Abstract NTRK chromosomal rearrangements yield oncogenic TRK fusion proteins that are sensitive to first-generation TRK inhibitors (larotrectinib, entrectinib) but the emergence of NTRK mutations limits their efficacy. Next-generation TRK inhibitors (repotrectinib, selitrectinib) have compact macrocyclic structures designed to limit the susceptibility to mutations. However, a detailed understanding of mutant potency and precise binding interactions are lacking. TRK inhibitors were evaluated in cellular models expressing TRKA/B/C fusions with wild-type (WT) and resistance mutations: solvent front (SFM), gatekeeper (GKM), activation loop (xDFG), and compound mutations. In cell proliferation assays, differential potencies against wild type TRKA/B/C fusions were observed: larotrectinib (IC50 23.5 - 49.4 nM), entrectinib (IC50 0.3 - 1.3 nM), selitrectinib (IC50 1.8 - 3.9 nM), and repotrectinib (IC50 < 0.2 nM). First generation TRK inhibitors with extended structures had reduced potency against resistance mutations. Larotrectinib had minimal activity (IC50 >600 nM) against all TRK mutations and entrectinib had >400-fold decreased against all mutations except GKM where there was a range of potencies (IC50 < 0.2 - 60.4 nM). Repotrectinib and selitrectinib were less affected by resistance mutations however repotrectinib was approximately 10-fold more potent than selitrectinib against SFM and compound mutations and 100-fold more potent against GKM. For TRKA/B/C xDFG mutations, repotrectinib had moderate potency (IC50 11.8 - 67.6 nM) while selitrectinib was less potent (IC50 124 - 341 nM). Co-crystal structures of repotrectinib with TRKA and TRKA harboring a SFM provide insight into how subtle differences in macrocyclic inhibitor structure and conformational profiles affect potency and mutant selectivity. Analysis of the first protein structure of a kinase harboring a common SFM (TRKA G595R) revealed unexpected intramolecular interactions which provide insight into its prevalence. Repotrectinib, but not selitrectinib, caused tumor regression in LMNA-NTRKA xenograft models harboring GKM, SFM, or GKM+SFM compound mutations. In the clinic, tumor regression was observed with repotrectinib treatment of a larotrectinib-resistant cholangiocarcinoma patient with both LMNA-TRKA GKM and SFM mutations. Repotrectinib has shown responses in patients with TRK driven tumors with or without resistant mutations in the ongoing global registrational TRIDENT-1 study and has been granted 3 fast track designations. Taken together, the current data characterizes TRK inhibitor potency against resistance mutations and highlights structural characteristics of repotrectinib that enable potent inhibition of TRK proteins and evasion of drug resistance mediated by TRK mutations. Citation Format: Alexander Drilon, Dayong Zhai, Evan Rogers, Wei Deng, Xi Chen, Paul Sprengeler, Siegfried H. Reich, Brion W. Murray. Molecular characteristics of repotrectinib that enable potent inhibition of TRK fusion proteins and broad mutant selectivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1119.