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Results indicate that kinases from protein tyrosine kinase families mediate phosphorylation of the T-cells activation linker, which involves the recruitment of the Vav guanine nucleotide exchange factor by Itk kinase. Data show that Vav specifically binds to the T-cells activation linker tyrosine residue Y-171.

The two potent and selective Itk inhibitors that blocked T-cell activation in vitro and suppressed inflammation in a murine asthma/allergy model are described. The analysis represent the first description of selective inhibitors to probe human Itk function and its associated pathway, and support the hypothesis that Itk is a therapeutic target for immunosuppressive and inflammatory diseases.

Pim-1 has emerged as an attractive target for developing therapeutic agents for treating disorders involving abnormal cell growth, especially cancers. Herein we present lead optimization, chemical synthesis and biological evaluation of pyrazolo[1,5-a]pyrimidine compounds as potent and selective inhibitors of Pim-1 starting from a hit from virtual screening. These pyrazolo[1,5-a]pyrimidine compounds strongly inhibited Pim-1 and Flt-3 kinases. Selected compounds suppressed both the phosphorylation of BAD protein in a cell-based assay and 2-dimensional colony formation in a clonogenic cell survival assay at submicromolar potency, suggesting that cellular activity was mediated through inhibition of Pim-1. Moreover, these Pim-1 inhibitors did not show significant hERG inhibition at 30 μM concentration. The lead compound proved to be highly selective against a panel of 119 oncogenic kinases, indicating it had an improved safety profile compared with the first generation Pim-1 inhibitor SGI-1776.

A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.

Nonreceptor protein tyrosine kinases including Lck, ZAP-70, and Itk play essential roles in T-cell receptor (TCR) signaling. Gene knockout studies have revealed that mice lacking these individual kinases exhibit various degrees of immunodeficiency; however, highly selective small molecule inhibitors of these kinases as potential immunosuppressive agents have not been identified. Here we discovered two novel compounds, BMS-488516 and BMS-509744, that potently and selectively inhibit Itk kinase activity. The compounds reduce TCR-induced functions including PLCgamma1 tyrosine phosphorylation, calcium mobilization, IL-2 secretion, and T-cell proliferation in vitro in both human and mouse cells. The inhibitors suppress the production of IL-2 induced by anti-TCR antibody administered to mice. BMS-509744 also significantly diminishes lung inflammation in a mouse model of ovalbumin-induced allergy/asthma. Our findings represent the first description of selective inhibitors to probe human Itk function and its associated pathway, and support the hypothesis that Itk is a therapeutic target for immunosuppressive and inflammatory diseases.

A series of novel anilino 5-azaimidazoquinoxaline analogues possessing potent in vitro activity against p56Lck and T cell proliferation have been discovered. Subsequent SAR studies led to the identification of compound 4 (BMS-279700) as an orally active lead candidate that blocks the production of proinflammatory cytokines (IL-2 and TNFalpha) in vivo. In addition, an expanded set of imidazoquinoxalines provided several descriptive QSAR models highlighting the influence of significant steric and electronic features. The H-bonding (Met319) contribution to observed binding affinities within a tightly congeneric series was found to be significant.