Antiviral treatments targeting the coronavirus disease 2019 are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as Remdesivir and Chroloquine in VeroE6/TMPRSS2 cells: the anti-inflammatory drug Cepharanthine and HIV protease inhibitor Nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry through the blocking of viral binding to target cells, whilst Nelfinavir suppressed viral replication partly by protease inhibition. Consistent with their different modes of action, synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation was highlighted. Mathematical modeling in vitro antiviral activity coupled with the calculated total drug concentrations in the lung predicts that Nelfinavir will shorten the period until viral clearance by 4.9-days and the combining Cepharanthine/Nelfinavir enhanced their predicted efficacy. These results warrant further evaluation of the potential anti-SARS-CoV-2 activity of Cepharanthine and Nelfinavir.

SummaryAntiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

medRxiv preprint Significance Statement (80/120) 47 Antiviral agents with different mechanisms of action have different curative effects 48 depending on precisely when therapy is initiated. Based on a model of viral 49 dynamics, parameterised using viral load data from SARS-CoV-2 infected patients 50 reported by Zou et al. (1), computer simulations were performed. We propose that 51 effective treatment of SARS-CoV-2 infection requires an appropriate choice of class-52 specific drugs and initiation timing as reported for treatment of other viral infections 53(2); otherwise, antivirals do not have a significant effect on the within-host viral 54 dynamics of SARS-CoV-2 and are wasted. 55

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Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 μM, IC90 = 2.31 μM, and IC99 = 4.39 μM in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

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Importance: Although the COVID-19 epidemic in some countries such as China are in the last phase by large effort for containment of the disease, another outbreaks can occur because huge susceptible population remains. Further, there remain countries in the early phase of outbreak with zero or limited number of cases in southern hemisphere countries. In those countries at risk of future outbreak, ascertaining whether cases are imported or the result of local secondary transmission is important for government to shape appropriate public health strategies. Objective: To develop a method to estimate timing of infection establishment, which helps differentiate imported and autochthonous cases. Design, Setting and Participants: Of the first 18 cases reported in Singapore, 12 were used in our study (1 case with insufficient data and 5 on anti-viral treatment were excluded from the analysis). The viral load data from these initial cases considered imported due to their travel history to Wuhan were analyzed. Another viral load data from 3 cases reported from Zhuhai, China, for whom exposed day were known, were also analyzed to determine the viral load threshold for infection establishment. Exposures: SARS-CoV-2 infection confirmed by the polymerase-chain-reaction (PCR) test. Main Outcomes and Measures: The timing of infection establishment of each case was assessed by analysing viral load data after symptom onset using a within-host viral dynamics model for SARS-CoV-2. Estimated timing of infection will indicate whether cases are imported or autochthonous transmission within Singapore. Results: Six among the 12 cases were clearly imported cases, whereas we could not rule out the possibility of secondary transmission for the rest of 6 cases, which collectively evidenced ongoing transmission in Singapore. For the 6 cases who could be the results of secondary transmission, further investigation to identify the source of infection within Singapore should be warranted (i.e., contact tracing). Conclusions and Relevance: In an early phase of outbreak due to entrance or re-entrance of the virus to countries/communities, collecting viral load data over time from cases from symptom onset is highly recommended, because viral load data are valuable to infer the timing of infection and distinguish between imported cases and ongoing local transmission.

Digestibility of maize starch granules with different amylose content (AL-0, 22, 54, 68, 80, or 90) was investigated. Measurement of the in vivo resistant starch (RS) content of the starches was performed using surgically prepared ileorectostomized rats. The rats were fed a purified diet containing one of the starches at 652.5 g/kg diet. The in vivo RS content was determined based on the fecal starch excretion. The dietary fiber (DF) value increased as a function of the amylose content in the starch and showed a positive linear correlation with the gelatinization temperature of the granules. In contrast, the in vitro RS content was likely to depend on both the surface area and amylose contents of the starch granules. The maximum in vitro RS content was obtained with AL-68 (54.4). In vivo RS content showed a significant correlation with the amount of in vitro RS but not in respect to the DF detected. The in vivo RS content of AL-68 (43.4) was higher than that found in AL-90 (37.8). A profound gap was observed for AL-54 between the amount of DF (6.4) and RS (in vitro = 46.6 and in vivo = 40.9) present. The results suggest that both in vitro and in vivo digestibility of maize starch is affected by the amylose content and surface area of the granules. The current evaluation suggests that the physiological occurrence of RS from maize starch might be predictable by reference to the in vitro RS value.