Preclinical evaluation of the SARS-CoV-2 Mpro inhibitor RAY1216 shows improved pharmacokinetics compared with nirmatrelvir
Xiaoxin Chen, Xiaodong Huang, Qinhai Ma, Petr Kuzmič, Biao Zhou, Sai Zhang, Jizheng Chen, Jinxin Xu, Bin Liu, Haiming Jiang, Wenjie Zhang, Chunguang Yang, Shiguan Wu, Jianzhou Huang, Haijun Li, Chaofeng Long, Xin Zhao, Hongrui Xu, Yanan Sheng, Yaoting Guo, Chuanying Niu, Lu Xue, Yong Xu, Jinsong Liu, Tianyu Zhang, James Spencer, Zhenzhen Zhu, Wenbin Deng, Xinwen Chen, Shu-Hui Chen, Nanshan Zhong, Xiaoli Xiong, Zifeng Yang
Nature Microbiology, doi:10.1038/s41564-024-01618-9
Although vaccines are available for SARS-CoV-2, antiviral drugs such as nirmatrelvir are still needed, particularly for individuals in whom vaccines are less effective, such as the immunocompromised, to prevent severe COVID-19. Here we report an α-ketoamide-based peptidomimetic inhibitor of the SARS-CoV-2 main protease (M pro ), designated RAY1216. Enzyme inhibition kinetic analysis shows that RAY1216 has an inhibition constant of 8.4 nM and suggests that it dissociates about 12 times slower from M pro compared with nirmatrelvir. The crystal structure of the SARS-CoV-2 M pro :RAY1216 complex shows that RAY1216 covalently binds to the catalytic Cys145 through the α-ketoamide group. In vitro and using human ACE2 transgenic mouse models, RAY1216 shows antiviral activities against SARS-CoV-2 variants comparable to those of nirmatrelvir. It also shows improved pharmacokinetics in mice and rats, suggesting that RAY1216 could be used without ritonavir, which is co-administered with nirmatrelvir. RAY1216 has been approved as a single-component drug named 'leritrelvir' for COVID-19 treatment in China. SARS-CoV-2 has become established in the human population through the coronavirus disease 2019 (COVID-19) pandemic and is likely to remain in circulation. Owing to multinational efforts, vaccines were rapidly rolled out in the early stage of the pandemic and proved successful in saving lives. However, probably due to population immune pressures established by infections and vaccinations, SARS-CoV-2 Omicron variants with highly mutated spike proteins quickly emerged 1 . Rapid emergence of highly mutated variants has shown the extraordinary capacity of the virus to escape humoral immunity, representing a great challenge to vaccines and therapeutic antibodies 2,3 . A number of small-molecule SARS-CoV-2 therapeutics have been developed 4 . This therapeutic strategy may be part of a solution to combat SARS-CoV-2 immune escape. Of note, the orally available drugs molnupiravir and Paxlovid have been approved for COVID-19 treatment
Article https://doi.org/10.1038/s41564-024-01618-9 before administration; the p.o. formulation was the same as that of the mice, and the rats were fed 4 h post-dose. Blood samples were taken via jugular vein cannula at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h after dosing and collected into tubes containing sodium heparin. Plasma samples were obtained and stored in the same manner as described in the mouse section above.
K18-hACE2 mouse pharmacokinetics To evaluate the pharmacokinetics of RAY1216 and PF-07321332 in the K18-hACE2 mouse, RAY1216 and PF-07321332 groups were set up and each group had five SPF-grade female 6-week-old K18-hACE2 mice (purchased from GemPharmatech). Each mouse was administrated with a single dose of 600 mg kg -1 RAY1216 or PF-07321332 p.o. daily for 5 days (the same dose used in the antiviral animal experiment). On the fifth day, blood samples were taken from the cheek at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h. The blood samples were processed and analysed with the same protocol as the ICR mouse samples.
LC-MS/MS analysis of mouse plasma samples For the mouse pharmacokinetic studies, 20 μl plasma samples (the blank sample used 20 μl blank plasma) were mixed with 200 μl of 50% methanol acetonitrile solution (50 ng ml -1 tolbutamide in MeOH); a double blank sample was prepared with 200 μl 50% methanol acetonitrile solution. Samples were centrifugated at 1,790 × g for 10 min at 4 °C. Then, 100 μl of the supernatant was transferred to a clean tube..
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'subject': [ 'Cell Biology', 'Microbiology (medical)', 'Genetics', 'Applied Microbiology and Biotechnology',
'Immunology', 'Microbiology'],
'container-title-short': 'Nat Microbiol',
'published': {'date-parts': [[2024, 3, 29]]},
'assertion': [ { 'value': '27 February 2023',
'order': 1,
'name': 'received',
'label': 'Received',
'group': {'name': 'ArticleHistory', 'label': 'Article History'}},
{ 'value': '22 January 2024',
'order': 2,
'name': 'accepted',
'label': 'Accepted',
'group': {'name': 'ArticleHistory', 'label': 'Article History'}},
{ 'value': '29 March 2024',
'order': 3,
'name': 'first_online',
'label': 'First Online',
'group': {'name': 'ArticleHistory', 'label': 'Article History'}},
{ 'value': 'Xiaoxin Chen, J.H., H.L. and C.L. are employees of Guangdong Raynovent Biotech, '
'which holds the patent of RAY1216. Guangdong Raynovent Biotech provided the '
'RAY1216 molecule used in this study and was responsible for the chemical '
'characterization of RAY1216 and the in vivo and in vitro pharmacokinetic '
'studies. The Raynovent company is not involved in the interpretation of the '
'other results reported in this study and provides no funding to the other '
'parties. P.K. is the author and distributor of the software package DynaFit. '
'DynaFit licenses are available free of charge to all academic, educational and '
'non-profit research institutions. The other authors declare no competing '
'interests.',
'order': 1,
'name': 'Ethics',
'group': {'name': 'EthicsHeading', 'label': 'Competing interests'}}]}
