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

Yvonne Grobben1, Nicole Willemsen-Seegers1, Joost C M Uitdehaag1

  • 1Netherlands Translational Research Center B.V., Oss, The Netherlands.

SLAS Discovery : Advancing Life Sciences R & D
|May 19, 2020
PubMed
Summary

We developed a novel fluorescence assay to discover Arginase-1 inhibitors, crucial for cancer immunotherapy. This high-throughput screening method enables the identification of small molecules targeting Arginase-1 activity in the tumor microenvironment.

Keywords:
ArginaseL-argininecancer immunotherapyhigh-throughput screening

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Area of Science:

  • Biochemistry
  • Pharmacology
  • Immunology

Background:

  • Arginase-1 is a key regulator of T-cell immunity within the tumor microenvironment.
  • Arginase-1 activity impacts cancer immunotherapy efficacy.
  • Targeting Arginase-1 with small molecules presents a therapeutic opportunity.

Purpose of the Study:

  • To develop a novel, homogeneous fluorescence-based activity assay for Arginase-1.
  • To enable high-throughput screening (HTS) for small-molecule Arginase-1 inhibitors.
  • To validate the assay's performance and utility in drug discovery.

Main Methods:

  • A homogeneous fluorescence-based assay utilizing the Arginase Gold probe was developed.
  • The assay measures L-arginine conversion to L-ornithine via a decrease in fluorescence signal.
  • Assay performance was validated through side-by-side comparisons with a colorimetric assay and automation for HTS.

Main Results:

  • The fluorescence assay demonstrated comparable potency and rank order to a colorimetric assay for reference inhibitors.
  • The assay was successfully automated for HTS in a 384-well format with a good Z'-factor.
  • The assay proved effective for hit confirmation and studying inhibitor binding kinetics.

Conclusions:

  • A novel, robust, and automatable fluorescence-based assay for Arginase-1 activity has been established.
  • This assay is suitable for high-throughput screening of small-molecule libraries to identify potential cancer immunotherapy drugs.
  • The assay facilitates the study of Arginase-1 inhibitor binding kinetics, aiding in drug development.