Targeting a cryptic allosteric site of SIRT6 with small-molecule inhibitors that inhibit the migration of pancreatic cancer cells

Abstract

SIRT6 belongs to the conserved NAD⁺-dependent deacetylase superfamily and mediates multiple biological and pathological processes. Targeting SIRT6 by allosteric modulators represents a novel direction for therapeutics, which can overcome the selectivity problem caused by the structural similarity of orthosteric sites among deacetylases. Here, developing a reversed allosteric strategy AlloReverse, we identified a cryptic allosteric site, Pocket Z, which was only induced by the bi-directional allosteric signal triggered upon orthosteric binding of NAD⁺. Based on Pocket Z, we discovered an SIRT6 allosteric inhibitor named JYQ-42. JYQ-42 selectively targets SIRT6 among other histone deacetylases and effectively inhibits SIRT6 deacetylation, with an IC₅₀ of 2.33 μmol/L. JYQ-42 significantly suppresses SIRT6-mediated cancer cell migration and pro-inflammatory cytokine production. JYQ-42, to our knowledge, is the most potent and selective allosteric SIRT6 inhibitor. This study provides a novel strategy for allosteric drug design and will help in the challenging development of therapeutic agents that can selectively bind SIRT6.

Keywords: ADPr, ADP-ribose; Allosteric inhibitor; BSA, bull serum albumin; CCK-8, Cell Counting Kit-8; Cell migration; Cytokine production; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; FDL, Fluor de Lys; H3K18, histone 3 lysine 18; H3K56, histone 3 lysine 56; H3K9, histone 3 lysine 9; HDAC, histone deacetylase; HPLC, high-performance liquid chromatography; IC50, half-maximum inhibitory concentration; IPTG, isopropyl-β-d-thiogalactoside; MD, molecular dynamics; Molecular dynamics simulations; NAD+, nicotinamide adenine dinucleotide; NAM, nicotinamide; PBS, phosphate buffer saline; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethanesulfonyl fluoride; Pancreatic cancer; RMSD, root-mean-square deviation; RT-qPCR, real-time quantitative PCR; Reversed allostery; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SIRT6; SIRT6, sirtuin 6.

Graphical abstract

Figure 1

The novel allosteric Pocket Z of SIRT6 was identified by computational methods (A) Conformational landscape of apo SIRT6. (B) Conformational landscape of holo SIRT6. (C) The novel allosteric site Pocket Z in S3′ within the conformational ensemble of holo SIRT6. (D) Energy coupling analysis of Pocket Z. (E) Pocket Z is located topologically distal to the orthosteric NAD⁺ binding site.

Figure 2

Validation of the predicted pocket in SIRT6. (A) Structural insights of Pocket Z. (B) Mutagenesis experiments of critical residues within Pocket Z. The deacetylation activity of different SIRT6 mutants was measured, and their relative activities were calculated as fold changes relative to that of the wild-type (WT) protein, which was set as 1. All assays were performed with at least three replicates, and the results are shown as the mean ± SD, n = 3 wells, from three independent experiments.

Figure 3

Discovery and biochemical characterization of JYQ-42 as an inhibitor of SIRT6 deacetylation. (A) Chemical structures of JYQ-1 and JYQ-42. (B) JYQ-42 can inhibit SIRT6 deacetylation on Ac-RHKK-Ac-AMC effectively. The inhibition activity of JYQ-42 on SIRT6 deacetylation was evaluated by the Fluor de Lys (FDL) assay. The IC₅₀ of JYQ-42 was 2.33 ± 0.17 μmol/L, and the data are presented as the mean ± SD, n = 3 wells, from three independent experiments. (C) The effects of SIRT6 deacetylation on Ac-KQTARK-Ac-STGGWW-NH₂ in the absence or presence of 50 μmol/L JYQ-42, as determined by HPLC. Data are representative of three independent experiments. (D) JYQ-42 inhibited SIRT6 deacetylation significantly on nucleosome substrates. Nucleosomes were extracted from HeLa cells, and the samples were analysed by Western blot analysis. Data are representative of three independent experiments.

Figure 4

JYQ-42 is an allosteric inhibitor of SIRT6. (A) The binding effect of JYQ-42 on SIRT6 was analysed by surface plasmon resonance (SPR). Three independent experiments were performed. (B) SPR binding curves fitted with steady-state model showing the binding affinity of JYQ-42. The K_d value of JYQ-42 was 22.03 ± 3.11 μmol/L, and three independent experiments were performed. (C) Molecular docking of SIRT6 with JYQ-42. The SIRT6-ADPr crystal structure (PDB ID: 3K35) was used for docking, and the best JYQ-42 binding pose with the smallest Glide/IFD score or lowest energy was chosen for further analysis. (D) Effects of allosteric-site mutations located in the JYQ-42 binding pocket on the inhibition of SIRT6 deacetylation, as determined by FDL assays. Data are presented as the mean ± SD, n = 3 wells, from three independent experiments.

Figure 5

JYQ-42 is a non-competitive inhibitor of SIRT6. (A) Competition assay between NAD⁺ and JYQ-42, as evaluated by the Fluor de Lys (FDL) assay. (B) Competition assay between Ac-RHKK-Ac-AMC and JYQ-42, as evaluated by the Fluor de Lys (FDL) assay. (C and D) Enzyme kinetics analysis of SIRT6 inhibition by JYQ-42 were performed with varying concentrations of NAD⁺(C) and Ac-RHKK-Ac-AMC (D) by HPLC. Double-reciprocal plots of the initial velocities (Lineweaver–Burk plots) showing non-competitive inhibition towards NAD⁺ and Ac-RHKK-Ac-AMC. Data are presented as the mean ± SD, n = 3 wells, from three independent experiments. (E) The sequence conservation of the allosteric pocket was analysed by using WebLogo. The amino acids are coloured according to their chemical properties as follows: hydrophobic: black; hydrophilic: blue; neutral: green.

Figure 6

JYQ-42 inhibits SIRT6 deacetylation and cell migration of pancreatic cancer cells. (A) Western blot analysis of SIRT6 and its substrates H3K9Ac, H3K18Ac and H3K56Ac in BXPC-3 cells treated with the indicated doses of JYQ-42 for 24 h. (B) Kinetic curves of the migration of BXPC-3 cells in the absence or presence of 5, 10, 20 μmol/L JYQ-42, as determined by xCELLigence RTCA-DP. Data are presented as the mean ± SD, n = 3 wells, from three independent experiments. ∗∗∗∗P < 0.0001, t-test (two-tailed and unpaired). (C) Representative images of wound-healing assays of BXPC-3 cells in the absence or presence of 10, 20, 40 μmol/L JYQ-42 obtained under phase contrast microscopy. Scale bar, 100 μm. The images shown are representative of triplicate experiments.

Figure 7

JYQ-42 inhibits the expression and secretion of inflammatory factors. (A) The SIRT6, IL6, IL8 and TNF-α mRNA levels in BXPC-3 cells in the absence or presence of 10, 20, 40 μmol/L JYQ-42 were measured by qPCR and quantified versus the GAPDH housekeeping gene. Data are presented as the mean ± SD, n = 3 wells, from three independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001, t-test (two-tailed and unpaired). (B–D) BXPC-3 cells were incubated for 24 h with 30 ng/mL PMA in the absence or presence of 10, 20, 40 μmol/L JYQ-42, and the concentrations of inflammatory factors (IL6, IL8, TNF-α) in supernatants were detected by ELISA. Data are presented as the mean ± SD, n = 3 wells, from three independent experiments. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, t-test (two-tailed and unpaired).