Author: Kawaoka, Yoshihiro ; Takamune, Nobutoki ; Bulut, Haydar ; Li, Mi ; Yamayoshi, Seiya ; Kishimoto, Naoki ; Mitsuya, Hiroaki ; Hasegawa, Kazuya ; Misumi, Shogo ; Murayama, Kazutaka ; Hayashi, Hironori ; Hattori, Shin-ichiro ; Wlodawer, Alexander ; Furusawa, Yuri ; Tamamura, Hirokazu

We generated SARS-CoV-2 variants resistant to three SARS-CoV-2 main protease (M pro ) inhibitors (nirmatrelvir, TKB245, and 5h), by propagating the ancestral SARS-CoV-2 WK521WT in VeroE6 TMPRSS2 cells with increasing concentrations of each inhibitor and examined their structural and virologic profiles. A predominant E166V-carrying variant (SARS-CoV-2 WK521E166V ), which emerged when passaged with nirmatrelvir and TKB245, proved to be resistant to the two inhibitors. A recombinant SARS-CoV-2 E166V was resistant to nirmatrelvir and TKB245, but sensitive to 5h. X-ray structural study showed that the dimerization of M pro was severely hindered by E166V substitution due to the disruption of the presumed dimerization-initiating Ser1’-Glu166 interactions. TKB245 stayed bound to M proE166V , whereas nirmatrelvir failed. Native mass spectrometry confirmed that nirmatrelvir and TKB245 promoted the dimerization of M pro , and compromised the enzymatic activity; the Ki values of recombinant M proE166V for nirmatrelvir and TKB245 were 117±3 and 17.1±1.9 µM, respectively, indicating that TKB245 has a greater (by a factor of 6.8) binding affinity to M proE166V than nirmatrelvir. SARS-CoV-2 WK521WT selected with 5h acquired A191T substitution in M pro (SARS-CoV-2 WK521A191T ) and better replicated in the presence of 5h, than SARS-CoV-2 WK521WT . However, no significant enzymatic or structural changes in M proA191T were observed. The replicability of SARS-CoV-2 WK521E166V proved to be compromised compared to SARS-CoV-2 WK521WT but predominated over SARS-CoV-2 WK521WT in the presence of nirmatrelvir. The replicability of SARS-CoV-2 WK521A191T surpassed that of SARS-CoV-2 WK521WT in the absence of 5h, confirming that A191T confers enhanced viral fitness. The present data should shed light on the understanding of the mechanism of SARS-CoV-2’s drug resistance acquisition and the development of resistance-repellant COVID-19 therapeutics.

01 Dec 2023·Chemical and Pharmaceutical Bulletin

Exploratory Studies of Effective Inhibitors against the SARS-CoV-2 Main Protease by Halogen Incorporation and Amide Bond Replacement

Article

Author: Azuma, Chika ; Higashi-Kuwata, Nobuyo ; Yamamoto, Kenichi ; Tamamura, Hirokazu ; Kobayakawa, Takuya ; Miura, Yutaro ; Mitsuya, Hiroaki ; Hattori, Shin-ichiro ; Shinohara, Kouki ; Bulut, Haydar ; Ishii, Takahiro ; Nishimura, Soshi ; Tsuji, Kohei

In the development of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs, its main protease (M^pro), which is an essential enzyme for viral replication, is a promising target. To date, the M^pro inhibitors, nirmatrelvir and ensitrelvir, have been clinically developed by Pfizer Inc. and Shionogi & Co., Ltd., respectively, as orally administrable drugs to treat coronavirus disease of 2019 (COVID-19). We have also developed several potent inhibitors of SARS-CoV-2 M^pro that include compounds 4, 5, TKB245 (6), and TKB248 (7), which possesses a 4-fluorobenzothiazole ketone moiety as a reactive warhead. In compounds 5 and TKB248 (7) we have also found that replacement of the P1-P2 amide of compounds 4 and TKB245 (6) with the corresponding thioamide improved their pharmacokinetics (PK) profile in mice. Here, we report the design, synthesis and evaluation of SARS-CoV-2 M^pro inhibitors with replacement of a digestible amide bond by surrogates (9-11, 33, and 34) and introduction of fluorine atoms in a metabolically reactive methyl group on the indole moiety (8). As the results, these compounds showed comparable or less potency compared to the corresponding parent compounds, YH-53/5h (2) and 4. These results should provide useful information for further development of M^pro inhibitors.

12 Oct 2023·Journal of medicinal chemistryQ1 · MEDICINE

Structure-Activity Relationship Studies of SARS-CoV-2 Main Protease Inhibitors Containing 4-Fluorobenzothiazole-2-carbonyl Moieties.

Q1 · MEDICINE

Article

Author: Ishii, Takahiro ; Shinohara, Kouki ; Azuma, Chika ; Higashi-Kuwata, Nobuyo ; Hattori, Shin-Ichiro ; Kobayakawa, Takuya ; Tamamura, Hirokazu ; Miura, Yutaro ; Mitsuya, Hiroaki ; Wada, Naoya ; Nakano, Hiroki ; Bulut, Haydar ; Tsuji, Kohei

The main protease (M^pro) of SARS-CoV-2 is an attractive target for the development of drugs to treat COVID-19. Here, we report the design, synthesis, and structure-activity relationship (SAR) studies of highly potent SARS-CoV-2 M^pro inhibitors including TKB245 (5)/TKB248 (6). Since we have previously developed M^pro inhibitors (3) and (4), several hybrid molecules of these previous compounds combined with nirmatrelvir (1) were designed and synthesized. Compounds such as TKB245 (5) and TKB248 (6), containing a 4-fluorobenzothiazole moiety at the P1' site, are highly effective in the blockade of SARS-CoV-2 replication in VeroE6 cells. Replacement of the P1-P2 amide with the thioamide surrogate in TKB248 (6) improved its PK profile in mice compared to that of TKB245 (5). A new diversity-oriented synthetic route to TKB245 (5) derivatives was also developed. The results of the SAR studies suggest that TKB245 (5) and TKB248 (6) are useful lead compounds for the further development of M^pro inhibitors.

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Indication	Highest Phase	Country/Location	Organization	Date
COVID-19	Discovery	JP	Tokyo Medical & Dental University	27 Oct 2023

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