Identification of existing pharmaceuticals and herbal medicines as inhibitors of SARS-CoV-2 infection

Abstract

The outbreak of COVID-19 caused by SARS-CoV-2 has resulted in more than 50 million confirmed cases and over 1 million deaths worldwide as of November 2020. Currently, there are no effective antivirals approved by the Food and Drug Administration to contain this pandemic except the antiviral agent remdesivir. In addition, the trimeric spike protein on the viral surface is highly glycosylated and almost 200,000 variants with mutations at more than 1,000 positions in its 1,273 amino acid sequence were reported, posing a major challenge in the development of antibodies and vaccines. It is therefore urgently needed to have alternative and timely treatments for the disease. In this study, we used a cell-based infection assay to screen more than 3,000 agents used in humans and animals, including 2,855 small molecules and 190 traditional herbal medicines, and identified 15 active small molecules in concentrations ranging from 0.1 nM to 50 μM. Two enzymatic assays, along with molecular modeling, were then developed to confirm those targeting the virus 3CL protease and the RNA-dependent RNA polymerase. Several water extracts of herbal medicines were active in the cell-based assay and could be further developed as plant-derived anti-SARS-CoV-2 agents. Some of the active compounds identified in the screen were further tested in vivo, and it was found that mefloquine, nelfinavir, and extracts of Ganoderma lucidum (RF3), Perilla frutescens, and Mentha haplocalyx were effective in a challenge study using hamsters as disease model.

Keywords: SARS-CoV-2; antiviral; cell-based and animal studies; drug repurposing.

Fig. 1.

Representative drugs showed antiinfective effects at 10 μM. These drugs are categorized according to their potential mode of action against SARS-CoV-2.

Fig. 2.

Dose–response relationships of 15 selected antiviral compounds. Vero E6 cells were pretreated with compounds at indicated doses followed by SARS-CoV-2 infection for 48 h. The percentage of viral titer determined by antinucleocapsid antibody after drug treatment (red) and cell viability (blue) were measured and expressed as mean ± SD of at least three independent experiments.

Fig. 3.

Structures of protease inhibitors and their IC₅₀ and K_i values for 3CL protease inhibition. The values were determined from three independent experiments using FRET-based enzymatic assays.

Fig. 4.

Computer simulation of (A) nelfinavir and (B) boceprevir binding to SARS-CoV-2 3CL protease (PDB ID code 6LU7). Pink dashed lines indicated interaction between compounds and protein.

Fig. 5.

SARS-CoV-2 spike protein sequence mutation analysis. (A) Analysis of spike protein mutation from the 196,276 sequences revealed 1,141 sites of mutation in the 1,273 amino acids of spike protein. The spike protein sequence of hCoV-19/Taiwan/4/2020 used in this study was identical to the original virus strain (UniProt Entry: P0DTC2). Single bottom line: S1 region (residues 13 to 685); double bottom line: S2 region (residues 686 to 1273); yellow bottom line: receptor binding domain (residues 319 to 541); green top line: N-glycosylation motifs; blue top line: O-glycosylation sites; pink: mutation residues; sequence representative: P0DTC2 (UniProt Entry). (B) Top and side view of the S protein indicating residue variants (from strictly conserved to highly variable: white to red), N-glycans (green stick), O-glycosylation sites (blue) in three-dimensional structure (PDB ID code 7CN9).

Fig. 6.

Evaluations of antiviral effect of Chinese herbal medicines in serial dilutions were presented as log₂(dilution fold). Anti–SARS-CoV-2 infection effects of selected Chinese herbal medicines as water extracts (1.0 g/20 mL H₂O) and RF3 dissolved in H₂O (0.25 mg/mL) are presented. The tested results of all Chinese herbal medicines are summarized in SI Appendix, Fig. S7.

Fig. 7.

In vivo anti–SARS-CoV-2 assay conducted in female golden Syrian hamsters. (A) Virus elimination effect of drugs and extracts. Hamsters were infected with SAR-CoV-2 by intranasal instillation at day 0, and treated with drugs and extracts orally twice a day (30 mg/kg/d for drugs and 200 mg/kg/d for extracts) continuously for 3 d. After 3 d, the lungs were collected to measure the virus load (n = 5), *P < 0.05; **P < 0.005. (B) Body-weight change after 3-d treatment, n = 5 for test group and n = 6 for the control group.