A study of SIRT1- and SIRT2‑Mediated Deacetylation

Significance of the Topic

The reversible deacetylation of lysine residues by sirtuin enzymes (SIRT1 and SIRT2) plays a critical role in regulating processes such as inflammation, metabolism, and neurodegeneration. Accurate and efficient assays for measuring sirtuin activity are essential to drug discovery efforts targeting these enzymes.

Objectives and Study Overview

This study demonstrates the use of the Agilent RapidFire high-throughput mass spectrometry system to perform label-free assays of SIRT1- and SIRT2-mediated deacetylation. The goals include determining linear reaction ranges, binding constants (Km), and inhibitor potencies (IC50) using native and multiply acetylated p53 peptides.

Methodology and Instrumentation

Recombinant SIRT1 and SIRT2 were expressed in E. coli, purified by affinity chromatography, and stored in Tris-based buffer. Acetylated p53 substrates (single and triple modification) were synthesized or purchased. Deacetylase reactions were conducted in Tris buffer with NAD+ and quenched with formic acid. RapidFire MS employed aqueous and organic solvents with formic acid and trifluoroacetic acid modifiers.

Used Instrumentation

• Agilent RapidFire high-throughput MS system
• RapidFire cartridge A
• MS/MS detection in positive ion mode targeting specific m/z transitions for each acetylation state

Key Results and Discussion

A series of enzyme titrations showed linear product formation over time for both SIRT1 and SIRT2 (R2 > 0.99). Km values were determined as 25 µM for SIRT1 and 8 µM for SIRT2 using the single-acetyl p53 peptide. Inhibitor assays with nicotinamide yielded IC50 values of 62 µM (SIRT1) and 11 µM (SIRT2), consistent with literature reports. Analysis of the triply acetylated peptide allowed simultaneous monitoring of 3ac→2ac→1ac→0ac transitions, illustrating the power of direct MS detection to resolve multiple modification states.

Benefits and Practical Applications

• Label-free detection eliminates artifacts from fluorescent or radioactive tags
• Direct measurement of substrates and products simplifies assay design
• Capability to distinguish successive deacetylation events enhances mechanistic insights
• High throughput supports screening of enzyme modulators in drug discovery

Future Trends and Opportunities

Expansion of RapidFire MS assays to additional epigenetic enzymes and multiplexed peptide libraries is anticipated. Integration with automated screening platforms and advanced data analytics could further accelerate lead identification and optimization.

Conclusion

The RapidFire high-throughput MS approach provides robust, label-free assays for sirtuin activity, enabling accurate kinetic and inhibition measurements. This methodology offers a valuable toolset for drug discovery programs targeting deacetylase enzymes.

References

1. Yamamoto H, Schoonjans K, Auwerx J. Sirtuin Functions in Health and Disease. Mol. Endocrinol. 2007;21(8):1745–55.
2. Porcu M, Chiarugi A. The Emerging Therapeutic Potential of Sirtuin-Interacting Drugs: from Cell Death to Lifespan Extension. Trends Pharmacol. Sci. 2005;26(2):94–103.
3. Marcotte PA et al. Fluorescence Assay of SIRT Protein Deacetylases Using an Acetylated Peptide Substrate and a Secondary Trypsin Reaction. Anal. Biochem. 2004;332(1):90–9.