Discovery and characterization of novel allosteric FAK inhibitors

Focal adhesion kinase (FAK) regulates cell survival and proliferation pathways. Here we report the discovery of a highly selective series of 1,5-dihydropyrazolo[4,3-c][2,1]benzothiazines that demonstrate a novel mode of allosteric inhibition of FAK. These compounds showed slow dissociation from unphosphorylated FAK and were noncompetitive with ATP after long preincubation. Co-crystal structural analysis revealed that the compounds target a novel allosteric site within the C-lobe of the kinase domain, which induces disruption of ATP pocket formation leading to the inhibition of kinase activity. The potency of allosteric inhibition was reduced by phosphorylation of FAK. Coupled SAR analysis revealed that N-substitution of the fused pyrazole is critical to achieve allosteric binding and high selectivity among kinases.

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► We discovered novel allosteric FAK inhibitors from high-throughput screening.
► These compounds showed slow dissociation from unphosphorylated FAK.
► The inhibitor binding in allosteric site disrupted ATP pocket formation.
► N-free pyrazole compound potentially bound with hinge region in DFG-in form.
► N-substitution of the pyrazole is critical to achieve allosteric binding.