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.     

Reversing the discovery paradigm: a new approach to the combinatorial discovery of fluorescent chemosensors

We report here a new approach to the discovery of fluorescent chemosensors in which a new signaling mechanism allows a core fluorophore to be used in a combinatorial search for new binding events, thus reversing the reigning discovery paradigm.     

High-throughput approaches for the discovery and optimization of new olefin polymerization catalysts

The discovery of new olefin polymerization catalysts is currently a time-intensive trial-and-error process with no guarantee of success. A fully integrated high-throughput screening workflow for the discovery of new catalysts for polyolefin production has been implemented at Symyx Technologies. The workflow includes the design of the metal-ligand libraries using custom-made computer software, automated delivery of metal precursors and ligands into the reactors using a liquid-handling robot, and a rapid primary screen that serves to assess the potential of each metalligand-activator combination as an olefin polymerization catalyst. "Hits" from the primary screen are subjected to secondary screens using a 48-cell parallel polymerization reactor. Individual polymerization reactions are monitored in real time under conditions that provide meaningful information about the performance capabilities of each catalyst. Rapid polymer characterization techniques support the primary and secondary screens. We have discovered many new and interesting catalyst classes using this technology.     

A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors

Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS.     

A new approach to mineralization of biocompatible hydrogel scaffolds: an efficient process toward 3-dimensional bonelike composites

As a first step toward the design and fabrication of biomimetic bonelike composite materials, we have developed a template-driven nucleation and mineral growth process for the high-affinity integration of hydroxyapatite with a poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel scaffold. A mineralization technique was developed that exposes carboxylate groups on the surface of cross-linked pHEMA, promoting high-affinity nucleation and growth of calcium phosphate on the surface, along with extensive calcification of the hydrogel interior. Robust surface mineral layers a few microns thick were obtained. The same mineralization technique, when applied to a hydrogel that is less prone to surface hydrolysis, led to distinctly different mineralization patterns, in terms of both the extent of mineralization and the crystallinity of the apatite grown on the hydrogel surface. This template-driven mineralization technique provides an efficient approach toward bonelike composites with high mineral-hydrogel interfacial adhesion strength.     

High-throughput prediction of blood-brain partitioning: a thermodynamic approach

A high-throughput in silico screening tool for potentially CNS active compounds was developed on the basis of the correlation of solvation free energies and blood-brain partitioning (log(cbrain/cblood) = log BB) data available from experimental sources. Utilizing a thermodynamic approach, solvation free energies were calculated by the fast and efficient generalized Born/surface area continuum solvation model, which enabled us to evaluate more than 10 compounds/min. Our training set involved a structurally diverse set of 55 compounds and yielded a function of log BB = 0.035Gsolv + 0.2592 (r = 0.85, standard error 0.37). Calculation of solvation free energies for 8700 CNS active compounds (CIPSLINE database) revealed that Gsolv is higher than -50 kJ/mol for the 96% of these compounds which can be used as suitable criteria for the identification of compounds preferable for CNS penetration.     

High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detection

Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC(50) value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases.     

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.     

Pharmacophore and docking-based virtual screening approach for the design of new dual inhibitors of Janus kinase 1 and Janus kinase 2

Janus kinase 1 and 2, non-receptor protein tyrosine kinases, are implicated in various cancerous diseases. Involvement of these two enzymes in the pathways that stimulate cell proliferation in cancerous conditions makes them potential therapeutic targets for designing new dual-targeted agents for the treatment of cancer. In the present study, two separate pharmacophore models were developed and the best models for JAK1 (AAADH.25) and JAK2 (ADRR.92) were selected on the basis of their external predictive ability. Both models were subjected to a systematic virtual screening (VS) protocol using a PHASE database of 1.5 million molecules. The hits retrieved in VS were investigated for ADME properties to avoid selection of molecules with a poor pharmacokinetic profile. The molecules considered to be within the range of acceptable limits of ADME properties were further employed for docking simulations with JAK1 and JAK2 proteins to explore the final hits that possess structural features of both pharmacophore models as well as display essential interactions with both of them. Thus, the new molecules obtained in this way should show inhibitory activity against JAK1 and JAK2 and may serve as novel therapeutic agents for the treatment of cancerous disease conditions.     

Searching for cyclin-dependent kinase inhibitors using a new variant of the cope elimination

beta-Piperidinoethylsulfides are oxidized by m-chloroperbenzoic acid to intermediates containing both N-oxide and sulfone functions. These undergo a Cope-type elimination to a vinylsulfone that can be captured by amines to afford beta-aminoethylsulfones. When a beta-aminoethylsulfone group is linked to the 4-position of a phenyl group attached at N-2 of O6-cyclohexylmethylguanine, the resulting derivatives are inhibitors of the cyclin-dependent kinase CDK2. One of the most potent inhibitors (IC50 = 45 nM) contained a N-3-hydroxypropyl group on the aminoethylsulfonyl substituent. The crystal structure of this inhibitor bound to CDK2/cyclin A was determined and shows an unusual network of hydrogen bonds. The synthetic methodology developed can be utilized in multiple-parallel format and has numerous potential applications in medicinal chemistry.     

