Discovery of the First-in-Class G9a/GLP Covalent Inhibitors

Abstract

The highly homologous protein lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9), have been implicated in various human diseases. To investigate functions of G9a and GLP in human diseases, we and others reported several noncovalent reversible small-molecule inhibitors of G9a and GLP. Here, we report the discovery of the first-in-class G9a/GLP covalent irreversible inhibitors, 1 and 8 (MS8511), by targeting a cysteine residue at the substrate binding site. We characterized these covalent inhibitors in enzymatic, mass spectrometry based and cellular assays and using X-ray crystallography. Compared to the noncovalent G9a/GLP inhibitor UNC0642, covalent inhibitor 8 displayed improved potency in enzymatic and cellular assays. Interestingly, compound 8 also displayed potential kinetic preference for covalently modifying G9a over GLP. Collectively, compound 8 could be a useful chemical tool for studying the functional roles of G9a and GLP by covalently modifying and inhibiting these methyltransferases.

Figure 1.

Design of G9a/GLP covalent inhibitors. (A) Chemical structures of UNC0638 and UNC0642. The cyclohexyl group of UNC0638 (highlighted in blue) was replaced by various covalent warheads. (B) Cocrystal structure of G9a in complex with UNC0638 (PDB ID 3RJW).

Figure 2.

Representative results of mass spectrometry based analysis of G9a incubated with compound 1, 6, or 8. The G9a–inhibitor adduct was quantified after 1 h incubation of G9a (5 μM) with DMSO or the indicated compound (50 μM) at room temperature. Molecular weight of G9a, 34 314 Da; molecular weight of G9a + compound 1, 34 811 Da; molecular weight of G9a + compound 8, 34 810 Da.

Figure 3.

Concentration-dependent inhibition of G9a and GLP by compounds 1 and 8 in SAHH-coupled enzymatic assays. The assays were performed after 10 min of preincubation with G9a (A) and GLP (B). Data are the means ± SD from four independent experiments.

Figure 4.

ITC titrations of UNC0642, compound 1, and compound 8 into G9a and GLP. Representative ITC titrations are shown for UNC0642 (A), compound 1 (B), and compound 8 (C) with G9a (left) and GLP (right). The calculated values represent the means ± SD from two independent experiments.

Figure 5.

Time-dependent inhibition and covalent modification of G9a and GLP by compound 8. (A) Inhibitory activity of compound 8 in G9a and GLP radioactivity-based scintillation proximity biochemical assays following different preincubation times (5, 15, 45 min). Data are the means ± SD from four replicates. (B) Quantification of time-dependent protein–inhibitor adduct formation using mass spectrometry based analysis. Data are the means ± SD from two independent experiments. (C) Representative mass spectrometric analysis results of G9a (left) and GLP (right) treated with compound 8 for 30 min (upper) or 60 min (lower). Molecular weight of G9a, 34 314 Da; molecular weight of G9a + compound 8, 34 810 Da; molecular weight of GLP, 34 687 Da; molecular weight of GLP + compound 8, 35 183 Da. In (B) and (C) 5 μM G9a or GLP and 50 μM compound 8 were used.

Figure 6.

Design of noncovalent inhibitor control of compound 8.

Figure 7.

(A) Representative mass spectrometric analysis results of G9a incubated with compound 9 or compound 8 (as a positive control). After treatment with the indicated compound (or DMSO) for 1 h, the formation of the protein–inhibitor adduct was quantified by using mass spectrometry. Data are shown as G9a + DMSO (top), G9a + compound 8 (middle), and G9a + compound 9 (bottom). (B) ITC titrations of compound 9 into G9a and GLP. The calculated values represent the means ± SD from two independent experiments. (C) Concentration-dependent inhibition of G9a and GLP by compound 9 in SAHH-coupled biochemical assays. The calculated values represent the means ± SD from four independent experiments.

Figure 8.

