This vector was transformed into One Shot BL21(DE3) competent cells (Thermo Scientific, Waltham, MA) and plated onto LB-AMP plates (InvivoGen, San Diego, CA). for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we developed and initiated a high-throughput cell-based screen that incorporates the essential viral papain-like protease (PLpro) and its peptide cleavage site into a luciferase complementation assay to evaluate the efficacy of known drugs encompassing approximately 15,000 clinical-stage or US Food and Drug Administration (FDA)-approved small molecules. Confirmed inhibitors were also tested to determine their cytotoxic properties. Here, we report the identification of four clinically relevant drugs that exhibit selective inhibition of the SARS-CoV-2 viral PLpro. using Invitrogen Maxiprep kits (Invitrogen, Waltham, MA) and Colec10 fully sequenced to confirm the correct sequence. MaxCyte Transient Transfection The MaxCyte transfection system was chosen over lipid-based methods due to its superior scalability and affordability.12 Briefly, 293T cells were grown in Dulbeccos modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) and 1% Anti-anti (all media reagents from Life Technologies, Carlsbad, CA). At Methacycline HCl (Physiomycine) 70C90% confluence, the 293T cells are harvested and resuspended in MaxCyte Electroporation Buffer at 1e8?cells/mL. DNA is usually added to the cells in the following ratios: 37% PLpro or vacant vector for high control cells, 55% FLuc reporterCPLpro, and 9% renilla plasmid (may be used for built-in cytotoxicity analysis, but we did not). The cells are electroporated using MaxCyte cassettes and the MaxCyte device per the manufacturers instructions. The cells are incubated for 20?min prior to seeding in flasks for a 4?h Methacycline HCl (Physiomycine) incubation. The cells were harvested and stored in liquid nitrogen to be used during high-throughput screening (HTS). PLpro 1536-Well Luciferase Assay The PLpro and vacant vector cells were thawed and counted. Compounds were pre-spotted onto fresh assay plates with either 5?nL (for 10?mM stocks of ReFRAME) or 20?nL (for 1?mM or 2.5?mM stocks of Pathogen Box or Target Mol). The cells were seeded at 2500 cells/well or 5e5?cells/mL in 293T growth medium using a BioRaptr FRD (Flying Reagent Dispenser; LGR, Carlsbad, CA) at 5 L/well. The plates were briefly spun at 1000 rpm and incubated for 48?h at 37?C, 5% CO2, and 95% relative humidity (RH). After a 48?h incubation, the plates were removed from the incubator and allowed to equilibrate to room temperature for 15?min. ONE-Glo Methacycline HCl (Physiomycine) (Promega, Madison, WI) luciferase reagent was added at 5 L/well with the BioRaptr FRD, and the plates were again briefly spun. After a 10?min incubation at room heat, the luminescence was measured using a ViewLux (PerkinElmer, Waltham, MA) for 30?s. The high control was vacant vector + FLuc wells, and the low control and data wells had PLpro?+ FLuc + compound or vehicle (DMSO). Post-HTS Confirmation Assay Following the completion of screening all three libraries, the most active and selective drugs were subjected to testing under the following conditions. HEK293T cells were transiently transfected in 6-well plates using jetPRIME transfection reagent (Polyplus, Illkirch-Graffenstaden, France), according to the manufacturers instructions, at the same ratios used in the MaxCyte transfection. After 4?h, transfection complexes were removed, and cells were reseeded into 96-well plates containing compounds at a density of 20,000 cells per well. Plates were then incubated at 37?C for 48?h. FLuc and renilla luciferase (RLuc) luminescence were detected using the Promega Dual Glo kit according to the manufacturers instructions. This procedure was done using both the SARS1 and SARS2 reporter systems, using plasmids with their analogous peptides based on the details referenced in the plasmid methods. Histidine-Tagged Small Ubiquitin-Like Modifier (His-SUMO) SARS-CoV-2 PLpro (1564C1877) Expression and Purification As a further test of specificity, we also characterized the most potent and selective drugs using a targeted biochemical SARS2 enzyme activity assay. First, we had to produce the enzyme. The SARS-CoV-2 PLpro (1564C1877, “type”:”entrez-nucleotide”,”attrs”:”text”:”MN908947.3″,”term_id”:”1798172431″,”term_text”:”MN908947.3″MN908947.3) amino acid sequence was codon optimized for expression, subcloned, and sequence verified (GenScript, Piscataway, NJ) into the pE-SUMOpro AMP vector (LifeSensors, Malvern, PA). This vector was transformed into One Shot BL21(DE3) competent cells (Thermo Scientific, Waltham, MA) and plated onto LB-AMP plates (InvivoGen, San Diego, CA). Transformants were inoculated in 100 mL terrific broth (TB) medium supplemented with 50 g/mL carbenicillin and incubated overnight at 37?C with shaking to saturation (OD600? 2). The overnight culture (~1:50 dilution) was used to inoculate fresh TB medium supplemented with Methacycline HCl (Physiomycine) 50 g/mL carbenicillin. A 3?L culture was incubated at 37?C with shaking to OD600 ~0.4, induced by adding IPTG to a final concentration of 0.5 mM, and cultured for an additional 24?h at 20?C, again with shaking. Cells were harvested by centrifugation, and the cell pellet was stored at ?80?C. The cell pellet was thawed on ice and resuspended in lysis buffer (50 mM HEPES, pH 8.0, 500 mM NaCl, 10 mM imidazole, 10% glycerol), DNase I (5 g/mL), and 1 SigmaFast protease inhibitor (Sigma Aldrich, St. Louis, MO).