Systematic analysis and molecular profiling of EGFR allosteric inhibitor cross-reactivity across the proto-oncogenic ErbB family kinases by integrating dynamics simulation, energetics calculation and biochemical assay.

Abstract

Human ErbB family of proteins contains four receptor tyrosine kinases (EGFR, Her2, Her3 and Her4) and has been established as a group of attractive druggable targets against diverse cancers. Over the past decades, a variety of ATP-competitive inhibitors have been discovered to target the orthosteric site of EGFR, which, however, would eventually develop acquired drug resistance due to the missense mutations T790M/C797S occurring in orthosteric site. In recent years, a number of forth-generation inhibitors have been successfully designed to overcome the T790M/C797S-induced drug resistance by targeting EGFR allosteric site instead of orthosteric site. Considering that the four proto-oncogenic ErbB kinases share a high conservation in sequence, structure and function, we herein attempted to investigate the binding potency and cross-reactivity of cognate EGFR allosteric inhibitors over noncognate Her2, Her3 and Her4 kinases--they are closely related to gynecological tumors such as ovarian cancer but no allosteric inhibitors have been reported specifically for them to date. A systematic affinity profile of 12 allosteric inhibitors and 4 orthosteric inhibitors to the 4 ErbB kinases was created by integrating dynamics simulations, energetics calculations and biochemical assays, which was then used to derive a systematic inhibitor selectivity profile for EGFR over other three kinases. It is found that allosteric and orthosteric inhibitors exhibit moderate and modest cross-reactivity across the ErbB family, respectively, but the former generally has a higher binding potency than the latter due to the additional energy cost used for inducing kinase conformational change. Moreover, most allosteric inhibitors can be sensitized by Her2 T798M gatekeeper mutation, a counterpart of EGFR T790M gatekeeper mutation that has been previously reported to cause generic drug resistance for orthosteric inhibitors.