Abstract SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein (SMARCAL1) is an ATP-dependent SNF2 motor protein family annealing helicase that maintains genomic stability during DNA replication by catalyzing fork regression and Holliday junction migration. Loss of SMARCAL1 and the attendant DNA damage has been shown to increase tumor immunity via stimulation of the cGAS/STING pathway and suppression of PDL-1 expression, making it an attractive target for cancer immunotherapy. Here we describe development of HTS-compatible assays for screening and profiling SMARCAL1 inhibitors using the Transcreener ADP2 Assay to measure DNA-dependent ATPase activity. The Transcreener ADP2 Assay directly detects ADP in a competitive immunoassay with far-red, fluorescence polarization (FP), fluorescence intensity (FI) or time-resolved Förster-resonance-energy-transfer (TR-FRET) readouts. This format is homogenous (mix-and-read), compatible with diverse DNA substrates, and resistant to interference from screening compounds. A human SMARCAL1 construct comprising the core catalytic domain plus the HARP1, HARP2 domains, produced in BaV-infected insect cells, showed robust ATPase activity in initial testing with synthetic forked DNA (fDNA) and sheared salmon sperm DNA (sssDNA); hydrolysis of ATP was completely dependent on the presence of DNA. The Km for ATP with either substrate was 110 μM, and the Km values for sssDNA and fDNA were 30 ng/ml and 38 pM, respectively. The enzyme showed remarkable catalytic rates (kcat) of 58.6 s-1 and 81.1 s-1 with the two respective DNA substrates, enabling the use of less than 100 pM enzyme to produce a robust signal under initial velocity conditions. The kinetic parameters determined using the three different assay readouts (FP, FI and TR-FRET) were in concordance. The FP assay was validated for HTS by screening a collection of 1280 bioactives, yielding a Z’ 0.9 and identification of hits that were confirmed in dose response experiments. Citation Format: Matthew Blackburn, Robert Lowery. Development and validation of HTS assays for discovery of SMARCAL1 ATPase inhibitors abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6426.
Blackburn et al. (Fri,) studied this question.