Novel inhibitors of severe acute respiratory syndrome coronavirus entry that act by three distinct mechanisms

Abstract

Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS coronavirus (SARS-CoV) and for which there are currently no approved treatments. We report the discovery and characterization of small-molecule inhibitors of SARS-CoV replication that block viral entry by three different mechanisms. The compounds were discovered by screening a chemical library of compounds for blocking of entry of HIV-1 pseudotyped with SARS-CoV surface glycoprotein S (SARS-S) but not that of HIV-1 pseudotyped with vesicular stomatitis virus surface glycoprotein G (VSV-G). Studies on their mechanisms of action revealed that the compounds act by three distinct mechanisms: (i) SSAA09E2 {N-[[4-(4-methylpiperazin-1-yl)phenyl]methyl]-1,2-oxazole-5-carboxamide} acts through a novel mechanism of action, by blocking early interactions of SARS-S with the receptor for SARS-CoV, angiotensin converting enzyme 2 (ACE2); (ii) SSAA09E1 {[(Z)-1-thiophen-2-ylethylideneamino]thiourea} acts later, by blocking cathepsin L, a host protease required for processing of SARS-S during viral entry; and (iii) SSAA09E3 [N-(9,10-dioxo-9,10-dihydroanthracen-2-yl)benzamide] also acts later and does not affect interactions of SARS-S with ACE2 or the enzymatic functions of cathepsin L but prevents fusion of the viral membrane with the host cellular membrane. Our work demonstrates that there are at least three independent strategies for blocking SARS-CoV entry, validates these mechanisms of inhibition, and introduces promising leads for the development of SARS therapeutics.

Fig 1

Specificity of initial hits selected from the initial screen. Forty-four compounds identified as inhibitors during the initial screening of the Maybridge Hitfinder chemical library were tested again using either SARS/HIV pseudotyped virions (A) or control VSV/HIV pseudotyped virions (B) to determine whether the inhibitors specifically blocked SARS-CoV entry. Compounds A (SSAA09E1), B (SSAA09E2), and C (SSAA09E3) were selected for further characterization, as they were efficient inhibitors of SARS/HIV but not VSV/HIV entry (<20% decrease in luminescence). Additional compounds that decreased luminescence by ∼20 to 50% in VSV/HIV entry experiments were considered to be less specific SARS/HIV pseudotype inhibitors and will be characterized in future studies. Experiments were performed three independent times, and error bars represent standard deviations for the three measurements. RLU, relative light units.

Fig 2

Dose-response assays of inhibition of SARS/HIV and VSV/HIV pseudotype entry. ACE2-expressing 293T cells were infected with SARS/HIV (■) or control VSV/HIV (▲) pseudovirions in the presence of various concentrations of inhibitors. Panels A, B, and C present data for compounds SSAA09E2, SSAA09E1, and SSAA09E3, respectively. Data shown are mean values with standard deviations derived from three independent experiments.

Fig 3

Chemical names and structures of the selected entry inhibitors.

Fig 4

(A) Effects of compounds on cathepsin L activity. Purified recombinant cathepsin L (2 units/assay mixture) was incubated with a 25 μM concentration of a fluorogenic substrate for cathepsin L (Z-Phe-Arg-7-amido-4-methylcoumarin) in the presence or absence of various concentrations of SSAA09E1 (■), SSAA09E2 (◆), and SSAA09E3 (▲), as described in the text. Data shown are mean values with standard deviations derived from three independent experiments. CPD, compound. (B) Effect of SSAA09E1 on cathepsin B activity. The specificity of SSAA09E1 was evaluated by testing its ability to block cathepsin B activity. Purified recombinant cathepsin B (2 units/assay mixture) was incubated with a 25 μM concentration of a fluorogenic substrate for cathepsin B (Z-Arg-Arg-7-amido-4-methylcoumarin) (with the first Arg being the difference from the cathepsin L substrate) in the presence or absence of a known specific cathepsin B inhibitor (CA074) or SSAA09E1. Data shown are mean values with standard deviations derived from three independent experiments. RFU, relative fluorescence units.

Fig 5

Effects of inhibitors on interactions of SARS-S RBD with soluble ACE2. The purified SARS-S RBD was incubated with purified soluble ACE2 in the presence and absence of increasing concentrations (0 to 20 μM) of the three SARS-CoV entry inhibitors. Immunoprecipitation and immunoblot analyses were carried out as described in the text. Experiments were independently confirmed three times.

Fig 6

Effects of compounds on SARS-S-mediated cell-to-cell fusion. The fusion assay was performed by overlaying TZM-bl cells transfected with a plasmid encoding ACE2 on 293T cells transfected with plasmids encoding SARS-S and Tat. After 3 h of incubation, 2 μg/ml trypsin was added to induce fusion. Cell fusion was determined at 6 h postinduction, by measuring luciferase activity as described in the text. To investigate the effects of the inhibitors on fusion of the SARS-S envelope with the host cellular membrane, compounds SSAA09E1 (■), SSAA09E2 (◆), and SSAA09E3 (▲) were added either after the cell overlay but before fusion induction (A) or before cell overlay and fusion induction (B). Experiments were performed three times, and error bars represent standard deviations from the means.

Fig 7

Time-of-addition experiment to validate inhibition mechanism. ACE2-expressing 293T cells infected with SARS/HIV pseudovirions in the presence of the inhibitors (10 μM) were tested for suppression of luciferase activity. Compounds were added at 0, 0.2, 0.5, 1, 3, 6, 9, 12, and 24 h postinfection (hpi), and luciferase activity was measured at 48 hpi as described in the text. A cathepsin L inhibitor [Z-FY(t-Bu)-DMK] (50 nM) was used as a control. Experiments were performed twice in triplicate, and error bars represent standard deviations from the means.

Fig 8

Effects of compounds on postentry steps of SARS-CoV life cycle. Compounds were tested for the ability to block SARS-CoV replication in a replicon-based system. HEK 293T cells were transfected with a plasmid encoding a SARS-CoV replicon in the presence of 20 μM SSAA09E1, SSAA09E2, or SSAA09E3 as described in the text. SSYA10-001 is a SARS-CoV replication inhibitor that targets the nsp13 helicase. Total RNA was isolated at 48 h posttransfection and analyzed by RT-qPCR as described in the text and as we have previously published (41, 55). Experiments were repeated three times, each time in triplicate, and error bars represent standard deviations for three independent experiments.

Fig 9

Stages of SARS-CoV entry inhibited by novel SARS-CoV small-molecule inhibitors. Following interaction of SARS-S with the ACE2 receptor on the permissive cell surface, the virus is endocytosed. After endocytosis of the virus, cathepsin L cleaves SARS-S to S1 and S2, allowing subsequent fusion of the viral membrane with the endosomal membrane. Our data suggest that SSAA09E2 prevents viral entry by blocking the interaction of SARS-S with the ACE2 receptor, SSAA09E1 impedes viral entry by inhibiting cathepsin L processing of the SARS-S envelope in the endosome, and SSAA09E3 inhibits viral entry by preventing fusion of the viral membrane with the host cellular membrane.