Molecular Characteristics of Repotrectinib That Enable Potent Inhibition of TRK Fusion Proteins and Resistant Mutations

Abstract

NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to TRK inhibitors (larotrectinib, entrectinib) but often mutate, limiting the durability of response for NTRK+ patients. Next-generation inhibitors with compact macrocyclic structures (repotrectinib, selitrectinib) were designed to avoid resistance mutations. Head-to-head potency comparisons of TRK inhibitors and molecular characterization of binding interactions are incomplete obscuring a detailed understanding of how molecular characteristics translate to potency. Larotrectinib, entrectinib, selitrectinib, and repotrectinib were characterized using cellular models of wild-type TRKA/B/C fusions and resistance mutant variants with a subset evaluated in xenograft tumor models. Crystal structures were determined for repotrectinib bound to TRKA (wild-type, solvent front mutant). TKI-naïve and pretreated case studies are presented. Repotrectinib was the most potent inhibitor of wild-type TRKA/B/C fusions and was more potent than selitrectinib against all tested resistance mutations, underscoring the importance of distinct features of the macrocycle structures. Co-crystal structures of repotrectinib with wild-type TRKA and the TRKA G595R SFM variant elucidated how differences in macrocyclic inhibitor structure, binding orientation, and conformational flexibility affect potency and mutant selectivity. The SFM crystal structure revealed an unexpected intramolecular arginine sidechain interaction. Repotrectinib caused tumor regression in LMNA-NTRK1 xenograft models harboring GKM, SFM, xDFG, and GKM+SFM compound mutations. Durable responses were observed in TKI-naïve and -pretreated patients with NTRK+ cancers treated with repotrectinib (NCT03093116). This comprehensive analysis of first-and second-generation TRK inhibitors informs the clinical utility, structural determinants of inhibitor potency, and design of new generations of macrocyclic inhibitors.

NTRK Fusion in Non-Small Cell Lung Cancer: Diagnosis, Therapy, and TRK Inhibitor Resistance

“…Repotrectinib (TPX-0005) is a novel next-generation ALK, ROS1, and pan-TRK inhibitor, which is designed to overcome resistance mutations and potently inhibit wildtype TRK fusions. Repotrectinib is highly potent and selective against wildtype ALK, ROS1, and TRK fusion proteins, as well as their solvent-front substitutions in preclinical studies, including TRKA G595R, TRKB G639R, and TRKC G623R ( 97 , 98 ). Similarly, a dramatic response to repotrectinib was observed in a patient with NTRK3 fusion-positive mammary analog secretory carcinoma harboring NTRK3 G623E mutation.…”

The Challenge and Opportunity of NTRK Inhibitors in Non-Small Cell Lung Cancer

“…Similar to other TKIs, the durability of the first generation NTRK inhibitors is often limited by mutations that affect NTRK inhibitors’ binding interactions, including both primary and acquired resistance [ 29 , 30 ]. Thus, 2nd generation NTRK inhibitors were developed to overcome these resistance mutations.…”

“…The compact macrocylic structure helps accommodate the drug resistance structure from mutations in distinct regions of the active site of the TRK kinase domain and possibly compound mutations with multiple mutations in an active site. The crystal structures of larotrectinib, entrectinib, selitrectinib, and repotrectinib were studied in cellular modules of TRKA/B/C fusions and resistant variants with a subtype evaluated in xenograft tumor models [ 30 ]. In this study, the potency against TRKA/B/C fusions is highest in repotrectinib (IC50 < 0.2 nmol/L), followed by selitrectinib (1.8–3.9 nmol/L), entrectinib (0.3–1.3 nmol/L), and larotrectinib (23.5–49.4 nmol/L).…”

“…As expected, there would be drug resistance to both 1st and 2nd generation NTRK inhibitors. NTRK drug resistance can be classified into on-target resistance, which means drug resistance mediated by mutation of the TRK kinase domain, and off-target resistance, which activates bypass or downstream pathways [ 29 , 30 , 31 ].…”

Diagnosis and management of TRK fusion cancer

“…Although some data show that repotrectinib, a ROS1/TRK/ALK tyrosine kinase inhibitor, can overcome acquired resistance to prior TRK inhibition, evidence remains limited so far, generally relating only to single patients. [45][46][47] Several other next-generation TRK inhibitors are under early investigation in patients with NTRK gene fusions; these include multikinase inhibitors (cabozantinib, merestinib, and sitravatinib) and the ROS1/TRK tyrosine kinase inhibitor taletrectinib (DS-6051b). 48,49…”