Mechanism-based inhibitors of SIRT2: structure–activity relationship, X-ray structures, target engagement, regulation of α-tubulin acetylation and inhibition of breast cancer cell migration

Abstract

Sirtuin 2 (SIRT2) is a protein deacylase enzyme that has been reported to remove both acetyl groups and longer chain acyl groups from lysine residues in post-translationally modified proteins. It affects diverse biological functions in the cell and has been considered a drug target in relation to both neurodegenerative diseases and cancer. Therefore, access to good chemical tool compounds are essential for the continued investigation of the complex function of this enzyme. Here, we report a collection of probes that are potent, selective, stable in serum, water soluble, amenable to cell culture experiments, and inhibit both SIRT2 deacetylation and demyristoylation. Compared to the current landscape of SIRT2 inhibitors, this is a unique ensemble of features built into a single compound.We expect the developed chemotypes to find broad applications in the interrogation of SIRT2 functions in both healthy and diseased cells to provide a foundation for the development of new therapeutics in the future.

Mitochondria-Targeted Inhibitors of the Human SIRT3 Lysine Deacetylase

“…This mechanism has been utilized to develop so-called mechanism-based inhibitors, by the use of substrate-mimicking chemotypes that form stalled intermediates in the active site of the sirtuin. [26][27][28][29][30][31][32][33] Many mechanism-based inhibitors exhibit high potency and, in several cases, high selectivity toward specific sirtuin subtypes. However, due to the shared mechanism and similar substrate preferences between SIRT1-3, it has been difficult to target SIRT3 selectively.…”

“…Among several demonstrated examples of mitochondrial targeting of various payloads, [34] a particularly appealing approach for our strategy was the mitochondria-targeting peptides developed by Kelley and co-workers. [35][36][37] Based on recent investigations of mechanismbased peptide inhibitors of other sirtuins, [30,33] we hypothesized that mitochondria-targeting peptide tags could be elaborated into potent inhibitors of the SIRT3 that would exhibit selectivity in cells…”

“…It was hypothesized that such inhibitors could be designed by incorporating a modified lysine residue that would enable sirtuin inhibition. Based on insight from previous structure-activity relationship (SAR) studies targeting SIRT2 and SIRT5, including selectivity screenings and co-crystal structures, [30,33] we positioned the ADP-ribose-binding thiocarbonyl-containing lysine residue as the N-terminal amino acid (see Scheme S1 and S2 for syntheses). First, we addressed the effect of mitochondria-targeting peptide length combined with e-N-thioacetylation or e-N-thiomyristoylation of the N-terminal lysine residue.…”

“…Those studies indicated a high degree of freedom for the selection of functionalities at this position and that potency is substantially more reliant on inhibitor backbone-enzyme interactions. [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),…”

“…1 H NMR (600 MHz, DMSO-d6) δ 9.37 (t, J = 5.7 Hz, 1H, NHε,Lys), 9.16 (t, J = 5.5 Hz, 1H, NHalkyne), 8.14 (d, J = 7.8 Hz, 1H, NHa,Arg), 8.10 (d, J = 8.6 Hz, 1H, NHa,Lys), 8.02-7.94 (m, 2H, H2Phenyl, H6Phenyl), 7.88-7.80 (m, 2H, H3Phenyl, H5Phenyl), 7.76 (d, J = 8.3 Hz, 1H, NHa,Cha), 7.53 (t, J = 5.8 Hz, 1H, NHε,Arg), 7.28 (d, J = 2.1 Hz, 1H, CONH2,A), 6.96-6.90 (m, 1H, CONH2,B), 4.26-4.18 (m, 1H, Ha,Cha), 4.08 (dd, J = 5.5, 2.5 Hz, 2H, CH2,alkyne), 4.05-3.98 (m, 1H, Ha,Arg), 3.82-3.77 (m, 1H, Ha,Lys), 3.13 (t, J = 2.5 Hz, 1H, CHalkyne), 3.10-2.99 (m, 4H, CH2,d,Arg, CH2,ε,Lys), 1.73-1.00 (m, 21H), 0.94-0.71 (m, 2H) (Hb,g,d,Lys, Hb,g,Arg,Hb,d,e,x,Cha, CHg,Cha) 13. C NMR (151 MHz, DMSO-d6) δ 174.0 (COCha), 170.6 (COArg), 170.6 (COLys), 164.9 COalkyne), 158.8-157.6 (m, residual COTFA), 156.7 (NHC(=NH)NH2), 156.1 (d, J = 35.9 Hz, COCF3), 143.C1Phenyl), 136.9 (C4Phenyl), 127.9 (C2, C6Phenyl), 126.6 (C2, C6Phenyl), 117.0 (d, J = 299.0 Hz, residual CF3,TFA), 115.9 (d, J = 288.2 Hz, CF3), 80.9 (CCHalkyne), 73.1 (CHCalkyne), 55.7 (Ca,Lys), 52.0 (Ca,Arg), 50.0 (Ca,Cha), 39.overlap with solvent peak, Cε,Lys), 39.5 (overlap with solvent peak, Cb,Cha),33.4 (Cg,Cha), 33.2, 32.4 (Cb,Lys), 31.7, 28.9 (Cb,Arg), 28.7 (CH2,alkyne), 27.8, 26.0, 25.8, 25.5, 24.8 (Cg,Arg), 22.3 (Cg,Lys) (Cd,Lys, Cd,e,x,Cha). Analytical HPLC gradient 5-95% eluent II in eluent I (20 min total runtime), tR 14.3 min (>98%, UV230).…”