Biomedical subjects

Ilya Okun

Publications and source records attributed to Ilya Okun.

5 recordsLinked to original sources

PubMed2006

Screening for caspase-3 inhibitors: effect of a reducing agent on identified hit chemotypes.

When studying cysteinyl proteases in general and caspases in particular, it is generally accepted that a reaction buffer must contain a reducing agent to prevent essential cysteinyl groups from spontaneous oxidation. Dithiothreitol (DTT) and beta-mercaptoethanol (beta-MCE) are 2 of the most broadly used reducing agents. While screening a library of small molecules against caspase-3, the authors have found that the nature of the reducing agent used, DTT or beta-MCE, dramatically affects screening results and leads to identification of nonoverlapping hits. Screening in DTT-containing buffer revealed few novel classes of small molecules that selectively and reversibly inhibit caspase-3 but failed to identify isatin sulfonamides recently found to be potent and selective caspase-3 inhibitors (false negatives). On the other hand, screening in the presence of beta-MCE failed to identify a series of hit compounds, 1,3-dioxo-2,3-dichloro-1H-pyrrolo[3,4-c]quinolines, discovered with DTT, whereas isatin sulphonamides in these conditions exhibited strong caspase-3 inhibition. In this work, the authors show that thiol-containing reducing agents can affect catalytic activity of caspase-3 and modify its thermostability in a redox-potential-independent manner. The authors speculate that the differential structural modifications of caspase-3 seen with different reducing agents represent structurally different caspase-3 conformations and are responsible for its differential sensitivity to small molecules of different chemotypes. Hence, selection of the reducing agent may dramatically affect the quality of high-throughput screening campaigns.

Caspase 3↗

PubMed2006

Screening for caspase-3 inhibitors: a new class of potent small-molecule inhibitors of caspase-3.

From the authors' 650,000 compound collection, they have selected approximately 15,000 potential small-molecule protease inhibitors, which were subjected to high-throughput screening against caspase-3. The screening yielded a series of hits that belong to 11 different scaffolds. Based on the structure of one of the hits, a new class of the small-molecule inhibitors with a double electrophilic warhead, 8-sulfonyl-pyrrolo[3,4-c]quinoline-1,3-diones (SPQ), was synthesized and tested in follow-up mechanistic and anti-apoptosis assays. Mechanistic analysis of a representative compound of this class, CD-001-0011, showed that the compound exhibited a high potency (IC (50)=130 nM), was reversible though noncompetitive, and had a broad selectivity profile to other caspases belonging to groups I to III. The compound was effective in preventing staurosporine induced apoptosis in a few cell lines and retinoic acid-induced apoptosis in zebrafish.

Animals↗

PubMed2004

Efficient optimization strategy for marginal hits active against abl tyrosine kinases.

Primary high-throughput screening of commercially available small molecules collections often results in hit compounds with unfavorable ADME/Tox properties and low IP potential. These issues are addressed empirically at follow-up lead development and optimization stages. In this work, we describe a rational approach to the optimization of hit compounds discovered during screening of a kinase focused library against abl tyrosine kinase. The optimization strategy involved application of modern chemoinformatics techniques, such as automatic bioisosteric transformation of the initial hits, efficient solution-phase combinatorial synthesis, and advanced methods of knowledge-based libraries design.

Algorithms↗

PubMed2002

Serotonin antagonist profiling on 5HT2A and 5HT2C receptors by nonequilibrium intracellular calcium response using an automated flow-through fluorescence analysis system, HT-PS 100.

Characterization of the potencies of agonists and antagonists in cell-based assays can be complicated by nonequilibrium conditions of functional response. We assessed the potencies of a series of serotonin (5HT) antagonists by inhibition of intracellular calcium response in HEK 293 cells expressing 5HT(2A) or 5HT(2C) receptors. An automated system, HT-PS 100, was used to profile the antagonists in two experimental setups: coadministration of agonist and antagonist to cells and preincubation of the cells with antagonist prior to agonist administration. We showed that the antagonist potencies (pIC(50) values) determined in the preincubation configuration were close to or exceeded those measured in the coadministration configuration. Closeness of the potencies determined in the two configurations supposedly reflected a rapid antagonist-receptor equilibration, whereas a significantly higher preincubation potency implied slow antagonist dissociation from the receptor. Schild analysis of the inhibition of serotonin-induced cell response by a competitive 5HT(2A) antagonist, spiperone, showed a typical competitive inhibition pattern when both the agonist and antagonist were applied simultaneously. Contrary to this, an insurmountable diminishing of the maximal cell response to serotonin was observed when the cells were preincubated with spiperone. We conclude that a combination of the coadministration and preincubation experimental setups is necessary for appropriate mechanistic interpretation and quantitative assessment of the antagonist activity when using transient functional readouts.

Calcium↗

PubMedDate not supplied

Property-based design of GPCR-targeted library.

The design of a GPCR-targeted library, based on a scoring scheme for the classification of molecules into "GPCR-ligand-like" and "non-GPCR-ligand-like", is outlined. The methodology is a valuable tool that can aid in the selection and prioritization of potential GPCR ligands for bioscreening from large collections of compounds. It is based on the distillation of knowledge from large databases of GPCR and non-GPCR active agents. The method employed a set of descriptors for encoding the molecular structures and by training of a neural network for classifying the molecules. The molecular requirements were profiled and validated by using available databases of GPCR- and non-GPCR-active agents [5736 diverse GPCR-active molecules and 7506 diverse non-GPCR-active molecules from the Ensemble Database (Prous Science, 2002)]. The method enables efficient qualification or disqualification of a molecule as a potential GPCR ligand and represents a useful tool for constraining the size of GPCR-targeted libraries that will help speed up the development of new GPCR-active drugs.

Databases, Factual↗