Dethioacylation by Sirtuins 1–3: Considerations for Drug Design Using Mechanism-Based Sirtuin Inhibition

Abstract

The sirtuin enzymes are potential drug targets for intervention in a series of diseases. Efforts to inhibit enzymes of this class with thioamide-and thiourea-containing, substrate-mimicking entities have produced a number of high-affinity binders. However, less attention has been dedicated to the investigation of the stability of these inhibitors under various conditions. Here, we provide evidence of unprecedented degree of cleavage of short-chain e-N-thioacyllysine modifications meant to target these sirtuins and further provide insights into the serum stability of compounds containing both thioamides and thioureas. Our study questions the utility short-chain thioamide-based inhibitors of sirtuins for drug development and points to mono-alkylated thiourea-based chemotypes as being more stable in human serum.

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

“…3a). Specifically, we synthesized X Nav1.5 SYN constructs that contained one of the following four variants: (i) mutations K1479R and Y1495F (termed [NM] Syn ) to prevent acetylation and phosphorylation, respectively; (ii) a thio-acetylated Lys analog at position 1479 (tAcK1479) that mimics PTM but displays increased metabolic stability against sirtuins compared to regular acetylation 28,29 ; (iii) a phosphonylated Tyr analog at position 1495 (phY1495) that provides a non-hydrolysable phosphate mimic; or (iv) both tAcK1479 and phY1495 to mimic a dual PTM scenario (Fig. 3b).…”

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

“…[30,33] Based on this series, we decided to proceed with the 3-phenylpropionyl group (c; Figure 2) and the alkynecontaining group (a, Figure 2), which is amenable for incorporation of fluorophores or other tags using Cu(I)-catalyzed azide-alkyne Huisgen 3+2 cycloaddition "click" chemistry. [39][40] Inspired by previous studies of SIRT1-3 [27,33,[41][42][43][44][45] and the structures of the active sites in SIRT1-3 ( Figure S3),…”

“…The most potent inhibitors of SIRT3 were compounds 1, 4, and 6, with 1 and 4 showing the lowest degree of off-target inhibition of SIRT1 and 2 ( Figure 2B). However, because e-N-thioacetyllysine residues have been shown to be processed by HDAC8 [46] and SIRT1-3, [43] the e-N'-methylthiourea functionality was chosen for further investigations.…”

“…inhibitors EX-527 [56] and 19 [31,43] as positive controls ( Figure 4E,F and Figure S13). Although, compounds 17, EX-527, and 19 are equipotent against SIRT1 in vitro, [43] the effect on p53 acetylation (K382) by compound 17 is significantly lower than the positive control compounds ( Figure 4E,F), suggesting a high degree of selectivity for SIRT3 over SIRT1 in cells. Significance of the CETSA shifts were calculated using unpaired t-test of Tm values from independent experiments.…”

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

“…32,35 Based on this series, we proceeded with the 3-phenylpropionyl group (c; Figure 2) and the alkyne-containing group (a, Figure 2, Scheme S6), which is amenable for incorporation of fluorophores or other tags using Cu(I)-catalyzed azide-alkyne Huisgen 3+2 cycloaddition "click" chemistry. 45,46 Inspired by previous studies of SIRT1-3 29,35,[47][48][49][50][51] and the structures of the active sites in SIRT1-3 ( Figure S3), we next analyzed a number of thiocarbonyl binding motifs (1-10; Figure 2A). The most potent inhibitors of SIRT3, which did not at the same time inhibit the other two isoforms to a more substantial extent, were compounds 1 and 4 ( Figure 2B).…”