Abstract
SignificanceWhile immensely successful, drugging kinases by active site inhibitors has faced major challenges. Selectivity issues leading to side effects and emergence of resistance mutations rendered treatments targeting active sites ineffective. Double-drugging via active and allosteric sites is a recently developed approach to overcome these obstacles. Using Aurora A and Abelson kinase, we provide a quantitative biophysical evaluation of double-drugging by rationally selecting inhibitor combinations with positive cooperativity. The results shed light on the interplay of kinase conformational equilibria and inhibitor-dose requirements for effective inhibition. Due to our rational selection of a positively cooperative drug combination for Abl, we deliver a fully closed, inactive Abl structure, including regulatory SH3 and SH2 domains. Collectively, this biophysical framework aids future rational double-drug designs.
Selective orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as “double-drugging”, has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Förster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing cooperativity. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe a fully closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light on the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies.