Rational design of substrate-based multivalent inhibitors of the histone acetyltransferase Tip60

Tip60, the 60 kDa HIV-1 Tat-interactive protein, is a key member of the MYST family of histone acetyltransferases (HATs) and plays critical roles in apoptosis and DNA repair. Potent and selective inhibitors of Tip60 are valuable tools for studying the functions of this potential drug target. In this work, we designed, synthesized and evaluated a new set of substrate-based inhibitors containing multiple binding modalities. In addition to the coenzyme A (CoA) moiety and the histone H3 peptide backbone, mono- and tri-methyl marks were incorporated at Lys 4 and/or Lys 9 sites in the H3 peptide substrate. The biochemical assay results showed that the presence of methyl group(s) on the substrate resulted in more potent inhibitors of Tip60, relative to the parent H3-CoA bisubstrate inhibitor. Importantly, by comparing the inhibitory properties of the ligands against full-length Tip60 and the HAT domain, we determined that the K4me1 and K9me3 marks contributed to the potency augmentation by interacting with the catalytic region of the enzyme.