2. Academic Validation
  3. Discovery of potent substrate-type lysine methyltransferase G9a inhibitors for the treatment of sickle cell disease

Discovery of potent substrate-type lysine methyltransferase G9a inhibitors for the treatment of sickle cell disease

  • Eur J Med Chem. 2025 May 11:293:117721. doi: 10.1016/j.ejmech.2025.117721.
Yosuke Nishigaya 1,  Shohei Takase 2,  Tatsunobu Sumiya 3,  Ko Kikuzato 4,  Takashi Hiroyama 5,  Yuki Maemoto 2,  Komei Aoki 2,  Tomohiro Sato 6,  Hideaki Niwa 7,  Shin Sato 7,  Kentaro Ihara 7,  Akiko Nakata 8,  Seiji Matsuoka 8,  Noriaki Hashimoto 3,  Ryosuke Namie 3,  Teruki Honma 6,  Takashi Umehara 7,  Mikako Shirouzu 7,  Hiroo Koyama 4,  Yukio Nakamura 5,  Minoru Yoshida 9,  Akihiro Ito 10,  Fumiyuki Shirai 11
Affiliations

Affiliations

  • 1 Watarase Research Center, Discovery Research Headquarters, Kyorin Pharmaceutical Co. Ltd., 1848 Nogi, Shimotsuga-gun, Tochigi, 329-0114, Japan. Electronic address: yousuke.nishigaya@mb.kyorin-pharm.co.jp.
  • 2 School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan.
  • 3 Watarase Research Center, Discovery Research Headquarters, Kyorin Pharmaceutical Co. Ltd., 1848 Nogi, Shimotsuga-gun, Tochigi, 329-0114, Japan.
  • 4 Drug Discovery Chemistry Platform Unit, Japan.
  • 5 Cell Engineering Division, RIKEN BioResource Research Center, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan.
  • 6 Drug Discovery Computational Chemistry Platform Unit, Japan.
  • 7 Drug Discovery Structural Biology Platform Unit, RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan.
  • 8 Drug Discovery Seed Compounds Exploratory Unit, Japan.
  • 9 Drug Discovery Seed Compounds Exploratory Unit, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan; the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan.
  • 10 Drug Discovery Seed Compounds Exploratory Unit, Japan; Chemical Genomics Research Group, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan; School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan.
  • 11 Drug Discovery Chemistry Platform Unit, Japan; Center for One Medicine Innovative Translational Research (COMIT), Institute for Advanced Study, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. Electronic address: fumiyuki.shirai@riken.jp.
PMID: 40367677 DOI: 10.1016/j.ejmech.2025.117721
Abstract

Structurally novel inhibitors of the lysine methyltransferase G9a have attracted considerable interest as potential drug candidates for Cancer and genetic diseases. Here, a detailed account of potency optimization from early leads 8 and 9 to compound 16g is presented. Our search for an alternative scaffold for the 4-oxo-4,5,6,7-tetrahydro-1H-indole moiety of compounds 8 and 9 via parallel synthesis led to the identification of the 4-pyridin-4-ylamino phenyl substructure in compound 16g. This substructure was found to bind to the enzyme in a horizontally flipped manner compared with compound 8 in X-ray crystallographic analysis. Compound 16g is a highly potent G9a inhibitor (IC50 = 0.0020 μM) and structurally distinct from Other G9a inhibitors reported in the literature. Importantly, compound 16g exhibited dose-dependent induction of γ-globin mRNA in HUDEP-2, leading to elevated γ-globin protein levels and F cell numbers in CD34+ bone marrow (BM)‒derived hematopoietic cells. Kinetic studies using surface plasmon resonance (SPR) analysis suggested that compound 16g interacts with G9a via a unique binding mode, as indicated by the markedly higher dissociation constant (KD) compared to those of compounds 8 and 9. Interestingly, X-ray crystallographic studies revealed that the binding motif of compound 16g was quite different from our previous series, including RK-701, and somewhat resembles that of endogenous substrates. Insights obtained in this lead optimization exercise on the association/dissociation constants as well as the binding motifs are expected to help in designing future G9a inhibitors for the treatment of sickle cell disease.

Keywords

Drug discovery; Histone methylation; Lysine methyltransferase G9a; Sickle cell disease; Substrate-type inhibitors; γ-globin mRNA.

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