tetano
Editor, Senior Moderator
SLAS Discov
. 2024 Aug 20:100181.
doi: 10.1016/j.slasd.2024.100181. Online ahead of print. SARS-CoV-2 M[SUP]pro[/SUP] inhibitor identification using a cellular gain-of-signal assay for high-throughput screening
Renee Delgado[SUP] 1 [/SUP], Jyoti Vishwakarma[SUP] 1 [/SUP], Seyed Arad Moghadasi[SUP] 2 [/SUP], Yuka Otsuka[SUP] 3 [/SUP], Justin Shumate[SUP] 3 [/SUP], Ashley Cuell[SUP] 1 [/SUP], Megan Tansiongco[SUP] 4 [/SUP], Christina B Cooley[SUP] 5 [/SUP], Yanjun Chen[SUP] 1 [/SUP], Agnieszka Dabrowska[SUP] 1 [/SUP], Rahul Basu[SUP] 1 [/SUP], Paulina Duhita Anindita[SUP] 6 [/SUP], Dahai Luo[SUP] 6 [/SUP], Peter I Dosa[SUP] 7 [/SUP], Daniel A Harki[SUP] 7 [/SUP], Thomas Bannister[SUP] 3 [/SUP], Louis Scampavia[SUP] 3 [/SUP], Timothy P Spicer[SUP] 3 [/SUP], Reuben S Harris[SUP] 8 [/SUP]
Affiliations
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, M[SUP]pro[/SUP] (also called 3C-like protease, 3CL[SUP]pro[/SUP]), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, M[SUP]pro[/SUP] inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified M[SUP]pro[/SUP] and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported M[SUP]pro[/SUP] inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known M[SUP]pro[/SUP] inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.
Keywords: Antiviral drugs; SARS-CoV-2 main protease (M(pro)/3CL(pro)); cell-based ultra-high throughput screening (uHTS); protease inhibitors.
. 2024 Aug 20:100181.
doi: 10.1016/j.slasd.2024.100181. Online ahead of print. SARS-CoV-2 M[SUP]pro[/SUP] inhibitor identification using a cellular gain-of-signal assay for high-throughput screening
Renee Delgado[SUP] 1 [/SUP], Jyoti Vishwakarma[SUP] 1 [/SUP], Seyed Arad Moghadasi[SUP] 2 [/SUP], Yuka Otsuka[SUP] 3 [/SUP], Justin Shumate[SUP] 3 [/SUP], Ashley Cuell[SUP] 1 [/SUP], Megan Tansiongco[SUP] 4 [/SUP], Christina B Cooley[SUP] 5 [/SUP], Yanjun Chen[SUP] 1 [/SUP], Agnieszka Dabrowska[SUP] 1 [/SUP], Rahul Basu[SUP] 1 [/SUP], Paulina Duhita Anindita[SUP] 6 [/SUP], Dahai Luo[SUP] 6 [/SUP], Peter I Dosa[SUP] 7 [/SUP], Daniel A Harki[SUP] 7 [/SUP], Thomas Bannister[SUP] 3 [/SUP], Louis Scampavia[SUP] 3 [/SUP], Timothy P Spicer[SUP] 3 [/SUP], Reuben S Harris[SUP] 8 [/SUP]
Affiliations
- PMID: 39173830
- DOI: 10.1016/j.slasd.2024.100181
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, M[SUP]pro[/SUP] (also called 3C-like protease, 3CL[SUP]pro[/SUP]), is a bona fide drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, M[SUP]pro[/SUP] inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified M[SUP]pro[/SUP] and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported M[SUP]pro[/SUP] inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known M[SUP]pro[/SUP] inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.
Keywords: Antiviral drugs; SARS-CoV-2 main protease (M(pro)/3CL(pro)); cell-based ultra-high throughput screening (uHTS); protease inhibitors.