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SARS-CoV-2 3CLpro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376

Heilmann et al., Science Translational Medicine, doi:10.1126/scitranslmed.abq7360
Jan 2023  
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In Vitro and In Silico study showing selection of resistant mutations with nirmatrelvir use.
Several mutations were identified that confer resistance to 3CLpro inhibitors nirmatrelvir, ensitrelvir, and GC376. Authors note that most of these have already been found in existing SARS-CoV-2 sequences.
Authors argue for highly selective use because extensive, unselective use is expected to rapidly lead to emergence of drug resistance.
Heilmann et al., 11 Jan 2023, peer-reviewed, 15 authors.
In Vitro studies are an important part of preclinical research, however results may be very different in vivo.
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SARS-CoV-2 3CL pro mutations selected in a VSV-based system confer resistance to nirmatrelvir, ensitrelvir, and GC376
Emmanuel Heilmann, Francesco Costacurta, Seyed Arad Moghadasi, Chengjin Ye, Matteo Pavan, Davide Bassani, Andre Volland, Claudia Ascher, Alexander Kurt, Hermann Weiss, David Bante, Reuben S Harris, Stefano Moro, Bernhard Rupp, Luis Martinez-Sobrido, Dorothee Von Laer
Protease inhibitors are among the most powerful antiviral drugs. Nirmatrelvir is the first protease inhibitor specifically developed against the SARS-CoV-2 protease 3CL pro that has been licensed for clinical use. To identify mutations that confer resistance to this protease inhibitor, we engineered a chimeric vesicular stomatitis virus (VSV) that expressed a polyprotein composed of the VSV glycoprotein (G), the SARS-CoV-2 3CL pro , and the VSV polymerase (L). Viral replication was thus dependent on the autocatalytic processing of this precursor protein by 3CL pro and release of the functional viral proteins G and L, and replication of this chimeric VSV was effectively inhibited by nirmatrelvir. Using this system, we applied nirmatrelvir to select for resistance mutations. Resistance was confirmed by retesting nirmatrelvir against the selected mutations in additional VSV-based systems, in an independently developed cellular system, in a biochemical assay, and in a recombinant SARS-CoV-2 system. We demonstrate that some mutants are cross-resistant to ensitrelvir and GC376, whereas others are less resistant to these compounds. Furthermore, we found that most of these resistance mutations already existed in SARS-CoV-2 sequences that have been deposited in the NCBI and GISAID databases, indicating that these mutations were present in circulating SARS-CoV-2 strains.
KTSAVLQSGFRKME-EDANS. IC 50 and EC 50 calculations and statistical analysis for all assays were performed with GraphPad Prism 9 (see the "Statistical analysis" section). Nanopore sequencing of recombinant SARS-CoV-2 (rWA1) expressing mCherry To validate the sequence of the recombinant SARS-CoV-2 (rWA1) expressing mCherry, we used the Nanopore sequencing "Midnight protocol," version 6 (57) . Primer pools generating 1200-base pair (bp) overlapping amplicons were purchased from Integrated DNA Technologies, as referenced in the abovementioned protocol. The sequencing reactions were prepared using the Rapid Barcoding Kit SQK-RBK110.96 (Oxford Nanopore Technologies) and were performed in a sequencer (MinION Mk1B) using a proprietary flow cell (R9.4.1, Oxford Nanopore Technologies). Electrical signals were translated into nucleotide sequences (basecalling). Sequenced reads were sorted into separate files for each sample (demultiplexing). Demultiplexing was done using the super highaccuracy model in Guppy 6.1.5. Output sequences generated socalled fastq files, and sequences below 200 and above 1200 bp were removed. Sequences between 200 and 1200 bp were assembled with the algorithm epi2me-labs/wf-artic v0.3.18 in Nextflow 22.04.4. The SARS-CoV-2 lineage pangolin 4.1.1 was used to map the sequences. A visualization application (Nextclade 2.4.0) was used to check mutations. Protein structure preparation for molecular modeling The three-dimensional structure of..
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