Ultrafast Histone Deacetylase Selectivity Screening Using the Agilent RapidFire High-Throughput Mass Spectrometry System

Importance of the Topic

The ability to rapidly screen histone deacetylase isoforms for selectivity is critical in early drug discovery to minimize off-target effects and accelerate lead optimization.

Study Objectives and Overview

This study developed full biochemical assays for four deacetylase enzymes (SIRT1, SIRT3, HDAC1, HDAC6) and evaluated the use of Agilent RapidFire high-throughput mass spectrometry in multiplexed selectivity screening of reference inhibitors.

Methodology and Instrumentation

Enzyme assays were performed at room temperature using acetylated peptide substrates representative of p53 and histone 4. Reactions were carried out in Tris buffer with NAD+ for sirtuins and quenched with formic acid. Multiplexed samples were pooled post-reaction and analyzed at high speed by solid phase extraction (SPE) coupled to tandem mass spectrometry.

Instrumentation Used

• Agilent RapidFire 365 SPE system with C4 cartridge
• Agilent 6490 iFunnel Triple Quadrupole mass spectrometer
• Agilent MassHunter acquisition and analysis software

Key Results and Discussion

Kinetic parameters (Km) and inhibition constants (IC50) determined in multiplexed mode matched those obtained in individual analyses within two-fold and fell within 95% confidence intervals.

Multiplexed data acquisition reached an effective rate of ~3 seconds per sample, enabling ~10 000 MS measurements per 8-hour shift. Inhibition curves for suramin, nicotinamide, valproic acid, and tubacin demonstrated isoform specificity without loss of data quality.

Benefits and Practical Applications

This SPE-MS approach combines label-free detection with throughput comparable to optical assays, reducing artifacts and accelerating selectivity screening in drug discovery, quality control, and enzyme profiling.

Future Trends and Applications

Further advances may include expanded multiplex panels, integration with automated liquid handling, real-time data analysis with machine learning, and application to other enzyme families for comprehensive profiling.

Conclusion

The Agilent RapidFire high-throughput MS platform enables ultrafast, reliable selectivity screening of histone deacetylase isoforms, delivering high-quality kinetic and inhibition data at speeds rivaling optical methods.

Reference

Regna NL; Reilly CM. Isoform-Selective HDAC Inhibition in Autoimmune Disease. J. Clin. Cell. Immunol. 2014, 5:207.