P-loop Conformation Governed Crizotinib Resistance in G2032R-Mutated ROS1 Tyrosine Kinase: Clues from Free Energy Landscape

Tyrosine kinases are regarded as excellent targets for chemical drug therapy of carcinomas. However, under strong purifying selection, drug resistance usually occurs in the cancer cells within a short term. Many cases of drug resistance have been found to be associated with secondary mutations in drug target, which lead to the attenuated drug-target interactions. For example, recently, an acquired secondary mutation, G2032R, has been detected in the drug target, ROS1 tyrosine kinase, from a crizotinib-resistant patient, who responded poorly to crizotinib within a very short therapeutic term. It was supposed that the mutation was located at the solvent front and might hinder the drug binding. However, a different fact could be uncovered by the simulations reported in this study. Here, free energy surfaces were characterized by the drug-target distance and the phosphate-binding loop (P-loop) conformational change of the crizotinib-ROS1 complex through advanced molecular dynamics techniques, and it was revealed that the more rigid P-loop region in the G2032R-mutated ROS1 was primarily responsible for the crizotinib resistance, which on one hand, impaired the binding of crizotinib directly, and on the other hand, shortened the residence time induced by the flattened free energy surface. Therefore, both of the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the kinase resistance.Author Summary: Cancers can eventually confer drug resistance to the continued medication. In most cases, mutations occurred in a drug target can attenuate the binding affinity of the drugs. Here, we studied the drug resistance mechanisms of the mutations G2032R in the ROS1 tyrosine kinase in fusion-type NSCLC. It is well known that the phosphate-binding loop (P-loop) plays a vital role in the binding of competitive inhibitors in tyrosine kinases, and numerous mutations have been found occurred around the P-loop, which may affect the binding/unbinding process of a drug. Free energy surfaces were constructed to characterize the impact of the mutation to the binding/unbinding process of a well-known NSCLC drug, crizotinib. Two advanced free energy calculation methods, namely funnel based well-tempered metadynamics and umbrella sampling based absolute binding free energy calculation achieved consistent results with the experimental data, suggesting that the rigid P-loop of the mutated target was mainly responsible for the crizotinib resistance to ROS1 tyrosine kinase.