| Autor: |
Zachary A. Gurard-Levin, Brian McMillan, Douglas A. Whittington, Brian Doyon, Michael D. Scholle, Jacques Ermolieff, Madhavi Bandi, Mu-Sen Liu, Alvaro Amor, William D. Mallender |
| Jazyk: |
angličtina |
| Rok vydání: |
2024 |
| Předmět: |
|
| Zdroj: |
SLAS Technology, Vol 29, Iss 5, Pp 100173- (2024) |
| Druh dokumentu: |
article |
| ISSN: |
2472-6303 |
| DOI: |
10.1016/j.slast.2024.100173 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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