Poster: Development and Validation of HTS Assays for Discovery of SMARCAL1 ATPase Inhibitors
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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.
In this poster, you will learn how we:
- Showed robust ATPase activity in initial testing with synthetic forked DNA (fDNA) and sheared salmon sperm DNA (sssDNA).
- Robust detection of ATPase activity with Z’ values exceeding 0.8 and 0.9.
- Achieved remarkable catalytic rates with two respective DNA substrates, enabling less than 100 pM enzyme to produce a robust signal under initial velocity conditions.
