Discovery of Potent and Selective Inhibitors for G9a-Like Protein (GLP) Lysine Methyltransferase

Abstract

G9a-like protein (GLP) and G9a are highly homologous protein lysine methyltransferases (PKMTs) sharing approximately 80% sequence identity in their catalytic domains. GLP and G9a form a heterodimer complex and catalyze mono- and dimethylation of histone H3 lysine 9 and nonhistone substrates. Although they are closely related, GLP and G9a possess distinct physiological and pathophysiological functions. Thus, GLP or G9a selective small-molecule inhibitors are useful tools to dissect their distinct biological functions. We previously reported potent and selective G9a/GLP dual inhibitors including UNC0638 and UNC0642. Here we report the discovery of potent and selective GLP inhibitors including 4 (MS0124) and 18 (MS012), which are >30-fold and 140-fold selective for GLP over G9a and other methyltransferases, respectively. The cocrystal structures of GLP and G9a in the complex with either 4 or 18 displayed virtually identical binding modes and interactions, highlighting the challenges in structure-based design of selective inhibitors for either enzyme.

Figure 1

Structures of selected G9a/GLP inhibitors.

Figure 2

X-ray cocrystal structures of (A) GLP (green) (PDB 5TUZ) and (B) G9a (blue) (PDB 5TUY) in complex with 4 in the presence of SAM. Water molecule is illustrated as a red sphere. Main interactions are shown in yellow dashed lines. (C) Overlay of (A) and (B).

Figure 3

Selectivity of 4 and 18 against methyltransferases. Selectivity of 4 (A) and 18 (B) against a panel of 29 PKMTs, PRMTs, DNMTs, and RNMTs was determined at two compound concentrations of 1 μM (blue bars) and 10 μM (red bars).

Figure 4

Binding confirmation of 4 and 18. Isothermal titration calorimetry (ITC) was used to confirm binding of 4 to (A) GLP with a K_d of 40 ± 5 nM and to (B) G9a with a K_d of 445 ± 40 nM. Similarly, ITC was used to confirm the binding of 18 to (C) GLP with a K_d of 46 ± 15 nM and to (D) G9a with a K_d of 610 ± 68 nM. ITC experiments for GLP and G9a were performed in triplicate.

Figure 5

Mechanism of GLP inhibition by 4 and 18. Mechanism of inhibition of GLP activity by 4 and 18 was explored by IC₅₀ determination at various peptide (A and C, respectively) or SAM (B and D, respectively) concentrations while the concentration of the other substrate was kept constant. Increase in peptide concentration in assays resulted in significant increase in IC₅₀ values, indicating that both compounds are peptide competitive (A,C). Decrease in IC₅₀ values upon increasing SAM concentration indicates that both compounds are SAM uncompetitive inhibitors (B,D).

Figure 6

X-ray cocrystal structures of GLP and G9a in complex with 18. (A) Structure of GLP–SAM–18 (green) (PDB 5TTG). (B) Structure of G9a–SAM–18 (blue) (PDB 5TTF). Water molecule is illustrated as a red sphere. Main interactions are shown in yellow dashed lines. (C) Overlay of (A) and (B). (D) Overlay of (A) and GLP–SAH–2 (purple) (PDB 3MO5).

Scheme 1. Synthesis of 4 and 7–22^a

^aReagents and conditions: (a) 1-alkylpiperidin-4-ylamine, K₂CO₃, DMF, rt, 80–90%; (b) R′ amines, 4 N HCl in dixoane, i-PrOH, microwave, 160 °C, 75–85%.

Scheme 2. Synthesis of 41–48^a

^aReagents and conditions: (a) EtI or i-PrBr, K₂CO₃, acetone, reflux; (b) HNO₃, Ac₂O, 0 °C to rt, 81–94% over 2 steps; (c) Fe dust, NH₄OAc, H₂O, reflux, 60–70%; (d) Bu₄NBr₃, ethyl acetate, 0 °C to rt, 80–90%; (e) Zn(CN)₂, Pd(pph₃)₄, DMF, microwave, 130 °C, 50–72%; (f) Br₂, Ac₂O, 50 °C, 64%; then HCl, EtOH, 95%; (g) Br₂, K₂CO₃, DCM, –15 °C, 70%; (h) CuCN, DMF, sealed tube, 160 °C, 56%; (i) ClCO₂Me, DIEA, DCM/DMF, 0°C to rt; (j) H₂O₂, NaOH, H₂O/MeOH, reflux; (k) PhNEt₂, POCl₃, reflux, 30–40% over 3 steps; (l) 1-methylpiperidin-4-amine, K₂CO₃, DMF, rt; (m) morpholine, 4 N HCl in dixoane, i-PrOH, microwave, 160 °C, 75–85% over 2 steps.

Scheme 3. Synthesis of 53 and 54^a

^aReagents and conditions: (a) acid chloride, DIEA, DCM/DMF, 0 °C to rt, 90–95%; (b) H₂O₂, NaOH, H₂O/MeOH, reflux, 46–55% over 2 steps; (c) PhNEt₂, POCl₃, reflux, 46–55% over 2 steps; (d) 1-methylpiperidin-4-amine, DIEA, i-PrOH, microwave, 160 °C, 80–90%.