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Development of a Novel Label-Free and High-Throughput Arginase-1 Assay Using Self-Assembled Monolayer Desorption Ionization Mass Spectrometry.
- Source :
-
SLAS discovery : advancing life sciences R & D [SLAS Discov] 2021 Jul; Vol. 26 (6), pp. 775-782. Date of Electronic Publication: 2021 Mar 23. - Publication Year :
- 2021
-
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 IC <subscript>50</subscript> measured to be in line with reported results (IC <subscript>50</subscript> = 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.
- Subjects :
- Arginase analysis
Arginase antagonists & inhibitors
Arginine metabolism
Biological Assay
Biotin metabolism
Dimerization
Drug Discovery methods
Enzyme Activation drug effects
Enzyme Inhibitors analysis
Enzyme Inhibitors isolation & purification
Enzyme Inhibitors pharmacology
Humans
Kinetics
Ornithine metabolism
Small Molecule Libraries chemistry
Arginase metabolism
High-Throughput Screening Assays methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
Subjects
Details
- Language :
- English
- ISSN :
- 2472-5560
- Volume :
- 26
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- SLAS discovery : advancing life sciences R & D
- Publication Type :
- Academic Journal
- Accession number :
- 33754845
- Full Text :
- https://doi.org/10.1177/24725552211000677