key: cord-0709847-8agznchi authors: Vuong, Wayne; Khan, Muhammed Bashir; Fischer, Conrad; Arutyunova, Elena; Lamer, Tess; Shields, Justin; Saffran, Holly A.; McKay, Ryan T.; van Belkum, Marco J.; Joyce, Michael; Young, Howard S.; Tyrrell, D. Lorne; Vederas, John C.; Lemieux, M. Joanne title: Feline coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication date: 2020-05-05 journal: bioRxiv DOI: 10.1101/2020.05.03.073080 sha: 17eb00a5f04d63f01618189d06933ee398c42673 doc_id: 709847 cord_uid: 8agznchi The COVID-19 pandemic, attributed to the SARS-CoV-2 coronavirus infection, resulted in millions infected worldwide and an immediate need for antiviral treatments. The main protease (Mpro) in SARS-CoV-2 is a viable drug target because of its essential role in the cleavage of the virus polypeptide and subsequent viral replication. Feline infectious peritonitis, a fatal infection in cats caused by a coronavirus, was successfully treated previously with a dipeptide-based protease inhibitor. Here we show this drug, GC376, and its analog GC373, are effective inhibitors of the Mpro from both SARS-CoV and SARS-CoV-2 with IC50 values in the nanomolar range. Crystal structures of the SARS-CoV and SARS-CoV-2 Mpro with these inhibitors have a covalent modification of the nucleophilic Cys145. NMR analysis reveals that inhibition proceeds via reversible formation of a hemithioacetal. GC373 and GC376 are potent inhibitors of SARS-CoV-2 in cell culture, with EC50 values near one micromolar and little to no toxicity. These protease inhibitors are soluble, non-toxic, and bind reversibly. They are strong drug candidates for the treatment of human coronavirus infections because they have already been successful in animals (cats). The work here lays the framework for their use in human trials for the treatment of COVID-19. 6 domain swapping and facilitates dimer formation 18 . Molecular replacement with structure 6Y7M.PDB revealed electron density in the Fo-Fc map at the catalytic cysteine for both inhibitors GC373 (PDB Code 6WTK) and GC376 (PDB Code 6WTJ). In both structures the peptidyl inhibitor is covalently attached to Cys-145 as a hemithioacetal, showing that as expected the bisulphite group leaves GC376 (Extended data, Figure S6 ). In contrast to the MERS M pro -GC376 structure, the SARS-CoV-2 M pro electron density indicated the formation of only one enantiomer for this inhibitor 12 . A strong hydrogen bond network is established from side chain of His163 and backbone amide of His164, and Glu166, with backbone contributions from Gly143, Ser144 and Cys145 defining the oxyanion hole (Figure 3) . Together this provides strong binding and a low IC 50 for the inhibitor. The glutamine surrogate in the substrate P1 position interacts with the side chain of His163, while the Leu in P2 inserts into a hydrophobic pocket, representing the S2 subsite of the enzyme. Similar to what was observed in the MERS-M pro -GC376 structure 12 , the benzyl ring and the b-lactam of the Gln surrogate forms a stacked hydrophobic interaction, which stabilizes the inhibitor in the active site of the protease. A close examination of the subsite for SARS-CoV-2 M pro reveals regions to allow for future inhibitor development (Extended data, Figure S6 ). To confirm the formation of a covalent hemithioacetal as a single enantiomer in the active site, GC373 was prepared with 13 C label (>99%) at the aldehyde carbon and mixed in 7.8fold excess with the M pro protease from SARS-CoV-2 in deuterated buffer. HSQC NMR analysis (700 MHz) showed appearance of a single crosspeak signal (one isomer only) for the hemithioacetal carbon at 76 ppm ( 13 C) and 5.65 ppm ( 1 H) in accordance with previous chemical shift reports for hemithioacetals 7 (Extended data, Figure S7 ). Recent crystal structural analysis reported differences in the residues residing between the dimer interface of SARS-CoV-2 M pro when compared with the SARS-CoV M pro13 . Previous mutagenesis studies, which altered residues at the dimer interface of SARS-CoV M pro , enhanced catalytic activity 3.6 fold 19 . In agreement with this, our analysis shows that the catalytic turnover