X-ray cocrystal structures of compound 1 in complex with G9a and GLP. (A) Cocrystal structure of compound 1 bound to G9a (PDB ID 7T7L). Cys1098 was covalently modified by compound 1. (B) Cocrystal structure of compound 1 bound to GLP (PDB ID 7T7M). Cys1186 was covalently modified by compound 1. (C) Overlay of the crystal structures of G9a in complex with compound 1 (in orange) and GLP in complex with compound 1 (in cyan). G9a and GLP structures that were not covalently modified were used for overlay. The dashed arrow indicates the slight downward movement of the α helix of G9a (in orange) compared to that of GLP (in cyan).

Figure 9.

Selectivity of compound 8 against 21 other methyltransferases. Data are the means ± SD from two independent experiments.

Figure 10.

Effective reduction of H3K9me2 by compound 8 in cells in a concentration- and time-dependent manner. (A) Effects of compound 1, compound 8, compound 9, and UNC0642 on reducing the H3K9me2 level in MDA-MB-231 cells. MDA-MB-231 cells were treated with DMSO or the indicated compound at the indicated concentration for 24, 48, or 72 h. Western blot results are representative from at least two independent experiments. (B) Effects of compound 1, compound 8, and UNC0642 on reducing the H3K9me2 level in K562 cells. K562 cells were treated with DMSO or the indicated compound at the indicated concentration for 48 h. Western blot results are representative from at least two independent experiments. (C) Quantification of Western blot results shown in (B) and biological repeats. Blots were quantified by Image Studio and analyzed by using unpaired student t test. ns, P > 0.05; **, P < 0.01; ***, P < 0.001.

Scheme 1.. Synthesis of Compounds 1 and 2^a

^aReagents and conditions: (a) NaN₃, CuI, VC, N¹,N²-dimethylethane-1,2-diamine, EtOH/H₂O, microwave, 130 °C, 30 min, 70%; (b) acid chloride, DMF, 60 °C, 2 h, 40–45%.

Scheme 2.. Synthesis of Compounds 3 and 4^a

^aReagents and conditions: (a) TBTU, TEA, DCM, rt, overnight, 49%; (b) H₂O₂, NaOH, MeOH/H₂O, reflux, 30 min, 98%; (c) pyrrolidine, DIEA, DMSO, microwave, 100 °C 1 h, 32%; (d) POCl₃, PhNMe₂, CHCl₃, 80 °C, 2 h; (e) (1-isopropylpiperidin-4-yl)amine, K₂CO₃, DMF, 80 °C, 1 h, 30% over two steps; (f) HCl, 1,4-dioxane, rt, 30 min; (g) acid chloride, DIEA, DCM, rt, 2 h, 60–70% over two steps.

Scheme 3.. Synthesis of Compound 5^a

^aReagents and conditions: (a) Sc(OTf)₃, DCM, rt, 2 h, 90%; (b) TFA, DCM, rt, 30 min, 80%; (c) intermediate 10, DIEA, IPA, microwave, 130 °C, 30 min, 53%.

Scheme 4.. Synthesis of Compound 6^a

^aReagents and conditions: (a) methylamine, HCl, IPA, microwave, 160 °C, 15 min, 82%; (b) acryloyl chloride, DMF, 60 °C, 2 h, 40%.

Scheme 5.. Synthesis of Compound 7^a

^aReagents and conditions: (a) Pd₂(dba)₃, K₃PO₄, BINAP, 1,4-dioxane, microwave, 160 °C, 30 min, 27%.

Scheme 6.. Synthesis of Compounds 8 and 9^a

^aReagents and conditions: (a) 4-methoxylbenzylamine, Pd(OAc)₂, BINAP, t-BuONa, THF, microwave, 80 °C, 30 min, 48%; (b) (1-isopropylpiperidin-4-yl)amine, Brettphos-G1-Pd, Brettphos, LHMDS, 1,4-dioxane, microwave, 160 °C, 30 min, 70%; (c), TFA, 50 °C, 1 h, 90%; (d) acid chloride, DIEA, CHCl3, rt, overnight, 30–40%.