High-Throughput Fluorescence-Based Activity Assay for Arginase-1

Abstract

Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z′-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.

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

“…2B ). The kinetic values are consistent with several reports using alternative assay methodologies under similar assay conditions (pH 7.4–7.5: K M 1.5 mM, k cat 190 s −1 ; 31 K M 2.3 mM, k cat 300 s −1 ; 32 K M 1.9 mM; 33 and K M 2.5 mM 14 ). Arginase activity was then measured with arginine at the K M concentration (5 mM final) in the presence of 1% DMSO to mimic the presence of compounds and revealed linear enzyme activity up to 100 nM enzyme ( Fig.…”

“…The data point to an IC 50 of 181 nM (132–247 nM) for nor-NOHA and 581 nM (504–664 nM) for ABH ( Fig. 3B ), which are in line with the reported IC 50 s of 119 nM and 554 nM, respectively, 14 using similar assay conditions, supporting the biochemical relevance of the SAMDI-MS assay.…”

“…The SAMDI-MS assay offers distinct advantages over label-dependent approaches that use colorimetric or fluorescent quenching to report on arginase activity. First, SAMDI-MS eliminates the requirement for an expensive fluorescent probe, saving on assay costs, while the MS readout provides a superior robustness (Z-factor = 0.8, relative to 0.62 for fluorescent approaches 14 ) and more than a 10-fold increase in assay window (S/B > 20, relative to 1.52 using fluorescent approaches and similar assay conditions 14 ) ( Fig. 4 ).…”

“…26 The approximate 65% false-positive and -negative hit rate due to compound autofluorescence or interference with the fluorescent probe exemplifies the caveat of optical readouts. 14 Characterizing small molecules with label-free SAMDI-MS ensures that only the most promising compounds progress toward the clinic, saving time and resources to drive drug discovery.…”

“…Recently, a fluorescent probe termed Arginase Gold was developed that exhibits a decrease in fluorescence due to quenching on conversion of arginine to ornithine. 14 While the probe enables a homogeneous, high-throughput arginase assay capable of analysis in kinetic mode, the robustness (Z-factor = 0.62) and small assay window in particular [signal-to-background (S/B) ratio = 1.52] 14 challenge the distinction between a hit and non-hit, and fluorescent assays are susceptible to false-positive and -negative results.…”