Mimicking H3 Substrate Arginine in the Design of G9a Lysine Methyltransferase Inhibitors for Cancer Therapy: A Computational Study for Structure-Based Drug Design

Abstract

G9a protein methyltransferase is a potential epigenetic drug target in different cancers and other disease conditions overexpressing the enzyme. G9a is responsible for the H3K9 dimethylation mark, which epigenetically regulates gene expression. Arg8 and Lys9 of the H3 substrate peptide are the two crucial residues for substrate-specific recognition and methylation. Several substrate competitive inhibitors are reported for the potent inhibition of G9a by incorporating lysine mimic groups in the inhibitor design. In this study, we explored the concept of arginine mimic strategy. The hydrophobic segment of the reported inhibitors BIX-01294 and UNC0638 was replaced by a guanidine moiety (side-chain moiety of arginine). The newly substituted guanidine moieties of the inhibitors were positioned similar to the Arg8 of the substrate peptide in molecular docking. Additionally, improved reactivity of the guanidine-substituted inhibitors was observed in density functional theory studies. Molecular dynamics, molecular mechanics Poisson-Boltzmann surface area binding free energy, linear interaction energy, and potential mean force calculated from steered molecular dynamics simulations of the newly designed analogues show enhanced conformational stability and improved H-bond potential and binding affinity toward the target G9a. Moreover, the presence of both lysine and arginine mimics together shows a drastic increase in the binding affinity of the inhibitor towards G9a. Hence, we propose incorporating a guanidine group to imitate the substrate arginine's side chain in the inhibitor design to improve the potency of G9a inhibitors.

Figure 1

(A) Scheme depicting the catalytic reaction performed by G9a lysine methyltransferase: substrate H3K9 peptide is dimethylated by G9a in which the methyl group donor SAM is converted to SAH. (B) Substrate-competitive G9a inhibitors. The majority of the reported inhibitors possess a lysine mimicking group.

Figure 2

SBDD strategy for developing G9a inhibitors based on G9a–H3 substrate peptide interactions. (A) H3 substrate peptide-bound G9a. Only residues Lys9 and Arg8 are shown for clarity. (B) Lysine mimic strategy: introduction of lysine mimic side chain similar to substrate peptide Lys9 on quinazoline ring improves the potency toward G9a in the inhibitor UNC0638. (C) Arginine mimic strategy: introduction of the guanidine group to mimic the substrate peptide Arg8 to improve the activity of BIX-01294 and UNC0638.

Figure 3

Docked orientation of newly designed guanidine analogues in G9a (PDB ID: 3RJW). Superimposition of glide XP docked orientation of the (A) BIX-01294 (green) and BIX-01294 guanidine analogue (magenta) and (C) UNC0638 (green) and UNC0638 guanidine analogue (magenta). Superimposition of inducted fit docked orientation of (B) BIX-01294 (green) and BIX-01294 guanidine analogue (magenta) and (D) UNC0638 (green) and UNC0638 guanidine analogue. The black circle highlights the guanidine group’s position in the newly designed analogues with respect to the hydrophobic group in the original ligands (BIX-01294 and UNC0638). Docked complex PDB files are given as the Supporting Information, PDB-S1–S4.

Figure 4

Number of frames H-bonds formed between G9a residues and the inhibitors (BIX-01294 and BIX-01294 guanidine analogue) during MD simulation.

Figure 5

Number of frames H-bonds formed between G9a residues and the inhibitors (UNC0638 and UNC0638 guanidine analogue) during MD simulation.

Figure 6

Results from SMD simulations of G9a inhibitors and their guanidine analogues. Each plot shows the nonbonded interaction energy terms (in kcal) between G9a–inhibitor complexes and the force (in pN) required during dissociation of the inhibitors and the guanidine analogues from the G9a (Video files S1–S4 of the representative SMD runs are given in the Supporting Information plots for multiple runs are given in Figure S6 of the Supporting Information).