| Abstract: | Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 μM in inhibiting Mpro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mpro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti−SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.The current worldwide impact of the COVID-19 pandemic has been so profound that it is often compared to that of the 1918 influenza pandemic (1, 2). As of February 4, 2021, the total global COVID-19 cases had surpassed 100 million, among which more than 2 million had succumbed to death (3). Important political figures who have been diagnosed COVID-19 positive include the US president Donald Trump and the UK prime minister Boris Johnson. A modeling study has predicted that this pandemic will continue to affect everyday life, and the circumstances may require societies to follow social distancing until 2022 (4). Finding timely treatment options is of tremendous importance to alleviate catastrophic damages of COVID-19. However, the short time window that is required to contain the disease is extremely challenging for a conventional drug discovery process that requires typically many years to finalize a drug and therefore might not achieve its goal before the pandemic ceases. In January 2020, we did a comparative biochemical analysis between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that has caused COVID-19, and SARS-CoV-1 that led to an epidemic in China in 2003, and proposed that remdesivir was a viable choice for the treatment of COVID-19 (5). We were excited to see that remdesivir was finally approved for emergency use in the United States and for use in Japan for people with severe symptoms. With only one medicine in stock that provides very limited benefits to COVID-19 patients (6), the virus may easily evade it, leaving us once again with no medicine to use. Given the rapid spread and the high fatality rate of COVID-19, finding alternative medicines is imperative. Drug repurposing stands out as an attractive option in the current situation. If an approved drug can be identified to treat COVID-19, it can quickly proceed to clinical trials and be manufactured at a large scale using its existing good manufacturing practice (GMP) lines. Previously, encouraging results were obtained from repurposing small-molecule medicines, including teicoplanin, ivermectin, itraconazole, and nitazoxanide (7–10). These antimicrobial agents showed antiviral activity against Ebola, Chikungunya, enterovirus, and influenza viruses, respectively (11). However, a common drawback of a repurposed drug is its low efficacy level. One way to circumvent this problem is to combine multiple existing medicines to accrue a synergistic effect. To be able to discover such combinations, breaking down the druggable targets of SARS-CoV-2 to identify drugs that do not cross-act on each other’s targets is a promising strategy. For example, a recent study showed that triple combination of interferon β-1b, lopinavir−ritonavir, and ribavirin was safe and superior to lopinavir−ritonavir alone for treating COVID-19 patients (12).In our January paper (5), we recommended four SARS-CoV-2 essential proteins, including Spike, RNA-dependent RNA polymerase, the main protease (Mpro), and papain-like protease, as drug targets for the development of anti−COVID-19 therapeutics. Among these four proteins, Mpro that was previously called 3C-like protease provides the most facile opportunity for drug repurposing, owing to the ease of its biochemical assays. Mpro is a cysteine protease that processes itself and then cleaves a number of nonstructural viral proteins from two very large polypeptide translates that are made from the viral genomic RNA in the human cell host (13). Its relatively large active site pocket and a highly nucleophilic, catalytic cysteine residue make it likely to be inhibited by a host of existing and investigational drugs. Previous work has disclosed some existing drugs that inhibit Mpro (14). However, complete characterization of existing drugs on the inhibition of Mpro is not yet available. Since the release of the first Mpro crystal structure, many computational studies have been carried out to screen existing drugs in their inhibition of Mpro, and many potent leads have been proposed (15–18). However, most of these lead drugs are yet to be confirmed experimentally. To investigate whether some existing drugs can potently inhibit Mpro, we have docked a group of selected Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines to the active site of Mpro and selected about 30 hit drugs to characterize their inhibition on Mpro experimentally. Our results revealed that a number of FDA/EMA-approved small-molecule medicines have high potency in inhibiting Mpro, and bepridil inhibits cytopathogenic effect (CPE) induced by the SARS-CoV-2 virus in Vero E6 and A549/ACE2 cells with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study encourages further preclinical testing of bepridil in animal models, paving the way to its clinical use against COVID-19. |
