A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor
<p dir="ltr">The main protease, M<sup>pro</sup>, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of ge...
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| مؤلفون آخرون: | , , , , , |
| منشور في: |
2022
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| الموضوعات: | |
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إضافة وسم
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| _version_ | 1864513518729953280 |
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| author | Anupriya M. Geethakumari (17052375) |
| author2 | Wesam S. Ahmed (10170053) Saad Rasool (9764093) Asma Fatima (17329974) S. M. Nasir Uddin (18420792) Mustapha Aouida (417652) Kabir H. Biswas (5705864) |
| author2_role | author author author author author author |
| author_facet | Anupriya M. Geethakumari (17052375) Wesam S. Ahmed (10170053) Saad Rasool (9764093) Asma Fatima (17329974) S. M. Nasir Uddin (18420792) Mustapha Aouida (417652) Kabir H. Biswas (5705864) |
| author_role | author |
| dc.creator.none.fl_str_mv | Anupriya M. Geethakumari (17052375) Wesam S. Ahmed (10170053) Saad Rasool (9764093) Asma Fatima (17329974) S. M. Nasir Uddin (18420792) Mustapha Aouida (417652) Kabir H. Biswas (5705864) |
| dc.date.none.fl_str_mv | 2022-09-28T03:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1038/s42004-022-00731-2 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/A_genetically_encoded_BRET-based_SARS-CoV-2_Mpro_protease_activity_sensor/25662624 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Biochemistry and cell biology Engineering Materials engineering Main protease (Mpro) SARS-CoV-2 replication Anti-SARS-CoV-2 agents Bioluminescence resonance energy transfer (BRET) Live cells In vitro assays mNeonGreen |
| dc.title.none.fl_str_mv | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <p dir="ltr">The main protease, M<sup>pro</sup>, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting M<sup>pro</sup> proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the M<sup>pro</sup> N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with M<sup>pro</sup> in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in M<sup>pro</sup> completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of M<sup>pro</sup> by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of M<sup>pro</sup> activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 M<sup>pro</sup> and functional genomics application to determine the effect of sequence variation in M<sup>pro</sup>.</p><p><br></p><h2>Other Information</h2><p dir="ltr">Published in: Communications Chemistry<br>License: <a href="https://creativecommons.org/licenses/by/4.0" target="_blank">https://creativecommons.org/licenses/by/4.0</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1038/s42004-022-00731-2" target="_blank">https://dx.doi.org/10.1038/s42004-022-00731-2</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_a8001609443b5ab23cd7a858bad0569d |
| identifier_str_mv | 10.1038/s42004-022-00731-2 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/25662624 |
| publishDate | 2022 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensorAnupriya M. Geethakumari (17052375)Wesam S. Ahmed (10170053)Saad Rasool (9764093)Asma Fatima (17329974)S. M. Nasir Uddin (18420792)Mustapha Aouida (417652)Kabir H. Biswas (5705864)Biological sciencesBiochemistry and cell biologyEngineeringMaterials engineeringMain protease (Mpro)SARS-CoV-2 replicationAnti-SARS-CoV-2 agentsBioluminescence resonance energy transfer (BRET)Live cellsIn vitro assaysmNeonGreen<p dir="ltr">The main protease, M<sup>pro</sup>, is critical for SARS-CoV-2 replication and an appealing target for designing anti-SARS-CoV-2 agents. Therefore, there is a demand for the development of improved sensors to monitor its activity. Here, we report a pair of genetically encoded, bioluminescence resonance energy transfer (BRET)-based sensors for detecting M<sup>pro</sup> proteolytic activity in live cells as well as in vitro. The sensors were generated by sandwiching peptides containing the M<sup>pro</sup> N-terminal autocleavage sites, either AVLQSGFR (short) or KTSAVLQSGFRKME (long), in between the mNeonGreen and NanoLuc proteins. Co-expression of the sensors with M<sup>pro</sup> in live cells resulted in their cleavage while mutation of the critical C145 residue (C145A) in M<sup>pro</sup> completely abrogated their cleavage. Additionally, the sensors recapitulated the inhibition of M<sup>pro</sup> by the well-characterized pharmacological agent GC376. Further, in vitro assays with the BRET-based Mpro sensors revealed a molecular crowding-mediated increase in the rate of M<sup>pro</sup> activity and a decrease in the inhibitory potential of GC376. The sensors developed here will find direct utility in studies related to drug discovery targeting the SARS-CoV-2 M<sup>pro</sup> and functional genomics application to determine the effect of sequence variation in M<sup>pro</sup>.</p><p><br></p><h2>Other Information</h2><p dir="ltr">Published in: Communications Chemistry<br>License: <a href="https://creativecommons.org/licenses/by/4.0" target="_blank">https://creativecommons.org/licenses/by/4.0</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1038/s42004-022-00731-2" target="_blank">https://dx.doi.org/10.1038/s42004-022-00731-2</a></p>2022-09-28T03:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1038/s42004-022-00731-2https://figshare.com/articles/journal_contribution/A_genetically_encoded_BRET-based_SARS-CoV-2_Mpro_protease_activity_sensor/25662624CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/256626242022-09-28T03:00:00Z |
| spellingShingle | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor Anupriya M. Geethakumari (17052375) Biological sciences Biochemistry and cell biology Engineering Materials engineering Main protease (Mpro) SARS-CoV-2 replication Anti-SARS-CoV-2 agents Bioluminescence resonance energy transfer (BRET) Live cells In vitro assays mNeonGreen |
| status_str | publishedVersion |
| title | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| title_full | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| title_fullStr | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| title_full_unstemmed | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| title_short | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| title_sort | A genetically encoded BRET-based SARS-CoV-2 Mpro protease activity sensor |
| topic | Biological sciences Biochemistry and cell biology Engineering Materials engineering Main protease (Mpro) SARS-CoV-2 replication Anti-SARS-CoV-2 agents Bioluminescence resonance energy transfer (BRET) Live cells In vitro assays mNeonGreen |