Phosphorylation State-Dependent High Throughput Screening of the c-Met Kinase.

High-throughput screening (HTS) of ~50,000 chemical compounds against phosphorylated and unphosphorylated c-Met, a tyrosine kinase receptor for hepatocyte growth factor (HGF), was carried out in order to compare hit rates, hit potencies and also to explore scaffolds that might serve as potential leads targeting only the unphosphorylated form of the enzyme. The hit rate and potency for the confirmed hit molecules were higher for the unphosphoryalted form of c-Met. While the target of small molecule inhibitor discovery efforts has traditionally been the phosphorylated form, there are now examples of small molecules that target unphosphorylated kinases.

Combined inhibition of Janus kinase 1/2 for the treatment of JAK2V617F-driven neoplasms: selective effects on mutant cells and improvements in measures of disease severity.

Purpose: Deregulation of the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is a hallmark for the Philadelphia chromosome-negative myeloproliferative diseases polycythemia vera, essential thrombocythemia, and primary myelofibrosis. We tested the efficacy of a selective JAK1/2 inhibitor in cellular and in vivo models of JAK2-driven malignancy.

Experimental Design: A novel inhibitor of JAK1/2 was characterized using kinase assays.

High-throughput determination of mode of inhibition in lead identification and optimization.

After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target.

Structural insights into the design of nonpeptidic isothiazolidinone-containing inhibitors of protein-tyrosine phosphatase 1B.

Structural analyses of the protein-tyrosine phosphatase 1B (PTP1B) active site and inhibitor complexes have aided in optimization of a peptide inhibitor containing the novel (S)-isothiazolidinone (IZD) phosphonate mimetic. Potency and permeability were simultaneously improved by replacing the polar peptidic backbone of the inhibitor with nonpeptidic moieties. The C-terminal primary amide was replaced with a benzimidazole ring, which hydrogen bonds to the carboxylate of Asp(48), and the N terminus of the peptide was replaced with an aryl sulfonamide, which hydrogen bonds to Asp(48) and the backbone NH of Arg(47) via a water molecule.

Structural basis for inhibition of protein-tyrosine phosphatase 1B by isothiazolidinone heterocyclic phosphonate mimetics.

Crystal structures of protein-tyrosine phosphatase 1B in complex with compounds bearing a novel isothiazolidinone (IZD) heterocyclic phosphonate mimetic reveal that the heterocycle is highly complementary to the catalytic pocket of the protein. The heterocycle participates in an extensive network of hydrogen bonds with the backbone of the phosphate-binding loop, Phe(182) of the flap, and the side chain of Arg(221). When substituted with a phenol, the small inhibitor induces the closed conformation of the protein and displaces all waters in the catalytic pocket.

Structure-based design and discovery of protein tyrosine phosphatase inhibitors incorporating novel isothiazolidinone heterocyclic phosphotyrosine mimetics.

Structure-based design led to the discovery of novel (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine (pTyr) mimetics that when incorporated into dipeptides are exceptionally potent, competitive, and reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). The crystal structure of PTP1B in complex with our most potent inhibitor 12 revealed that the (S)-IZD heterocycle interacts extensively with the phosphate binding loop precisely as designed in silico. Our data provide strong evidence that the (S)-IZD is the most potent pTyr mimetic reported to date.