Discovery of De Novo Macrocycle Inhibitors of Histone Deacetylase 11

Abstract

Histone deacetylase (HDAC) enzymes are epigenetic regulators that affect diverse protein function by removing acyl groups from lysine side chains throughout the proteome. The most recently discovered human isozyme, HDAC11, differs from other HDACs in substrate preference and tissue expression profile. Elucidation of the biological function of this enzyme has been scarce and only a few chemical probes to help advance this insight have been developed thus far. Here we discovered macrocyclic inhibitors that exhibit selectivity for HDAC11 and penetrate the cytoplasmic membrane in cultured cells as determined by the chloroalkane penetration assay. Our work establishes the combination of de novo macrocycle synthesis with incorporation of N-alkylated hydroxamic acid moieties as a viable strategy for targeting HDAC11. Further, this study demonstrates the potential of applying macrocyclic peptide-based library synthesis to directly furnish high-affinity, cell-permeating ligands. The discovered inhibitors comprise tool compounds for the investigation of the biological function of HDAC11.

Figure 1

Structures of selected HDAC inhibitors. (A) Examples of previously identified inhibitors of HDAC11. (B) Natural product derived inhibitors and generic structure of macrocyclic library members prepared in this work. S.I. = selectivity index, defined as K_i, HDAC1/K_i, HDAC11.

Scheme 1. Synthesis of Building Block 10

Reagents and conditions: (a) MEM-Cl, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), DMF, r.t., 16 h; (b) MeNHNH₂, CH₂Cl₂, r.t., 16 h; (c) Boc₂O, iPr₂NEt, 16 h; (d) 1-hexyl iodide, NaH, THF, 0 °C → r.t., 16 h; (e) TFA–CH₂C_l2 (1:4), 1h; (f) acryloyl chloride, iPr₂NEt, CH₂Cl₂, 0 °C → r.t., 16 h; (g) Grubbs II catalyst (0.08 equiv), CH₂Cl₂, reflux, 5 h; (h) H₂, Pd/C (10% w/w) MeOH, r.t., 3 h; (i) Fmoc-OSu, iPr₂NEt, 1,4-dioxane, r.t., 16 h.

Scheme 2. Synthesis of Macrocycle Library for Targeting HDAC11.

Reagents and conditions: (a) (i) Fmoc-AA–OH, HATU, N-methylmorpholine, N-methyl-2-pyrrolidone (NMP), 2 h, 0.5 μmol scale (performed twice); (ii) piperidine–DMF (1:4), (2 × 2 min); (b) TFA–iPr₃SiH–H₂O (95:2.5:2.5); (c) 1,4-butanedithiol, Et₃N, DMF, 5 h, 5 nmol scale; (d) Reconstitution in DMSO and acoustic dispensing into 1536-well microtiter plates; (e) (i) bis-eletrophile (L2–L6), NH₄HCO₃, MeCN–H₂O (1:1, v/v, pH 8), 2 h; (ii) β-mercaptoethanol quenching. For full lists of employed amino acid building blocks and synthesized compounds, please consult Supplementary Table S5.

Figure 2

Macrocyclic library screening, hit validation, and selectivity screen. (A) Scatter dot plot of the library screening against HDAC11; arrows mark compounds selected for validation. (B) Summary of the resynthesized hits. (C) Dose–response curves for the resynthesized hits against HDAC1 and HDAC11, trapoxin A is used as positive control compound. (D) Structures of 14-N^C6OH and its analogues and their potencies against HDAC11. (E) Selectivity profile of 14-N^C6OH (1 μM) and 14-NHOH (1 μM). The data in B–E represent mean ± standard deviation of at least two individual assays performed in duplicate.

Figure 3

Activity of 14-N^C6OH and analogues against enzymatic activities in mouse brain lysate. (A) Demyristoylation of the Ac-ETDKmyr-AMC substrate with mouse brain lysate. B) Deacetylation the Ac-LGKac-AMC substrate with mouse brain lysate. The data represent mean ± standard deviation of three individual assays performed in technical duplicate compared to DMSO as negative control. Trapoxin A was dosed at 1 μM concentration as positive control of nonselective inhibition of Zn²⁺-dependent HDACs. AMC = 7-amino-4-methylcoumarin.

Figure 4

Pulldown of native HDAC11 and complex partner HDAC6 from cell lysate. (A) Cartoon illustration of the pulldown experiment, where the biotinylated probe (18 or 19) is preincubated with streptavidin-coated magnetic beads, followed by T-47D whole cell lysate. (B) Relative expression of HDAC11 in cell lysate (20 μg) from selected cultured cells to identify a suitable cell line for pulldown of native HDAC11. (C) Pulldown of HDAC11 and HDAC6 with probes 18 and 19 (50 μM) from T-47D cell lysate including competition with 14-N^C6OH (10 μM) (n = 2). For synthesis and full structures of the probes, see Supporting Information [we note that probe 18 was only obtained with 77% purity based on analytical HPLC analysis (215 nm)]. For full blots and replicates, see Supplementary Figure S15.

Figure 5

Cell penetration, histone acetylation, and HDAC11 regulated YAP1 and SOX2 expression. (A) Schematic of the principle in CAPA: cultured HaloTag-expressing HeLa cells are treated with CA-labeled test compound (i), followed by CA-TAMRA probe (ii), and flow cytometry is then applied to determine the extent of competition for CA conjugation sites inside the cells. (B) Structure of synthesized chloroalkane probe 14-N^CAOH and CAPA results after 4 h treatment with compounds (n = 3). The data represent mean ± standard deviation of at least two individual assays performed in duplicate. See the Supporting Information for synthesis of 14-N^CAOH and the positive control compounds, Ac-Asuha(CA)-Trp-NH₂ (20) and CA-Trp-NH₂ (21). (C) Histone acetylation levels following treatment of HEK293T cells with 14-N^C6OH (5 μM), 14-NHOH (5 μM), or trapoxin A (50 nM), with blots above each protein being vinculin (vinc.) used as the loading control (n = 2). For full blots and replicates, see Supplementary Figures S17–19. (D) Regulation of the expression of YAP1 and SOX2 by inhibiting HDAC11 in T-47D cells (n = 3). See Supplementary Figures S21–23 for data from HEK293T cells and full blots of all replicates. Statistical significance was calculated using one-way ANOVA tests with adjusted p values in comparison to treatment with DMSO: ns denotes p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.