tetano
Editor, Senior Moderator
Antiviral Res
. 2022 Mar 9;105272.
doi: 10.1016/j.antiviral.2022.105272. Online ahead of print.
A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors
K Y Chen[SUP] 1 [/SUP], T Krischuns[SUP] 1 [/SUP], L Ortega Varga[SUP] 2 [/SUP], E Harigua-Souiai[SUP] 3 [/SUP], S Paisant[SUP] 1 [/SUP], A Zettor[SUP] 4 [/SUP], J Chiaravalli[SUP] 4 [/SUP], A Delpal[SUP] 5 [/SUP], D Courtney[SUP] 1 [/SUP], A O'Brien[SUP] 6 [/SUP], S C Baker[SUP] 6 [/SUP], E Decroly[SUP] 5 [/SUP], C Isel[SUP] 1 [/SUP], F Agou[SUP] 4 [/SUP], Y Jacob[SUP] 7 [/SUP], A Blondel[SUP] 2 [/SUP], N Naffakh[SUP] 8 [/SUP]
Affiliations
Abstract
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC[SUB]50[/SUB] values in the 4-8 μM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
Keywords: 3CLpro; Cell-based assay; High-throughput screening; SARS-CoV-2; Small molecule inhibitors; nsp5.
. 2022 Mar 9;105272.
doi: 10.1016/j.antiviral.2022.105272. Online ahead of print.
A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors
K Y Chen[SUP] 1 [/SUP], T Krischuns[SUP] 1 [/SUP], L Ortega Varga[SUP] 2 [/SUP], E Harigua-Souiai[SUP] 3 [/SUP], S Paisant[SUP] 1 [/SUP], A Zettor[SUP] 4 [/SUP], J Chiaravalli[SUP] 4 [/SUP], A Delpal[SUP] 5 [/SUP], D Courtney[SUP] 1 [/SUP], A O'Brien[SUP] 6 [/SUP], S C Baker[SUP] 6 [/SUP], E Decroly[SUP] 5 [/SUP], C Isel[SUP] 1 [/SUP], F Agou[SUP] 4 [/SUP], Y Jacob[SUP] 7 [/SUP], A Blondel[SUP] 2 [/SUP], N Naffakh[SUP] 8 [/SUP]
Affiliations
- PMID: 35278581
- DOI: 10.1016/j.antiviral.2022.105272
Abstract
Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for the screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified two molecules which show anti-nsp5 activity, both in our cell-based assay and in vitro on purified nsp5 protein, and inhibit SARS-CoV-2 replication in A549-ACE2 cells with EC[SUB]50[/SUB] values in the 4-8 μM range. The here described high-throughput-compatible assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.
Keywords: 3CLpro; Cell-based assay; High-throughput screening; SARS-CoV-2; Small molecule inhibitors; nsp5.