High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

• Published in: SLAS discovery Vol. 26; no. 8; pp. 974 - 983
• Main Authors: Scholle, Michael D., McLaughlin, Doug, Gurard-Levin, Zachary A.
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA Elsevier Inc 01.09.2021; SAGE Publications
• Subjects: 3C Viral Proteases - chemistry; affinity selection; ASMS; COVID-19 - virology; COVID-19 Drug Treatment; Drug Discovery; High-Throughput Screening Assays; HTS; Humans; label-free; Ligands; Mass Spectrometry; Rhinovirus - drug effects; SAMDI; SARS-CoV-2 - drug effects; SARS-CoV-2 - pathogenicity; Small Molecule Libraries - chemistry; Small Molecule Libraries - isolation & purification; Small Molecule Libraries - therapeutic use; label-free; SAMDI; drug discovery; ASMS; HTS; affinity selection
• ISSN: 2472-5552; 2472-5560
• DOI: 10.1177/24725552211023211

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.