Pseudotyped viruses can mimic the entry process mediated by exogenous viral envelope proteins that belong to the class I fusion type, while viral genome release and reporter protein expression in post fusion steps proceed much like those for the HIV virus. Our work suggests that these 7 drugs could enter into further in vivo studies and clinical investigations for COVID-19 treatment. Keywords: COVID-19, SARS-CoV-2, virus entry inhibitors, high throughput screening assay, Benzathine penicilline approved drug library, histamine receptor antagonists, clemastine Introduction Since the outbreak of COVID-19, there have been ~15 million confirmed cases worldwide, and this number continues to grow . Whole-genome sequencing of samples from patients with COVID-19 pneumonia Rabbit Polyclonal to CCRL2 resulted in the identification of a beta-coronavirus that had never been seen before that was different from but related to SARS-CoV and MERS-CoV . There are multiple sequence segments in the receptor-binding domain of the S protein of SARS-CoV-2 that have a high degree of homology with SARS-CoV [2, 3]. Moreover, some studies have confirmed that SARS-CoV-2 can bind to the ACE2 receptor on the cell surface, as does SARS-CoV, which then mediates virus infection [4C6]. Therefore, inhibitors targeting the S protein of SARS-CoV-2 may become potential drugs for the treatment of COVID-19 by preventing viral entry. Given the urgent need for specific antiviral drugs, drug repurposing is an effective strategy for identifying available drugs and promising molecular leads for novel drug development, which can shorten the time and reduce the costs compared with de novo drug discovery . Through this method, some FDA-approved drugs have already been tested in clinical trials, including Benzathine penicilline arbidol, favipiravir, and hydroxychloroquie [8C11], both domestically and internationally. However, most of these drugs have made a limited contribution to the control of the pandemic . It is widely accepted that SARS-CoV-2 is likely to persist as a pandemic for a longer period than anticipated 3 months ago. Therefore, there will remain a need to develop novel antiviral drugs with high activity and specificity that will be of critical benefit for our long-term control of this emerging disease. Several viral proteins have been suggested as Benzathine penicilline drug targets and are currently being investigated, such as the 3CL protease, which had been selected as a target for both SARS-CoV and SARS-CoV-2 virus [13, 14]. In the work described in this article, we adopted a method for screening a unique set of approved drugs with a clear known targeted cellular pathway that could possibly be effective against SARS-CoV-2 infection in vitro. We found that seven of the drugs screened in vitro were able to effectively inhibit SARS-CoV-2 infection via blockade of the viral entry step. Of these, antihistamine drugs might be of benefit to COVID-19 patients because of their anti-SARS2 activity and additional anti-inflammatory effects. We suggest that further in vivo studies and clinical investigations Benzathine penicilline should be undertaken for the seven anti-SARS2 drugs we report here. Materials and methods Cell lines Huh-7 cells were obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Vero E6 and 293?T cell lines were obtained from ATCC (Manassas, VA, USA). All cell lines were grown in Dulbeccos modified Eagle medium (DMEM, Thermo Fisher, Shanghai, China) supplemented with 10% fetal bovine serum (FBS) as described previously [15, 16]. Plasmid and pseudovirus production To generate expression plasmids for the SARS-CoV and SARS-CoV-2 spike proteins (SARS-S, SARS2-S), the coding sequence of each protein was obtained from the published viral genome (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”AY278491″,”term_id”:”30023963″,”term_text”:”AY278491″AY278491 for SARS-CoV, GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”MN908947″,”term_id”:”1798172431″,”term_text”:”MN908947″MN908947 for SARS-CoV-2) and optimized for mammalian expression. An optimized coding sequence was chemically synthesized (Inovogen, Chengdu,China) and cloned into the pcDNA3.1 vector (Thermo Fisher, Shanghai, China), which was verified by sequencing. To generate a MERS spike protein (MERS-S) expression plasmid, the codon-optimized gene of the full-length S protein of MERS-CoV (GenBank: “type”:”entrez-protein”,”attrs”:”text”:”AFS88936″,”term_id”:”407076737″,”term_text”:”AFS88936″AFS88936) with replacement of the N-terminal signal peptide (aa 1C17) with the CD5 signal sequence  was synthesized (Inovogen, Chengdu,China) and inserted into the pcDNA3.1 vector, and it was also verified by sequencing. To produce viral spike protein-pseudotyped HIV virions, the HIV backbone vector pNL4-3. Benzathine penicilline Luc. R-E- was used for pseudovirus packaging. 293?T cells were cotransfected with the respective envelope protein expression plasmid plus the pNL4.3LucR-E- plasmid using X-tremeGENE DNA HP Transfection Reagent (Roche) according to the manufacturers instructions. At 72?h post-transfection, supernatants were harvested and filtered through 0.45 m filters, after which the filtered supernatant was further concentrated 10-fold by ultracentrifugation with a filter cartridge with a 100?kDa cutoff, aliquoted,.
Pseudotyped viruses can mimic the entry process mediated by exogenous viral envelope proteins that belong to the class I fusion type, while viral genome release and reporter protein expression in post fusion steps proceed much like those for the HIV virus
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