Small molecules targeting severe acute respiratory syndrome human coronavirus

Abstract

Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel human coronavirus. Currently, no effective antiviral agents exist against this type of virus. A cell-based assay, with SARS virus and Vero E6 cells, was developed to screen existing drugs, natural products, and synthetic compounds to identify effective anti-SARS agents. Of >10,000 agents tested, approximately 50 compounds were found active at 10 microM; among these compounds, two are existing drugs (Reserpine 13 and Aescin 5) and several are in clinical development. These 50 active compounds were tested again, and compounds 2-6, 10, and 13 showed active at 3 microM. The 50% inhibitory concentrations for the inhibition of viral replication (EC(50)) and host growth (CC(50)) were then measured and the selectivity index (SI = CC(50)/EC(50)) was determined. The EC(50), based on ELISA, and SI for Reserpine, Aescim, and Valinomycin are 3.4 microM (SI = 7.3), 6.0 microM (SI = 2.5), and 0.85 microM (SI = 80), respectively. Additional studies were carried out to further understand the mode of action of some active compounds, including ELISA, Western blot analysis, immunofluorescence and flow cytometry assays, and inhibition against the 3CL protease and viral entry. Of particular interest are the two anti-HIV agents, one as an entry blocker and the other as a 3CL protease inhibitor (K(i) = 0.6 microM).

Figures

Fig. 1.

The fluorogenic substrate used for SARS-CoV 3CL protease inhibition assay. Enhanced fluorescence caused by cleavage of the fluorogenic substrate peptide was monitored at 538 nm with excitation at 355 nm.

Fig. 2.

The test for cytopathogenic effects. +++ indicates that the protective effect is obvious, and ++ and + indicate moderate and weak, respectively.

Fig. 3.

Representative compounds showed protective effect at 10 μM. Parentheses indicate the result of cytotoxicity. (A) indicates the compounds that are noninhibitory to Vero E6 at concentrations greater than four times the anti-SARS concentrations. (B) indicates compounds that are noninhibitory at the anti-SARS concentrations. However, growth retardation, to ≈80% of the control level, was observed when Vero E6 cells were treated with these compounds at two times the anti-SARS concentrations.

Fig. 4.

ELISA of SARS-CoV spike protein in response to compound 10 treatment. % of control = (OD of SARS-CoV Inf. - OD of Mock.Inf. [Conc. x])/(OD of SARS-CoV. Inf. - OD of Mock-Inf. [Conc. 0]). EC50 = 0.85 μM; S.I. = 80. Inf., infection.

Fig. 5.

Compound 10-inhibited cytopathic effect induced by SARS-CoV and the viral protein expression shown by IFA.

Fig. 6.

Flow cytometry analysis of the inhibitory effect of compound 10 on SARS-CoV. S, spike protein; PE, phycoerythrin (a red fluorescent dye). The PE value represents the level of viral spike protein expression.

Fig. 7.

The commercially available compounds whose structures have 80% similarity with Glycyrrihizin, Aescin, and Reserpine that showed anti-SARS-CoV activities

Fig. 8.

Computer modeling of compound 2 binding to SARS-CoV 3CL protease. One of the two hydroxyl groups forms hydrogen bonds with Glu-166 through its side chain and the backbone amide bond. The phenyl groups fit into two pockets, one of which is well defined by hydrophobic residues (Met-165, Pro-168, and Leu-167).

Fig. 9.

The Lopinavir-like structures and their IC50 against 3CL protease.