Development of therapeutics for SARS-CoV-2 infection

Division Of Basic Sciences - Nci

Investigators

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Abstract

We have examined a variety of compounds that have reportedly been active against SARS-CoV. We specifically selected and synthesized a panel of active compounds based on previously known structures and their activity against SARS-CoV. We have now identified two small molecule compounds, GRL-1720 and GRL-2420, both of which target the main protease (Mpro) of SARS-CoV-2 and potently block the infectivity, replication, and cytopathicity of SARS-CoV-2WK-521. As assessed using the quantitative VeroE6 cell-based assay with RNA-qPCR, the EC50 values of GRL-1720 and GRL-2420 were 15 and 4.2 microM, respectively, and apparent CC50 values were both 100 microM. Since the cytotoxicity of test compounds in cell-based assays is often mistakenly judged to be the apparent antiviral activity being estimated by the multitudes of toxicity-associated reduction of the number of virion copies produced in the assays. Indeed, none of such toxic agents did not become of clinical utility as antiviral agents. Thus, we carefully asked if the three compounds, GRL-1720, GRL-2420, and remdesivir exerted cytotoxicity at 1, 10, and 100 microM. VeroE6 cells cultured alone appeared to be robust and had spread at the bottom of the microtiter culture plates; however, VeroE6 cells exposed to SARS-CoV-2WK-521 and cultured in the absence of test compounds, many cells got granular patterns and got detached from the bottom of the culture plate, indicating the cells were infected and killed by the cytopathicity of the virus. In contrast, when the SARS-CoV-2WK-521-exposed cells were cultured in the presence of each of the three compounds at 10 microM, the cells appeared to be moderately protected and at 100 microM, all the cells appeared to be completely protected by each compound and all three images of the cells cultured with 100 microM of each compound appeared to be similar to the image of the cells cultured alone. In order to confirm and corroborate the potent activity seen in GRL-1720 and GRL-2420 by using the quantitative RNA-qPCR assay, which often does not differentiate the actual antiviral activity from the misleading and distractive 'antiviral effect' caused by the cytostatic effect and/or cytotoxicity of test compounds, we employed immunocytochemistry, which can allow us to examine the antiviral activity of test compounds at cellular level. For the primary antibody in our immunocytochemistry, we used the IgG fraction isolated from a COVID-9-convalescent patient who proved to have high titers of neutralizing antibodies as well as SARS-CoV-2-binding IgG antibodies. However, when VeroE6 cells were exposed to SARS-CoV-2WK-521 and cultured in the absence of test compound, the F-actin structure had been lost and a number of cells had been infected by the virus and stained in green. In contrast, the SARS-CoV-2WK-521-exposed VeroE6 cells were cultured in the presence of 10 microM GRL-1720, there was a significant reduction in the number of SARS-CoV-2WK-521-infected cells and there were essentially no infected cells when the cells were cultured in the presence of 100 microM GRL-1720. In the presence of 10 microM GRL-2420, there was further significant reduction in the number of infected cells and there were no infected cells when culture at 100 microM GRL-2420. Remdesivir also significantly reduced the number of infected cells at 10 and 100 microM, however, there was viral breakthrough in the culture and some infected cells were identified. In order to further examine the antiviral activity of remdesivir, GRL-1720, and GRL-2420, we tested the antiviral activity against SARS-CoV-2WK-521, virus-exposed VeroE6 cells were cultured in the presence of a wide range of concentrations of each compound. In the set of immunocytochemistry data, there was no virus breakthrough identified when all the fields with Cytation 5 were examined at concentrations 50 through 100 microM; however, there was obvious virus breakthrough even at 150 and 200 microM of remdesivir. The formation of tetrahedral ketals has been observed for covalently bound inhibitors of Mpro. The hydroxyl oxygen forms hydrogen bond interactions with the backbone amine nitrogens of Ser144 and Cys145 as well as with the sidechain hydroxyl of Ser144. There are several other polar interactions between GRL-2420 and Mpro. The benzothiazole nitrogen forms a hydrogen bond with the backbone NH of Gly143. GRL-2420 has a 4-methoxyindole-2-carbonyl as a P3-substituent. The indole NH forms a hydrogen bond interaction with the backbone carbonyl oxygen of Glu-166. The oxygen of the methoxy indole forms a hydrogen bond interaction with the side chain of Gln189. GRL-2420 has a 2-oxopyrrolidine group as P1-moiety, and its oxygen forms a hydrogen bond interaction with the backbone NH of Glu-166. There is an additional hydrogen bond from a peptide NH to the backbone carbonyl of His164. GRL-2420-Mpro covalently linked ketal converts to a carbonyl with non-covalent binding with Mpro in a reversible manner. In fact, in our LC/MS studies, we observed that the Mpro and GRL-2420 eluted separately indicating they did not form a covalent bond under the conditions in which LC/MS experiments were carried out. There are some differences in interactions of the P1' moiety compared to the covalently bound ketal form. The benzothiazole carbonyl oxygen forms hydrogen bond interactions with the backbone amines of Gly143 and Ser144 and a weaker polar interaction with the backbone amine hydrogen of Cys145. The later interaction is weaker since the distance between the carbonyl oxygen and amine hydrogen has increased to 3.31 angstrom and we employ a cut-off distance of 3.0 angstrom to determine strong hydrogen bonds. The corresponding distance between the hydroxyl oxygen and backbone amine hydrogen of Cys145 is 1.95 angstrom for the tetrahedral ketal. The polar interaction between the benzothiazole carbonyl and the side chain hydroxyl of Ser144 is also weakened. The hydrogen bond interaction between the benzothiazole nitrogen and Gly143 is lost. The hydrogen bond interaction from the P1-2-oxopyrrolidine moiety with the backbone NH of Glu-166 is maintained, as is the hydrogen bond interaction from the P3-4-methoxyindole to the side chain of Gln189. As part of the overall structural rearrangement, the indole NH and the backbone carbonyl oxygen of Glu-166 move away from each other. Overall, both the covalently bound tetrahedral ketal as well as the non-covalent bound carbonyl form of GRL-2420 make a number of stabilizing interactions with active site residues of Mpro. In order to ask whether GRL-1720 and GRL-2420 form covalent bond, ESI-QTOF/MS analysis was performed to study the nature of the inhibition after treatment of Mpro with GRL-1720 or GRL-2420. As compared with the mass of apoMpro, mass increments of +145.26 Da and +144.58 Da in the presence of GRL-1720, were observed. The increased size corresponds to the molecular weight of 1H-indpline-4-carbonyl group-binding. In the active site of Mpro, Cys145 and His41 appear to form a catalytic dyad. As a consequence, the sulfur atom gains nucleophilicity, and presumably exerts a nucleophilic attack on the electrophilic carbon atom of the polar carbonyl group from GRL-1720. The mass increment was seen when Mpro was treated with GRL-1720 for both 30 minutes and 3 hours. In contrast, no changes in the mass were identified in Mpro treated with GRL-2420 over 30 minutes as compared with Mpro unexposed to GRL-2420. No changes in the mass were seen when Mpro was exposed to GRL-2420 for 3 hours, either, suggesting that GRL-2420 does not form covalent bond with Mpro contrary to the modelling or that the compound forms reversible covalent bond or that GRL-2420 forms only non-covalent binding.