RAD51抑制剂JKYN-1的发现及其在非小细胞肺癌临床前模型中抗癌活性的表征

BACKGROUND: RAD51 is a recombinase that plays a central role in homologous recombination-mediated double-strand DNA break repair and is required for the maintenance of genome stability. In addition to this canonical function, RAD51 contributes to the tolerance of replication stress, a phenotype frequently observed in tumor cells, thereby establishing RAD51 as a potential pharmacologic target in multiple malignancies including non-small cell lung cancer (NSCLC). The development of direct small-molecule RAD51 inhibitors has been limited by suboptimal potency and solubility. Here, we report the discovery of JKYN-1, an optimized RAD51 inhibitor, and the characterization of its single-agent and combinatorial antitumor activity in preclinical models of NSCLC. METHODS: Using molecular docking, JKYN-1 was discovered through virtual chemical modifications of the previously reported RAD51 inhibitor IBR120. Target engagement in cells was evaluated by the cellular thermal shift assay (CETSA) and immunoblotting. CellTiter-Glo assay was used to measure cytotoxicity in cell lines and patient-derived organoids (PDOs) of NSCLC after 6-day treatment. RESULTS: JKYN-1 (patent application # CA3204011A1) is a derivative of IBR120, with improved solubility and potency. As assessed by CETSA, JKYN-1 bound to and destabilized RAD51 in H1975. Using immunoblotting, we demonstrated downregulation of RAD51 in response to JKYN-1 treatment in a concentration-dependent manner. Compared to the classical RAD51 inhibitor B02, JKYN-1 demonstrated higher cytotoxicity in A549 and H1975 cell lines with over 50% reduction in the IC 50 values (H1975, 3.2 vs 8.3 μM; A549, 4.9 vs >10 μM). In a panel of 8 EGFR -mutated NSCLC PDOs, JKYN-1 and its more soluble mesylate salt displayed sub-micromolar IC 50 values (0.1-0.88 μM) as opposed to B02 (2.4 to >10 μM). We subsequently evaluated the synergistic potential of JKYN-1 in combination with the EGFR inhibitor osimertinib in osimertinib-resistant models. Combination with JKYN-1 resulted in > 4-fold reduction in the IC 50 of osimertinib in the laboratory-evolved osimertinib-resistant H1975 cells, and > 2-fold reduction in the LPTO357 PDO. Mechanistically, both JKYN-1 and osimertinib induced downregulation of RAD51 in cell lines and PDOs in a concentration-dependent manner as assessed by immunoblotting, explaining in part the synergistic combinatorial activity. CONCLUSIONS: Our data identify JKYN-1 and its mesylate salt as promising RAD51 inhibitors with superior anticancer activity compared to existing inhibitors in preclinical models of NSCLC. Moreover, JKYN-1 can potentially overcome resistance to osimertinib in a subset of NSCLC models.