Comprehensive and unbiased detection methods are a prerequisite for high-throughput screening (HTS) campaigns within drug discovery research. Label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been introduced as an HTS-compatible readout for biochemical test systems to support the drug discovery process. So far, reported HTS applications were based on surface-modified systems or proof-of-concept studies. We present the utilization of a MALDI-TOF-based screening platform to identify inhibitors of human cyclic GMP-AMP synthase (cGAS), a mediator of innate immune response whose aberration has been causally correlated to a number of inflammatory disorders. In this context, the development and validation of a MALDI-TOF-based activity assay is reported to demonstrate fast, robust, and accurate detection of chemical cGAS inhibition by direct quantification of the physiological reaction product cyclic GMP-ATP (cGAMP). Results from a screen of a diverse library of more than 1 million small molecules in 1536-well format against the catalytic cGAS activity are presented with excellent assay performance and data quality. Identified hits were qualified in dose-response experiments and confirmed by RapidFire-MS measurements. Conclusively, the presented data provide the first proof of applicability of direct automated MALDI-TOF MS as a readout strategy for large-scale drug discovery HTS campaigns.
Keywords: MALDI-TOF; cGAS; drug discovery; high-throughput screening; mass spectrometry.