rate for SARS-CoV-2-M pro (135±6 min -1 ) is almost 5 times faster than SARS-CoV M pro (30±2 min -1 ) with our substrate Abz-SVTLQSG-Tyr NO2 R ( Table 1) . With this FRET substrate, we demonstrate a higher catalytic efficiency with SARS-CoV-2 M pro (1.8±0.4 min -1 µM -1 ) compared to SARS-CoV M pro (0.6±0.2 min -1 µM -1 ). This finding is in contrast to recent reports where no differences were observed in the catalytic efficiency between SARS-CoV M pro and SARS-CoV-2 M pro using a different substrate: 3011±294 s -1 M -1 (0.18±0.02 min -1 µM -1 ) and 3426±416.9 s -1 M -1 (0.2±0.03 min -1 µM -1 ), respectively) 13 . It remains to be determined whether this influences the virulence of SARS-CoV-2. To test the ability of GC373 and GC376 to inhibit SARS-CoV-2, plaque reduction assays were performed on infected Vero E6 cells in the absence or the presence of increasing concentrations of either GC373 ( Figure 4A ) or GC376 ( Figure 4B ) for 48 hours. The results were plotted as a percent inhibition of the number of plaque forming units per well. The EC 50 for GC373 was 1.5 µM while the EC 50 for GC376 was 0.92 µM. To examine cell cytotoxicity, ATP production was measured using the CellTiter-Glo assay on either Vero E6 cells or A549 cells incubated in the presence of the inhibitors for 24 hours. The CC 50 s of both GC373 and GC376 were greater than 200 µM. To further examine their antiviral activities, quantitative RT-PCR was performed on 8 supernatants from cells untreated, and GC373 ( Figure 4C ) or GC376 ( Figure 4D ) treated cell cultures. It was observed that both GC373 and GC376 are potent inhibitors of SARS-CoV-2 decreasing viral titers 3 logs, compared with a 2 log decrease in recently published results using other aldehyde compounds 16 . These results indicate that both GC373 and GC376 are potent inhibitors of SARS-CoV-2 with a therapeutic index of >200. Numerous drugs were designed originally to inhibit the SARS-CoV M pro 3 . However, the SARS outbreak of 2002 was controlled by public health measures and these compounds were never licensed. GC376 has been used to cure FIP in cats as the M pro of FIP is effectively inhibited by its breakdown product GC373. Analogs of these drugs also inhibited the MERS CoV M pro and blocked viral replication in cells at an EC 50 of 0.5 µM 12 . Our studies show GC376 and GC373 to be effective inhibitors of SARS-CoV-2 M pro . Clearly these drugs need to be advanced quickly into human trials for COVID-19. SARS-CoV-2 is the cause of COVID-19 and is a virus with a significant mutation rate 20 . Also, in some patients the virus has persisted longer than 2 months with some possibility of re-infection 21 . Vaccines are critically important, but still likely a year or more away as this virus will likely present vaccine challenges. There are many clinical trials testing drugs repurposed from their original indications. Remdesivir, a polymerase inhibitor developed as a treatment for Ebola virus 22 is showing very promising early results and will likely be confirmed in clinical trials 23 Figure 3 . GC373 binds in the active site pocket of SARS-CoV-2 M pro . A) GC373 forms a covalent bond with Cys145, and the oxyanion is stabilized by backbone H-bonds with Gly143, Ser144, and Cys145. B) H-bonds are established with GC373 and His163 side chain as well as backbone of residues H164 and E166, which is supported by the backbone of His41. SARS-CoV-2 M pro is represented in cartoon representation with the inhibitor in pink color. Inhibitor is colored in pink and interacting residues are colored in blue. P1, P2 and P3 of the peptidyl-inhibitor are indicated. PDB Code:6WTJ. All commercially available reagents and protected amino acids were purchased and used without further purification unless otherwise noted. All the solvents used for reactions were used without further purification unless otherwise noted. Dry solvents refer to solvents freshly distilled over appropriate drying reagents prior to use. Commercially available ACS grade solvents (> 99.0% purity) were used for column chromatography without any further purification. All reactions and fractions from column