Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of multi-functional enzymes such as Polymerase (POLB) that features lyase and polymerase activities. POLB plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. Knockout and rescue studies in BRCA1/2-mutant cancer cell lines reveal that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting the need for an assay that can simultaneously report on both enzymatic activities. Here, we describe the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. We will present the development of the multiplexed assay for POLB dual enzymatic activities, optimizing for kinetically balanced conditions, and a subsequent high-throughput screen of 200,000 diverse small molecules. We will discuss the confirmation of small molecule modulators identified in the screen in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.
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