Abstract：
Polymerase β (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes 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. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed 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 highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.
摘要：
聚合酶β（POLB），具有裂解酶和聚合酶的双重功能，在碱基切除修复（BER）途径中发挥关键作用，以维持基因组的稳定性。BRCA1/2突变癌细胞系中的POLB敲除和挽救研究表明，PARP抑制剂观察到的合成致死相互作用需要抑制裂解酶和聚合酶活性，强调POLB是一个有价值的治疗靶点。筛选酶抑制剂的传统生化测定集中在单一底物与产物的关系上,并限制了对利用多种底物或催化多步反应的酶如POLB的综合分析。该报告描述了第一个基于高通量质谱的屏幕，该屏幕使用双工自组装单层解吸电离（SAMDI）质谱方法在单个测定中测量POLB的两种不同的生化活性。开发了针对动力学平衡条件进行优化的POLB双酶活性的多重测定，并以双链形式筛选了200,000个不同小分子的集合。在传统的基于荧光的聚合酶链置换测定和使用SAMDI亲和选择质谱(ASMS)的正交无标记结合测定中确认了在筛选中鉴定的小分子调节剂。这项工作证明了高通量质谱方法在药物发现中的灵活性，并强调了SAMDI技术的新应用，为多路高通量筛选开辟了新途径。
