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Development of a microplate-based scintillation proximity assay for MraY using a modified substrate
S M Solapure1, P Raphael, C N Gayathri
1AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore, India. suresh.solapure@astrazeneca.com
Journal of Biomolecular Screening
|April 1, 2005
Summary
Researchers developed a novel, rapid chemical radiolabeling method for the MraY substrate, UDP-MurNAc-pentapeptide. This enables a highly sensitive, MraY-specific assay suitable for high-throughput screening (HTS) of new antibacterial agents.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- MraY is a validated antibacterial target involved in peptidoglycan synthesis.
- Conventional assays for MraY are laborious and lack sensitivity for high-throughput screening (HTS).
Purpose of the Study:
- To develop a simple, rapid, and sensitive assay for MraY suitable for HTS.
- To chemically radiolabel the MraY substrate, UDP-MurNAc-pentapeptide, for improved assay performance.
Main Methods:
- Chemical radiolabeling of UDP-MurNAc-pentapeptide.
- Development of a microplate-based homogeneous assay using wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads.
- Validation using specific MraY inhibitors and wild-type Escherichia coli membranes.
Main Results:
- Achieved >100-fold higher specific activity compared to conventional methods.
- Developed a homogeneous assay with high yields suitable for HTS.
- Demonstrated MraY specificity, avoiding the need for deconvolution of hits.
Conclusions:
- The new radiolabeling method and homogeneous SPA assay provide a robust platform for MraY inhibitor discovery.
- This assay is advantageous over existing methods due to its simplicity, sensitivity, specificity, and use of native enzyme sources.