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
  • 1. Watarase Research Center, Discovery Research Headquarters, Kyorin Pharmaceutical Co. Ltd., 1848 Nogi, Shimotsuga-gun, Tochigi, 329-0114, Japan. Electronic address: [email protected].
  • 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: [email protected].
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.
Products