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A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor

Furutani, Yutaka Hirano, Yoshinori Toguchi, Mariko Higuchi, Shoko Qin, Xian-Yang Yanaka, Kaori Sato-Shiozaki, Yumi Takahashi, Nobuaki Sakai, Marina Kongpracha, Pornparn Suzuki, Takehiro Dohmae, Naoshi Kukimoto-Niino, Mutsuko Shirouzu, Mikako Nagamori, Shushi Suzuki, Harukazu Kobayashi, Kaoru Masaki, Takahiro Koyama, Hiroo Sekiba, Kazuma Otsuka, Motoyuki Koike, Kazuhiko Kohara, Michinori Kojima, Soichi Kakeya, Hideaki Matsuura, Tomokazu 京都大学 DOI:10.1038/s41420-023-01755-w

2023.12.22

概要

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1–37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

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We also thank Chiemi Mishima-Tsumagari and Miyuki Kato-Murayama for protein

purification and Shuji Kobayashi for AhR signal analysis. HepG2-NTCP-C4 cells were

kindly gifted from Drs. Koichi Watashi and Takaji Wakita (NIID). This manuscript is

dedicated to the memory of Dr. Soichi Kojima (1961–2019).

AUTHOR CONTRIBUTIONS

Y.F.: Methodology, Data curation, Formal analysis, Investigation, Writing—original

draft, Funding acquisition,; XY.Q., Y.H., M.K.-N.: Data curation Investigation, Writing—

original draft; N.D., M.S., S.N. M.S., S.N., H.S., K.K., T.Masaki, H.K.: Methodology,

Supervision, Investigation, Writing—original draft; M.T., S.H., K.Y., Y. SS, N.T., M.S., P.K.,

T.S. K.S., M.O., K.K.: Data curation, Investigation; M.K.: Resources; S.K., H.K., T.Matsuura:

Methodology, Supervision, Writing - review and editing, Funding acquisition, Project

administration.

FUNDING

This work was mainly supported by AMED under Grant Number JP21fk0310112

(T.Matsuura, Y.F., Y.H., N.D., M.S., S.N., H.S., K.K., T.Masaki, H.K.) and 22fk0210112 (Y.F.)

and partly supported by Grant Number 22fk0310511 (Y.F.) and 22fk0210100 (Y.F.) and

a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports,

Science and Technology, Japan [17H06401 (H.K.), 23H04882 (H.K.)].

COMPETING INTERESTS

The authors declare no competing interests.

ADDITIONAL INFORMATION

Supplementary information The online version contains supplementary material

available at https://doi.org/10.1038/s41420-023-01755-w.

Correspondence and requests for materials should be addressed to Yutaka Furutani.

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ACKNOWLEDGEMENTS

The authors thank members of Department of Laboratory Medicine and Liver Cancer

Prevention Research Unit for kind discussion and technical and secretarial assistance.

Cell Death Discovery (2023)9:467

© The Author(s) 2023

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