Development of new methods toward efficient immobilization of chiral catalysts

BINOL moieties are efficiently immobilized onto the surface of a micelle-derived spherical polymer and a monolayer-protected Au cluster (Au-MPC). Ti-BINOLate complexes generated from the BINOL immobilized polymer and Au-MPC are found to promote catalytic asymmetric alkylation of benzaldehyde with Et₂Zn to afford the adduct in up to 84% ee and 86% ee, respectively. Au-MPC-supported multicomponent asymmetric catalyst was prepared and used in the Michael reaction of 2-cyclohexen-1-one with dibenzyl malonate affording the adduct in up to 67% yield and 98% ee.     

Cyclotrimerization of unsymmetrically bromo-substituted diynes: toward the synthesis of potential selective inhibitors of tyrosine kinase 2

A study on transition metal catalyzed alkyne cyclotrimerization of unsymmetrically bromo-substituted diynes with ethynyltrimethylsilane was carried out to prepare bicyclic bromobenzene key intermediates for the total synthesis of five potential tyrosine kinase 2 inhibitors. Two different pre-catalysts (Cp*RuCl(cod) and [Rh(cod)₂]BF₄/BINAP) and different reaction conditions have been examined.     

An efficient approach toward the synthesis of the A/B rings of ouabain

The synthesis of the highly functionalized A/B ring related to ouabain has been accomplished efficiently from commercially available α-tetralone. A key Birch reductive alkylation allows the building of an angularly substituted decalone that was adequately functionalized to produce the intermediate 2c.     

A combinatorial approach toward the generation of ambiphilic peptide-based inhibitors of protein:geranylgeranyl transferase-1

A combinatorial synthesis of oligopeptide analogues and their evaluation as protein:geranylgeranyl transferase inhibitors is presented. The combinatorial strategy is based on the random mutation, in each new generation, of one of any of the four amino acid building blocks of which the most effective compounds of the previous generation are assembled. In this way, a progressive improvement of the average inhibitory activity was observed until the fifth generation. The most active inhibitors were found to inhibit PGGT-1 in the low micromolar range (IC(50): 3.8-8.1 microM).     

Toward a fragment-based approach to MMPs inhibitors: an expedite and efficient synthesis of N-hydroxylactams

Matrix metalloproteinases (MMPs), a class of zinc-enzymes over-activated in many pathologies, such as arthritis and cancer, can be efficiently inhibited by a variety of molecules bearing zinc-binding groups (ZBGs). The hydroxamic acid moiety represents one of the most potent and widely exploited ZBG but the poor target selectivity and in vivo toxicity have tempered the initial enthusiasm for this class of potential therapeutics. These drawbacks might be circumvented, at least in part, by increasing the structural constraints around the hydroxamic moiety. Following this strategy we designed and prepared N-hydroxylactam molecules of different size through a synthetic protocol based on a ring closing metathesis amenable to a fragment-based approach potentially leading to a large molecular diversity.     

Design of new plasmepsin inhibitors: a virtual high throughput screening approach on the EGEE grid

Though different species of the genus Plasmodium may be responsible for malaria, the variant caused by P. falciparum is often very dangerous and even fatal if untreated. Hemoglobin degradation is one of the key metabolic processes for the survival of the Plasmodium parasite in its host. Plasmepsins, a family of aspartic proteases encoded by the Plasmodium genome, play a prominent role in host hemoglobin cleavage. In this paper we demonstrate the use of virtual screening, in particular molecular docking, employed at a very large scale to identify novel inhibitors for plasmepsins II and IV. A large grid infrastructure, the EGEE grid, was used to address the problem of large computation resources required for docking hundreds of thousands of chemical compounds on different plasmepsin targets of P. falciparum. A large compound library of about 1 million chemical compounds was docked on 5 different targets of plasmepsins using two different docking software, namely FlexX and AutoDock. Several strategies were employed to analyze the results of this virtual screening approach including docking scores, ideal binding modes, and interactions to key residues of the protein. Three different classes of structures with thiourea, diphenylurea, and guanidino scaffolds were identified to be promising hits. While the identification of diphenylurea compounds is in accordance with the literature and thus provides a sort of "positive control", the identification of novel compounds with a guanidino scaffold proves that high throughput docking can be effectively used to identify novel potential inhibitors of P. falciparum plasmepsins. Thus, with the work presented here, we do not only demonstrate the relevance of computational grids in drug discovery but also identify several promising small molecules which have the potential to serve as candidate inhibitors for P. falciparum plasmepsins. With the use of the EGEE grid infrastructure for the virtual screening campaign against the malaria causing parasite P. falciparum we have demonstrated that resource sharing on an eScience infrastructure such as EGEE provides a new model for doing collaborative research to fight diseases of the poor.     

Kinase-catalyzed modification of gold nanoparticles: a new approach to colorimetric kinase activity screening

Peptide-stabilized gold nanoparticles have been enzymatically biotinylated by a kinase-catalyzed reaction using biotin-ATP as a cosubstrate. Upon mixing with avidin-modified particles, solutions of biotinylated particles change color from red to blue, indicating aggregation of particles. On the basis of this reaction, we have developed a simple colorimetric test to monitor kinase inhibitor activity.