chromatography were monitored by thin layer chromatography (TLC) using glass plates ( capped under a blanket of argon and allowed to warm to rt. The reaction was then allowed to stir at rt for 3h. Reaction mixture was then quenched with the addition of 3 mL of 10% Na2S2O3 followed with stirring for 15 min. The layers were then separated and the DCM layer was washed sequentially with 10% Na2S2O3 (1 x 3 mL), sat. NaHCO3 (2 x 3 mL), H2O (2 x 3 mL), and brine (2 x 3 mL). The DCM layer was then dried over Na2SO4 and concentrated to furnish a yellow residue. The TLC of this crude is very messy (extensive streaking) but the product was successfully purified via flash column chromatography on silica using an eluent system of 2.5/97.5 MeOH/EtOAc. Elution of product was monitored by TLC and KMnO4 staining (Rf = 0.14, 2.5/97.5 MeOH/EtOAc). Concentration of product fractions furnishes an oil that solidifies to a yellow foam upon co-evaporation with Et2O (0.206 g, 0.511 mmol, 65%). Afterwards, the mixture was filtered to remove solids and the solids were thoroughly washed with additional volumes of EtOH. The combined washings were dried over Na2SO4 and filtered again. Concentration of the filtrate furnishes a yellow oil. To this oil was added 0.5 mL of Et2O, causing a white solid to crash out after mixing. The mixture was then centrifuged and the Et2O was removed. This step was repeated once more. The resultant white solid was then treated with Et2O (0.444 mL) and EtOAc (0.222 mL). Mixing for 5 min followed by centrifuge and removal of solvent furnishes the white bisulfite adduct as the product. The material was dried on hi-vac overnight to remove residual solvent (0.018 g, 0.037 mmol, 50% The pellet and triturated crude residue were pooled together and dissolved in 0.1% aqueous TFA. The FRET peptide substrate was purified using a C18 RP-HPLC column with The corrected initial velocity of the reaction was calculated as V = Vo / (corr%). Vo represents the initial velocity of each reaction. Kinetic constants (vmax and Km) were derived by fitting the corrected initial velocity Triplicate experiments were performed for each data point, and the value was presented as mean ± standard deviation (SD). Stock solutions of GC373 and GC376 were prepared with 10% aqueous DMSO. (Table S1 ). For the complex of SARS-CoV-2 M pro with GC376 and GC373, the dataset collected, was processed at a resolution of 1.9 Å and 2.0 Å and in space group C2 (Table S1 ). All three structures were determined by molecular replacement with the crystal structure of the free enzyme of the SARS-CoV2 M pro (PDB entry 6Y7M as search model, using the Phaser program from Phenix. Ligand Fit from Phenix was employed for the fitting of both inhibitors in the density of pre-calculated map from Phenix refinement, using the ligand code K36. Refinement of the three structures was performed with phenix.refine in Phenix software. Statistics of diffraction, data processing and model refinement are given in (Table S1 ). All spectra were run "locked" on the 2 H resonance signal and chemical shifts were referenced using the residual proton 1 HOD signal position 7 (i.e. 4.7 ppm) prior to saturation. One dimensional 1 H data were acquired using presaturation 8, 9 for residual 1 HOD solvent suppression, followed by a 90° excitation pulse and data acquisition. The saturation carrier position amplitude were manually optimized using a position sweep array and based on the calibrated high-power pulse, respectively. Saturation was applied directly on the water resonance (i.e. depending on water suppression efficacy and the ability to avoid analog to digital and/or receiver overloads with sufficient gain for data acquisition). The saturation was applied with a gammaB1 induced field strength of ~80 Hz 10 , and a duration of 2 seconds. The 90° pulse width was determined after tuning and matching each sample, using one-pulse nutation optimization 11 . Other specific 1D acquisition parameter settings were: sweep width of 14044 Hz, acquisition time 2.5 seconds, with 70224 total (i.e. real plus imaginary) data points. 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