The enormous success of kinase inhibitors in the treatment of cancer is being rapidly overshadowed by the emergence of drug resistance. Drug-resistant mutations increase the kinases’ binding affinity with ATP, shifting them to the active conformation, and/or introduce new steric hindrance that interferes with the inhibitor structural motifs outside the highly conserved ATP binding boundary. Most kinase inhibitors are oversized, and often have motifs close to or across the gatekeeper to the hydrophobic back pocket to gain high potency, rendering them vulnerable to the clinical gatekeeper mutations, such as ALK L1196M, EGFR T790M, and ABL T315I. Another common vulnerability of kinase inhibitors is interaction with the conserved glycine residue at the hinge C-terminal that forms a hydrophobic sandwich with the kinase β1 sheet. Kinase inhibitors often use an aromatic ring or a flat motif to go through this narrow sandwich to the solvent exposure area. Alterations at the conserved glycine commonly referred to as solvent-front mutations clash with the inhibitor motif inside the sandwich, underlying clinical resistance. For example, ALK G1202R confers a common resistance to crizotinib and other ALK inhibitors, ROS1 G2032R and TRKA G595R render resistances to crizotinib and entrectinib, respectively. Overall, the traditionally designed, oversized kinase inhibitors are destined to develop many different resistance profiles in the clinic. Continuously introducing new inhibitors to overcome new evolving mutations is becoming increasingly hard to sustain. Here, we propose targeting resistance-driven kinase active conformation with a compact molecule that is completely located inside the ATP binding boundary as a novel strategy to tackle the ever evolving mutation resistances. We designed TPX-0005, a novel three-dimensional macrocycle with a much smaller size (MW <370) than current ALK, ROS1, and TRK inhibitors in the clinic. Efficiently targeting the center of the ATP binding site avoids the steric interference with mutations outside the ATP binding boundary. As expected, TPX-0005 potently inhibited WT ALK (IC50 1.01 nM) and mutant ALKs including ALKG1202R (1.26 nM) and ALKL1196M (1.08 nM). TPX-0005 demonstrated even stronger potency against the closely related kinases ROS1 (ROS1 WT 0.07 nM and ROS1 G2032R 0.456 nM), and TRK family (TRKA 0.826 nM, TRKB 0.052 nM, and TRKC 0.096 nM). TPX-0005 potently inhibited the phosphorylation of LMNA-TRKA G595R (IC50 < 1 nM) in NIH3T3 LMNA-TRKA G595R cells. Taken together, TPX-0005 is a novel ALK/ROS1/TRK inhibitor and can effectively overcome a broad spectrum of acquired resistance mutations, especially the solvent front mutants ALK G1202R, ROS1 G2032R and TRKA G595R.

Citation Format: J. Jean Cui, Evan Rogers, Dayong Zhai, Wei Deng, Zhongdong Huang, Jeffrey Whitten, Yishan Li. Ending the endless acquired tyrosine kinase resistance mutations — Design of TPX-0005, a multi-target ALK/ROS1/TRK inhibitor with broad spectrum activity against wild-type and mutants including ALK G1202R, ROS1 G2032R and TRKA G595R. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2133.