Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry

Abstract

Arginase-1, an enzyme that catalyzes the reaction of L-arginine to L-ornithine, is implicated in the tumor immune response and represents an interesting therapeutic target in immuno-oncology. Initiating arginase drug discovery efforts remains a challenge due to a lack of suitable high-throughput assay methodologies. This report describes the combination of self-assembled monolayers and matrix-assisted laser desorption ionization mass spectrometry to enable the first label-free and high-throughput assay for arginase activity. The assay was optimized for kinetically balanced conditions and miniaturized, while achieving a robust assay (Z-factor > 0.8) and a significant assay window [signal-to-background ratio > 20] relative to fluorescent approaches. To validate the assay, the inhibition of the reference compound nor-NOHA (Nω-hydroxy-nor-L-arginine) was evaluated, and the IC50 measured to be in line with reported results (IC50 = 180 nM). The assay was then used to complete a screen of 175,000 compounds, demonstrating the high-throughput capacity of the approach. The label-free format also eliminates opportunities for false-positive results due to interference from library compounds and optical readouts. The assay methodology described here enables new opportunities for drug discovery for arginase and, due to the assay flexibility, can be more broadly applicable for measuring other amino acid–metabolizing enzymes.

Label-Free Screening of SARS-CoV-2 NSP14 Exonuclease Activity Using SAMDI Mass Spectrometry

“…The SAMDI-MS technology has recently proven to be a powerful approach for measuring SARS-CoV-2 3CLpro activity, 32 including simultaneously reporting on SARS-CoV-2 3CLpro and rhinovirus HRV3C protease activities. 33 In addition to the SARS-CoV-2 target, the SAMDI-MS technology has been reported to characterize dozens of biochemical activities, including posttranslational modifying enzymes, 24,34–37 RNA-modifying proteins, 30 and arginase, 29 highlighting its flexibility and broad drug discovery solutions. SAMDI-MS offers several solutions over other MS approaches, including traditional MALDI.…”

“…Moreover, the platform is amenable to any buffer system, including detergents, high salts, carrier proteins, organic additives, and even cell lysates. 19–21 SAMDI-MS has been reported to measure biochemical activities on peptides, 22–24 DNA, 25 and small molecules, 26–29 and has recently been validated on RNA substrates. 30 A major benefit of SAMDI-MS for RNA substrates is the ability to monitor the purity and integrity of RNA in every reaction well, where RNases present in most labs and/or enzyme preps can lead to RNA degradation and false positives/negatives in label-based approaches such as Forster resonance energy transfer.…”

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

“…Second, there remains a need for high-throughput and robust screening assays suitable for identifying ligand–target interactions. Here we expand the capabilities of the SAMDI technology, extensively reported as a powerful label-free and high-throughput assay for measuring biochemical and chemical reactions, 9,18–28 to offer a high-throughput ASMS assay with key benefits over traditional ASMS approaches.…”

Enzyme Cascade with Horseradish Peroxidase Readout for High-Throughput Screening and Engineering of Human Arginase-1

“…However, this method also requires sample treatment under harsh conditions and is limited to endpoint kinetics. 23 Mass spectroscopy-based arginase activity assays have been reported, relying on hydrophilic interaction chromatography combined with Rapid-Fire 24 or self-assembled monolayer desorption ionization (SAMDI); 25 however, these assays require large infrastructures and rely on multistep chromatographic separation and derivatization steps. Recently, Grobben et al 26 developed a fluorescent dye that was quenched by L-ornithine, resulting in fluorescence enhancement when the enzyme was inhibited.…”