Abstract 5671: Development of HTS enzymatic assays for VPS4A and related ATPases

VSP4A and VSP4B (Vacuolar Sorting Protein) are members of the meiotic clade ATPases, which function as oligomers and use ATP hydrolysis to drive protein translocation through their central pore. VPS4B is frequently deleted in several cancers, and VPS4A has been shown to be synthetic lethal with VPS4B in cells and animals, causing cell death accompanied by induction of inflammatory cytokines and anti-tumor responses. To accelerate development of selective VPS4A inhibitors while avoiding off target effects with related ATPases, we are assembling a panel of high throughput biochemical assays using the Transcreener ADP2 assay for homogenous detection of ATPase activity with a far-red fluorescence polarization (FP) readout. Here we describe development of assays for VPS4A, VPS4B, as well as Spastin, Katanin and Fidgetin, related meiotic clade ATPases that play important roles in microtubule metabolism. Using purified recombinant enzymes, we established reaction conditions, determined kinetic parameters, and optimized the assays for initial velocity detection of ATPase activity with a Z’ value greater than 0.7, ensuring a sufficient signal window for inhibitor screening and dose response assays. We validated the VPS4A assay for HTS by screening a collection of bioactives (Tocris 2.0) and confirming two hits in dose response mode: BVT948, a PTP inhibitor, and polygodial, an antifungal that has been shown to inhibit a mitochondrial ATPase, both inhibited VPS4A and VPS4B with low micromolar potency. We then profiled BVT948 and polygodial for inhibition of Spastin, Katanin and Fidgetin: both molecules showed modest selectivity (2-5-fold) vs. Katanin and Fidgetin but had similar or greater potency for inhibition of Spastin. These assays will aid efforts to exploit the VPS4A/4B synthetic lethality therapeutically, including screening and SAR-driven hit-to-lead to minimize off-target effects with related ATPases. Citation Format: Anibal Ramos Martinez, Camdon Yemm, Ha Pham, Robert Lowery. Development of HTS enzymatic assays for VPS4A and related ATPases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5671.