Identification of Diketopiperazine-Containing 2-Anilinobenzamides as Potent Sirtuin 2 (SIRT2)-Selective Inhibitors Targeting the “Selectivity Pocket”, Substrate-Binding Site, and NAD⁺-Binding Site

Abstract

The NAD+-dependent deacetylase SIRT2 represents an attractive target for drug development. Here, we designed and synthesized drug-like SIRT2-selective inhibitors based on an analysis of the putative binding modes of recently reported SIRT2-selective inhibitors and evaluated their SIRT2-inhibitory activity. This led us to develop a more drug-like diketopiperazine structure as a “hydrogen bond (H-bond) hunter” to target the substrate-binding site of SIRT2. Thioamide 53, a conjugate of diketopiperazine and 2-anilinobenzamide which is expected to occupy the “selectivity pocket” of SIRT2, exhibited potent SIRT2-selective inhibition. Inhibition of SIRT2 by 53 was mediated by the formation of a 53-ADP-ribose conjugate, suggesting that 53 is a mechanism-based inhibitor targeting the “selectivity pocket”, substrate-binding site, and NAD+-binding site. Furthermore, 53 showed potent antiproliferative activity toward breast cancer cells and promoted neurite outgrowth of Neuro-2a cells. These findings should pave the way for the discovery of novel therapeutic agents for cancer and neurological disorders.

Drug Discovery Researches on Modulators of Lysine-Modifying Enzymes Based on Strategic Chemistry Approaches

“…Since each KDAC isozyme is thought to have specific substrate proteins/lysine residues and to be associated with distinct diseases, isozymeselective KDAC inhibitors are of interest as chemical tools and therapeutic agents with few side effects. 93) In our group, we have reported several isoform-selective inhibitors against HDAC3, 40,94) HDAC6, 95,96) HDAC8, 36,41) and SIRT2 39,[97][98][99] by SBDD, LBDD, strategic chemistry approaches, and their combination. In this section, I present the studies on SIRT2selective inhibitors identified by combining SBDD with drug design based on an enzymatic mechanism.…”

“…In this section, I present the studies on SIRT2selective inhibitors identified by combining SBDD with drug design based on an enzymatic mechanism. 98,99) SIRT2 is classified as a cytoplasmic NAD + -dependent deacetylase 100) and is involved in the destabilization of microtubules through deacetylation of acetylated α-tubulin. 101) Although the functions of SIRT2 are not completely understood yet, they have been suggested to be associated with some diseases, including neurodegenerative disorders.…”

“…In 2012, we reported NCO-90 as a SIRT2 selective inhibitor with an IC 50 value within micromolar range 97) and recently, we have succeeded in determining a crystal structure of SIRT2 in complex with NCO-90 98) (Figs. 6A, B).…”

“…This result supported the hypothesis that targeting both the substrate binding pocket and the "selectivity pocket" represents a useful strategy to develop novel SIRT2 inhibitors. 98) Inspired by this result, we also designed mechanism-based SIRT2 inhibitors.…”

“…7A). This proposed mechanism drove us to design KPM-2 by replacing the amide oxygen of KPM-1 with a sulfur atom 98,105,111) (Figs. 7B, C).…”