Integrated Establishment of Human VEGFR2 V916M Gatekeeper Mutation and Rational Discovery of Staralogs as Wild Type–Sparing, V916M–Selective Inhibitors Using Analog–Sensitive Kinase Technology

The V916M mutation is located in the hinge section of human VEGFR2 kinase domain and set on the peripheral region of kinase active site, which has been pre-clinically observed to be harbored in some human tumor cells such as lung adenocarcinoma H441/H1975 and liver cancer HepG2. Here, we first established the V916M as a new kinase gatekeeper mutation that can elicit acquired drug resistance to an array of ATP-competitive, reversible kinase inhibitors at the molecular level, by integrating computational kinase–inhibitor interaction analyses and experimental kinase inhibition assays. The steric hindrance caused by the linearly extended, bulky side chain of mutant Met916 residue was found as the prime factor to confer the generic drug resistance to traditional first-line VEGFR2 inhibitors, which was revealed to induce a significant displacement in inhibitor binding mode and to reduce the inhibitor binding affinity, thus considerably impairing their inhibitory activity against the kinase by up to [Formula: see text] 16-fold. Analog-sensitive (AS) kinase technology was further employed to rationally exploit the sophisticated pan-kinase inhibitor Staurosporine and its 14 commercially purchasable chemical analogs (termed Staralogs) as wild type-sparing inhibitors of VEGFR2 V916M gatekeeper mutant. By excluding those noninhibitors (IC 50 = n.d.) of VEGFR2 WT and/or VEGFR2 V916M , most exhibit molecular selectivity for mutant over wild-type kinases. The Staralog UCN01 was determined to have moderate and high inhibitory potencies against VEGFR2 WT and VEGFR2 V916M (IC 50 WT = 682 ± 57 nM and IC 50 V916 = 75 ± 6 nM), respectively, which also possesses the strongest selectivity for mutant over wild-type kinases ([Formula: see text] 9.1-fold) in all investigated Staralogs. Computational modeling revealed that the side-chain thioether moiety of Met916 can form an orthogonal noncovalent interaction system with the ligand, which consists of a vertical [Formula: see text]–[Formula: see text] stacking with the aromatic ring of UCN01 nucleus and a horizontal sulfur-containing hydrogen bond with the 7-hydroxyl group of UCN01 substituent, thus conferring additional affinity to VEGFR2 V916M and selectivity for VEGFR2 V916M over VEGFR2 WT .