Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme**

Abstract

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ‐N‐carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism‐based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high‐affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5‐dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Therapeutic Potential and Activity Modulation of the Protein Lysine Deacylase Sirtuin 5

“…Among SIRT5 inhibitors, a great deal of work has been carried out starting from peptide substrate analogues, which has led to promising peptide-based inhibitors. These consist of thiourea-based molecules such as 2c , 135 3b , 3d , 155 and 3i , 148 which display submicromolar SIRT5 inhibition ( Table 2 ). These molecules were all administered as prodrugs to mask the negative charge of the carboxylic group and increase cellular permeability.…”

“… 147 In line with this, SIRT5 knockdown decreases colony formation and enhances apoptosis in a wide range of AML cell lines, and the pharmacological inhibition of SIRT5 impairs cell proliferation and induces apoptosis in SIRT5-dependent AML cells such as OCI-AML2, SKM-1, and MOLM-13 (see compounds 3b , 3d , and 3i , respectively, in section 4.1 ). 147 , 148 Similarly, SIRT5 expression is necessary for tumor insurgence and growth in both xenograft and syngeneic AML mouse models. 147 Finally, a recent study also revealed that the tumor suppressor p53 is succinylated at Lys120; 149 this residue was also previously identified as an acetylation site of KAT8, Tip60, and NAT10.…”

“…Starting from compound 3d , Rajabi et al recently developed a series of derivatives to investigate whether the bioisosteric substitution of the carboxylic acid moiety might retain the SIRT5 inhibitory potency. 148 Among the derivatives, 1,2,4-oxadiazol-5(4 H )-one ( 3f ), 1,2,4-oxadiazol-5(4 H )-thione ( 3g ), 2-hydroxyisoxazole ( 3h ), and tetrazole ( 3i ) displayed submicromolar IC 50 values for SIRT5-mediated deglutarylation (IC 50 ( 3f ) ≤ 0.05 μM, IC 50 ( 3g ) = 0.9 μM, IC 50 ( 3h ) = 0.29 μM, and IC 50 ( 3i ) ≤ 0.05 μM). The kinetics of SIRT5 inhibition by compounds 3f , 3h , and 3i was also evaluated.…”

“…3i -he displayed a higher efficacy in OCI-AML2 (IC 50 (OCI-AML2, 3d -et ) > 50 μM and IC 50 (OCI-AML2, 3i -he ) = 20 μM), while similar cell growth inhibition was observed for MOLM-13 (IC 50 (MOLM-13, 3d -et ) = 29 μM and IC 50 (MOLM-13, 3i -he ) = 24 μM). 148 …”

Insights on the Modulation of SIRT5 Activity: A Challenging Balance

“…This peptide became the lead compound for a series of optimization campaigns, carried out by different research groups. In 2022, Rajabi and co-workers investigated isostere and prodrug derivatives of 32, modified at the carboxylic function [55]. In a BioRxiv preprint, Bolding et al reported the study of fluorosulfate analogues, with po- Many other research groups investigated thiourea derivatives, albeit with more modest success.…”