Aryl Fluorosulfate Based Inhibitors That Covalently Target the SIRT5 Lysine Deacylase**

Abstract

The sirtuin enzymes are a family of lysine deacylases that regulate gene transcription and metabolism. Sirtuin 5 (SIRT5) hydrolyzes malonyl, succinyl, and glutaryl ϵ‐N‐carboxyacyllysine posttranslational modifications and has recently emerged as a vulnerability in certain cancers. However, chemical probes to illuminate its potential as a pharmacological target have been lacking. Here we report the harnessing of aryl fluorosulfate‐based electrophiles as an avenue to furnish covalent inhibitors that target SIRT5. Alkyne‐tagged affinity‐labeling agents recognize and capture overexpressed SIRT5 in cultured HEK293T cells and can label SIRT5 in the hearts of mice upon intravenous injection of the compound. This work demonstrates the utility of aryl fluorosulfate electrophiles for targeting of SIRT5 and suggests this as a means for the development of potential covalent drug candidates. It is our hope that these results will serve as inspiration for future studies investigating SIRT5 and general sirtuin biology in the mitochondria.

Emerging Roles of SIRT5 in Metabolism, Cancer, and SARS-CoV-2 Infection

“…The same group recently developed additional derivatives by introducing aryl fluorosulfate groups in order to obtain SIRT5 covalent inhibitors [ 171 ]. Starting from compound 8d , different derivatives have been designed to enhance water solubility.…”

“…These compounds were assessed for their influence on SIRT5-mediated deglutarylation at different incubation times (0–24 h). The parent compound 8k did not exhibit great variation over time (IC 50 (0 h) = 0.074 μM, IC 50 (24 h) = 0.103 μM), while compounds 8k - o exhibited time-dependent inhibition with IC 50 values at 0 h over 200 µM, which decreased to 18–36 µM after 4 h and 4.6–6.1 µM after 24 h incubation (IC 50 ( 8l ) = 6.1 μM, IC 50 ( 8m ) = 5.0 μM, IC 50 ( 8n ) = 4.6 μM, IC 50 ( 8o ) = 4.9 μM) [ 171 ]. LC-MS analysis indicated that 8l – o form covalent conjugates with SIRT5 and the presence of NAD + was shown to increase the rate of covalent formation.…”

“…In-gel fluorescence imaging experiments also demonstrated that 8m and 8o could only form adducts with SIRT5, but not SIRT1-4, 6, or 7. Moreover, incubation of HEK293T cells overexpressing SIRT5-FLAG with compounds 8m and 8o (5 h at 20 µM) efficiently enabled SIRT5 pull-down following a click-chemistry reaction with azide-containing biotin and incubating the products with streptavidin-coated magnetic beads [ 171 ]. Furthermore, evaluation in HeLa cells, following the same approach described for compounds 3a and 3i , indicated that compounds 8l and 8m could decrease SIRT5 activity at 200 µM, with compound 8m having a higher efficacy than compound 8l .…”

bioTCIs: Middle-to-Macro Biomolecular Targeted Covalent Inhibitors Possessing Both Semi-Permanent Drug Action and Stringent Target Specificity as Potential Antibody Replacements

“…After bio-panning using the warhead-introduced peptide library, cyclic peptidic TCIs against cysteine- and serine-proteases were obtained. Using another approach, Taki’s group introduced a latent-electrophilic aryl fluorosulfate (i.e., fosylate [ 72 ]; Ar-OSO 2 F) warhead [ 41 , 73 ] which is completely inert and activated only in a matchmaking (i.e., enzyme-like) microenvironment [ 38 , 39 , 46 ] created between the target protein and an appropriate peptide during the reactivity and affinity-based [ 74 ] co-selection process of the T7 phage display [ 51 ]. The fosylate warhead minimized the promiscuous reaction during the library’s construction/selection, and a TCI was obtained with only 2 rounds of bio-panning.…”

Reversible Dual-Covalent Molecular Locking of the 14-3-3/ERRγ Protein–Protein Interaction as a Molecular Glue Drug Discovery Approach

“…This is beneficial as the off-rate of small-molecule binding is reduced, enhancing target engagement. The targetable scope of nucleophilic amino acids has moreover expanded beyond cysteine, to encompass serine, lysine, − and arginine, providing greater control of anchoring sites on the protein and expanded possibilities for more diverse tool compounds that stabilize protein complexes. Covalent drug discovery has already shown significant success in targeting challenging proteins, ,− which makes the strategy promising to molecular glue tool compound development.…”