Evaluation of lysine modified peptides and their cell penetrating peptide conjugates with sirtuin activity inhibition properties and intracellular delivery.

Sirtuins are NAD+ dependent class of histone deacetylase enzymes responsible for post-translational modifications regulating transcription, cell cycle, metabolism, DNA repair and apoptosis. Substrates of sirtuins are histones, α-tubulin, p65, FOXO1 etc. all of which have multiple NƐ-acetyl lysine residues taking part in catalytic reactions of sirtuins with the help of NAD+. In this chapter, we present methods for Solid Phase Peptide Synthesis and solution phase cyclization of a series of substrate-based peptide inhibitors of sirtuins and their cyclic and CPP conjugated derivatives KP 1, Cyc KP 1, Tat KP 1, KP 2, Cyc KP 2, Tat KP 2, KP 3, Cyc KP 3 and Tat KP 3. Further, describe methods that could be used to study sirtuin inhibition activity and cytotoxicity properties of peptides and small molecules. We present data of three peptides from this family Tat KP 1, Tat KP 2 and Tat KP 3 that show promising yeast sirtuin and mammalian SIRT1 inhibition potential (IC50 6-12 µM) comparable to known sirtuin inhibitors suramin and splitomicin and also showed cytotoxicity against HeLa and BE(2)-C cells. Finally, the morphological studies using SEM and TEM showed membrane disruption potential and evidence of apoptosis of Tat conjugated derivatives. This study documents the one of the initial reports of Tat conjugation modification to enhance the sirtuin inhibition potential. Collectively, this chapter provides comprehensive protocols and practical guidance for the characterization and biological evaluation of sirtuin inhibitors which could be used to identify and characterize novel peptides inhibitors of sirtuins with cell penetrating properties.