P-2041. In Vitro Resistance Profiling of S-892216, a Second Generation SARS-CoV-2 3CLpro Inhibitor for the Treatment of COVID-19
Authors: Kawashima Sho , Nobori Haruaki , Okane Miyako , Hashimoto Takashi , Inoue Kae , Yamano Yuko , Funabiki Keiko , Watanabe Michie , Baba Kaoru , Fukao Keita , Kato Teruhisa 
Published: January 2025

Abstract

Background
S-892216 is a candidate for the treatment of COVID-19 that has 3CLpro inhibitory activity and antiviral activity. In this study, the susceptibility of S-892216 was evaluated against SARS-CoV-2 with 3CLpro substitution associated with reduced susceptibility to other 3CLpro inhibitors, ensitrelvir and nirmatrelvir. Additionally, in vitro selection of SARS-CoV-2 with reduced susceptibility to S-892216 was conducted.

Methods
Reverse genetics-derived SARS-CoV-2 (rgSARS-CoV-2) with 3CLpro substitutions including M49L and E166V were generated using circular polymerase extension reaction (CPER) system The susceptibility of S-892216 was assessed using rgSARS-CoV-2 infected VeroE6/TMPRSS2 cells. For the selection of SARS-CoV-2 with reduced susceptibility to S-892216 in vitro, VeroE6/TMPRSS2 cells infected with SARS-CoV-2 Omicron BE.1/BA.5-like variant (hCoV-19/Japan/TY41-702/2022) were cultured in the presence of S-892216 and passaged 10 times. Genotyping analysis of 3CLpro and its cleavage sites in the isolated SARS-CoV-2 were then conducted. The susceptibility of S-892216 and nirmatrelvir against the isolated viruses were assessed.

Results
Fold change (FC) values of S-892216 against rgSARS-CoV-2 with 3CLpro substitutions M49L and E166V were &lt; 2-fold. SARS-CoV-2 with 3CLpro substitutions of D48E, M49K, L50F, N221K, and P252L were isolated from the in vitro virus selection. The FC values of each compound against the isolated SARS-CoV-2 were 3.51- to 28.7-fold for S-892216 and 0.736- to 3.25- fold for nirmatrelvir.

Conclusion
The isolated SARS-CoV-2- strains with reduced susceptibility to S-892216 carried 3CLpro mutation - indicating that the target site is 3CLpro, did not show cross-resistance to nirmatrelvir. Furthermore, S-892216 did not show cross-resistance against nirmatrelvir and ensitrelvir-reduced susceptibility strains. These data suggest S-892216 might be effective if described ensitrelvir- and nirmatrelvir-resistant SARS-CoV-2 variants become relevant in the clinical setting.

Disclosures
Sho Kawashima, Shionogi & Co., Ltd.: Employee Haruaki Nobori, PhD, Shionogi & Co., Ltd.: Employee|Shionogi & Co., Ltd.: Stocks/Bonds (Private Company) Miyako Okane, n/a, Shionogi & Co., Ltd.: Stocks/Bonds (Private Company)|Shionogi TechnoAdvance Research CO., Ltd.: employee Takashi Hashimoto, n/a, Shionogi TechnoAdvance Research CO., Ltd.: employee Kae Inoue, n/a, Shionogi & Co., Ltd.: Stocks/Bonds (Private Company)|Shionogi TechnoAdvance Research CO., Ltd.: employee Yuko Yamano, n/a, Shionogi TechnoAdvance Research CO., Ltd.: employee Keiko Funabiki, n/a, Shionogi TechnoAdvance Research CO., Ltd.: ex-employee Michie Watanabe, n/a, Shionogi TechnoAdvance Research CO., Ltd.: employee Kaoru Baba, n/a, Shionogi TechnoAdvance Research: Employee Keita Fukao, MD, SHIONOGI and CO., LTD.: Stocks/Bonds (Private Company) Teruhisa Kato, PhD, Shionogi & Co., Ltd.: employee|Shionogi & Co., Ltd.: Stocks/Bonds (Private Company)