Severity: Warning
Message: file_get_contents(https://...@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09): Failed to open stream: HTTP request failed! HTTP/1.1 429 Too Many Requests
Filename: helpers/my_audit_helper.php
Line Number: 143
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 143
Function: file_get_contents
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 209
Function: simplexml_load_file_from_url
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3098
Function: getPubMedXML
File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
Severity: Warning
Message: Attempt to read property "Count" on bool
Filename: helpers/my_audit_helper.php
Line Number: 3100
Backtrace:
File: /var/www/html/application/helpers/my_audit_helper.php
Line: 3100
Function: _error_handler
File: /var/www/html/application/controllers/Detail.php
Line: 574
Function: pubMedSearch_Global
File: /var/www/html/application/controllers/Detail.php
Line: 488
Function: pubMedGetRelatedKeyword
File: /var/www/html/index.php
Line: 316
Function: require_once
The 3CL protease (3CLpro) is a viral cysteine protease of SARS-CoV-2 and is responsible for the main processing of the viral polyproteins involved in viral replication and proliferation. Despite the importance of 3CLpro as a drug target, the intracellular dynamics of active 3CLpro, including its expression and subcellular localization in SARS-CoV-2-infected cells, are poorly understood. Herein, we report an activity-based probe (ABP) with a clickable alkyne and an irreversible warhead for the SARS-CoV-2 3CL protease. We designed and synthesized two ABPs that contain a chloromethyl ketone (probe ) or 2,6-dichlorobenzoyloxymethyl ketone (probe ) reactive group at the P1' site. Labeling of recombinant 3CLpro by the ABPs in the purified and proteome systems revealed that probe displayed ligand-directed and selective labeling against 3CLpro. Labeling of transiently expressed active 3CLpro in COS-7 cells also validated the good target selectivity of probe for 3CLpro. We finally demonstrated that endogenously expressed 3CLpro in SARS-CoV-2-infected cells can be detected by fluorescence microscopy imaging using probe , suggesting that active 3CLpro at 5 h postinfection is localized in the juxtanuclear region. To the best of our knowledge, this is the first report investigating the subcellular localization of active 3CLpro by using ABPs. We believe that probe will be a useful chemical tool for acquiring important biological knowledge of active 3CLpro in SARS-CoV-2-infected cells.
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Source |
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http://dx.doi.org/10.1021/acschembio.4c00024 | DOI Listing |
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