molecule via CuAAC. In this way protein mimics for use as synthetic vaccines and beyond will become available.     

High-throughput kinase profiling: a more efficient approach toward the discovery of new kinase inhibitors

Selective protein kinase inhibitors have only been developed against a small number of kinase targets. Here we demonstrate that "high-throughput kinase profiling" is an efficient method for the discovery of lead compounds for established as well as unexplored kinase targets. We screened a library of 118 compounds constituting two distinct scaffolds (furan-thiazolidinediones and pyrimido-diazepines) against a panel of 353 kinases. A distinct kinase selectivity profile was observed for each scaffold. Selective inhibitors were identified with submicromolar cellular activity against PIM1, ERK5, ACK1, MPS1, PLK1-3, and Aurora A,B kinases. In addition, we identified potent inhibitors for so far unexplored kinases such as DRAK1, HIPK2, and DCAMKL1 that await further evaluation. This inhibitor-centric approach permits comprehensive assessment of a scaffold of interest and represents an efficient and general strategy for identifying new selective kinase inhibitors.     

Efficient solid-phase synthesis of peptide-based phosphine ligands: towards combinatorial libraries of selective transition metal catalysts

A new methodology for the solid-phase synthesis of peptide-based phosphine ligands has been developed. Solid supported peptide scaffolds possessing either primary or secondary amines were synthesised using commercially available Fmoc-protected amino acids and readily available Fmoc-protected amino aldehydes for reductive alkylation, in standard solid-phase peptide synthesis (SPPS). Phosphine moieties were introduced by phosphinomethylation of the free amines as the final solid-phase synthetic step, immediately prior to complexation with palladium(II), thus avoiding tedious protection/deprotection of the phosphine moieties during the synthesis of the ligands. The extensive use of commercial building blocks and standard SPPS makes this methodology well suited for the generation of solid-phase combinatorial libraries of novel ligands. Furthermore, it is possible to generate several different phosphine ligand libraries for every peptide scaffold library synthesised, by functionalising the scaffold libraries with different phosphine moieties. The synthesised ligands were characterised on solid support by conventional (31)P NMR spectroscopy and, cleaved from the support, as their phosphine oxides by HPLC, (1)H NMR, (31)P NMR and high resolution ESMS. Palladium(II) allyl complexes were generated from the resin bound ligands and to demonstrate their catalytic properties, palladium catalysed asymmetric allylic substitution reactions were performed. Good yields and moderate enantioselectivity was obtained for the selected combination of catalysts and substrate, but most importantly the concept of this new methodology was proven. Screening of ligand libraries should afford more selective catalysts.     

A new approach for the removal of protein impurities from purified biologicals using combinatorial solid-phase ligand libraries

The removal of last impurity traces from a purified protein is generally called polishing. It is an important step in downstream processing since protein impurities may generate undesirable side effects when the preparation is intended for research, diagnostic and more importantly therapeutic applications. Polishing is generally achieved by using orthogonal separation methods to previous steps, the most common being gel permeation chromatography. In spite of its polishing effectiveness, this technique suffers from a poor separation capacity and modest productivity as a result of low speed. Other approaches, for instance, based on anion exchange or on hydrophobic chromatography, that may be optimized for a given process cannot be used as generic methods. This document reports for the first time the use of a combinatorial solid-phase peptide library as a general method for the removal of impurity traces. Several examples of impurity trace removal are reported; starting material is either a pure protein spiked with serum proteins or with Escherichia coli extracts or current purified proteins still containing a small percentage of impurities. Among polished proteins are recombinant human albumin expressed in Pichia pastoris and human transferrin purified from whole plasma. This new method is used in neutral or even physiological pH and ionic strength conditions, with a remarkable capability to remove impurities. The process is as rapid as current adsorption chromatography procedures usable for the removal of a large number of protein impurities, with each one present in small amounts, such as host cell proteins.     

A versatile approach toward the ansamycin antibiotics

[STRUCTURE: SEE TEXT] The ansamycin antibiotics contain metacyclophanic macrolactams, many of which possess potent antitumor activity. Only a few total syntheses of this family of natural products have been reported, and modifications to increase potency have not been described. Therefore, a method was developed to prepare the trienomycin A core via resin-bound triphenylphosphonium salts, which serve as both a reagent and a traceless linker to afford olefinic products that undergo ring-closing metathesis (RCM) to give macrocyclic scaffolds of varying ring sizes.     

A combinatorial approach to studying protein complex composition by employing size-exclusion chromatography and proteome analysis

The genome sequences of numerous organisms are available now, but gene sequences alone do not provide sufficient information to accurately deduce protein functions. Protein function is largely dependent on the association of multiple polypeptide chains into large structures with interacting subunits that regulate and support each other. Therefore, the mapping of protein interaction networks in a physiological context is conducive to deciphering protein functions, including those of hypothetical proteins. Although several high-throughput methods to globally identify protein interactions have been reported in recent years, these approaches often have a high rate of nonspecific or artificial interactions detected. For instance, the fraction of false positives of the protein interactions identified by yeast two-hybrid assay has been predicted to be of the order of 50%. We have developed a strategy to globally map Bacillus subtilis protein-protein interactions in a physiological context by fractionating the cell lysates using size-exclusion chromatography (SEC), followed by proteome analysis. Components of both known and unknown protein complexes, multisubunits and multiproteins, have been identified using this strategy. In one case, the partners of the B. subtilis protein complex have been coexpressed in Escherichia coli, and the formation of the overexpressed protein complex has been further confirmed by a pull-down assay.     

Convergent approach toward the synthesis of the stereoisomers of C-6 homologues of 1-deoxynojirimycin and their analogues: evaluation as specific glycosidase inhibitors

A new and stereoselective strategy is developed to synthesize an appropriate template 9 to obtain C-6 homologues of 1-deoxyazasugars such as 1-deoxy-D-galactohomonojirimycin (5), 1-deoxy-4-hydroxymethyl-D-glucohomonojirimycin (6), and their enantiomers. The template 9 is also used to obtain neutral nonbasic pseudo-glyconolactam (8), C-4 amino, and methyl analogues of 1-deoxy-homonojirimycin as new analogues of 1-deoxyhomoazasugars. Compound 5 is found to be a potent and specific inhibitor to alpha-galactosidase (Ki = 1.7 microM). Similarly compounds 6 (Ki= 28 microM), ent-5 (Ki= 129 microM), and ent-6 (Ki= 12 microM) exhibited specific inhibition of beta-glucosidase.