High-Throughput Lead Discovery with Agilent RapidFire/MS Systems: Analysis of UDP-3-O-(R-3-hydroxymyristoyl)-NAcetylglucosamine Deacetylase (LpxC)

Significance of the topic

The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC is essential for lipid A biosynthesis in gram negative bacteria and a key antibiotic target. Conventional assays rely on surrogate substrates with poor kinetics and labeling requirements. High throughput native detection methods address these challenges and accelerate drug discovery.

Objectives and overview of the study

This study evaluates the Agilent RapidFire High throughput Mass Spectrometry System for screening LpxC inhibitors using the native substrate. It aims to demonstrate assay linearity, enzyme kinetics and compound screening performance under high throughput conditions.

Methodology and instrumentation

The RapidFire system couples solid phase extraction with mass spectrometry for label free, direct detection of native substrate and product. LpxC catalyzes deacetylation of UDP 3 O R 3 hydroxymyristoyl N acetylglucosamine to release a free amine and acetate. Assay development included determination of enzyme concentration linearity and Michaelis Menten kinetics. A random compound library was screened for inhibitors with follow up dose response confirmation.

Main results and discussion

Product formation exhibited a linear response to enzyme concentration and standard enzyme kinetics with a Km of 2.0 mM for the native substrate. Screening identified active compound mixtures which were deconvoluted by five point dose response assays yielding IC50 values for confirmed hits. Direct measurement of native analytes reduced false positives and simplified assay development compared to surrogate based methods.

Benefits and practical application of the method

• Label free native analyte detection without surrogate substrates
• High throughput sampling speeds of 6 to 10 seconds per sample
• Streamlined sample cleanup via integrated SPE
• Accurate kinetic measurements and reliable screening data
• Accelerated lead discovery for challenging enzyme targets

Future trends and possibilities for use

The RapidFire MS approach can be extended to other intractable enzyme assays and integrated with automated workflows and AI driven hit triaging. Miniaturization and multiplexing may further increase throughput. Expanded kinetic profiling and label free metabolomics applications offer additional discovery opportunities.

Conclusion

The Agilent RapidFire High throughput Mass Spectrometry System provides a robust label free platform for screening LpxC and other challenging targets. By enabling native substrate detection and rapid sample processing it enhances data quality and accelerates the drug discovery process.

References

1. Wang W et al A Fluorescence Based Homogeneous Assay for Measuring Activity of UDP 3 O R 3 Hydroxymyristoyl N acetylglucosamine Deacetylase Analytical Biochemistry 2001 290 338 346
2. Langsdorf E et al Screening for Antibacterial Inhibitors of the UDP 3 O R 3 Hydroxymyristoyl N Acetylglucosamine Deacetylase LpxC Using a High Throughput Mass Spectrometry Assay J Biomol Screen 2010 15 1 52 61