Rapid Optimization Enabled by Single-Molecule Tracking: Discovery of a Potent RUVBL1/2 Inhibitor to Evaluate the Targeting of MYC-Driven Cancers.

RuvB-like 1 (RUVBL1) and RuvB-like 2 (RUVBL2) are AAA ATPases that form hetero-oligomeric complexes involved in diverse cellular functions. Increasing evidence implicates the RUVBL1/2 complex as an essential cofactor of MYC, with RUVBL1/2 inhibition reducing c-MYC levels in vitro. Herein, we report a potent RUVBL1/2 inhibitor discovered through a Single-Molecule Tracking (SMT)-driven SAR campaign. Compared with a biochemical ADP-Glo assay, which exhibited limited dynamic range and poor reproducibility under our experimental conditions, the live-cell high-throughput RUVBL SMT assay provided robust and reproducible potency measurements and correlated strongly with cell viability and MYC degradation. Multiparameter optimization yielded compound 18, which demonstrated improved efficacy in a MYC-dependent Burkitt lymphoma xenograft model at a significantly lower dose than the RUVBL1/2 inhibitor CB-6644. This work establishes SMT as a powerful tool to facilitate the drug discovery SAR campaigns and evaluates the therapeutic potential of RUVBL1/2 inhibition in MYC-dependent cancers.