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Related Experiment Video

Updated: Jun 16, 2025

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Published on: November 1, 2019

Microplate-Based Enzymatic Activity Assay Protocol Powered by Activity-Based Probes.

Exequiel O J Porta¹, Karunakaran Kalesh^2,3, Jaime A Isern⁴

¹Department of Chemistry, Durham University, Durham, UK. e.porta@ucl.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)

|June 14, 2025

Summary

This summary is machine-generated.

This study introduces a novel method combining activity-based protein profiling (ABPP) with microplate assays for rapid enzyme inhibitor discovery. This approach accelerates the identification and characterization of potential drug candidates by efficiently screening compound libraries against target enzymes.

Keywords:

Activity-Based Probes Activity-Based Protein Profiling Chemical Probes Drug Discovery Inhibitors Microplates Plate-Based Assay Screening

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Last Updated: Jun 16, 2025

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

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Published on: November 1, 2019

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A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Published on: April 16, 2021

Area of Science:

Biochemistry
Chemical Biology
Drug Discovery

Background:

Enzyme identification and understanding inhibition mechanisms are crucial for drug discovery.
Current methods can be time-consuming and lack efficiency in screening inhibitors.

Purpose of the Study:

To develop and present a novel, accelerated methodology for enzyme inhibitor discovery.
To combine activity-based protein profiling (ABPP) with microplate assay technology for enhanced efficiency.

Main Methods:

Utilized competitive ABPP with a fluorophosphonate (FP)-based probe and pig liver esterase (PLE) as a model enzyme.
Immobilized streptavidin-tagged PLE onto biotinylated assay plates for parallel screening.
Employed a commercially derived compound library for inhibitor identification.

Main Results:

Successfully demonstrated a method for rapid identification of enzyme inhibitors.
Enabled simultaneous estimation of IC50 values for identified inhibitors.
Provided a comprehensive protocol for probe synthesis, enzyme preparation, and assay execution.

Conclusions:

The developed plate-based competitive ABPP assay offers a robust platform for discovering novel enzyme inhibitors.
This technique expedites the drug discovery process and deepens understanding of inhibitor-enzyme interactions.
The methodology is applicable to enzymes with unknown structures or substrate specificities.