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. 2008 Jul 4;283(27):18734-42.
doi: 10.1074/jbc.M802089200. Epub 2008 May 12.

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Andrew M Lee  1 , Jillian M Rojek, Christina F Spiropoulou, Anette T Gundersen, Wei Jin, Alex Shaginian, Joanne York, Jack H Nunberg, Dale L Boger, Michael B A Oldstone, Stefan Kunz
Affiliations

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Andrew M Lee et al. J Biol Chem. .

Abstract

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15-35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC(50)=500-800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC(50) of 200-350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.

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Figures

FIGURE 1.
FIGURE 1.
HTS of combinatorial chemical libraries for inhibitors of LASV entry. A, for HTS of combinatorial chemical libraries, recombinant retroviruses containing a luciferase reporter in their genome were pseudotyped with the envelope GP of LASV. Because the GP of arenaviruses is necessary and sufficient for viral entry, these pseudotypes allow HTS for entry inhibitors using the luciferase reporter for rapid and reliable readout of infection. Inhibition of LASV pseudotype infection is detected as a reduced luciferase signal. B, example of HTS of a combinatorial chemical library. LASV pseudotypes were preincubated with compounds (concentration was 50 μm for individual compounds and 5 μm for individual compounds in mixtures of 4–10) for 45 min and then added to monolayers of HeLa cells in 96-well plates. After 1 h, the inoculum was removed, and the cells were washed and incubated for 48 h. Infection was quantified using luciferase reporter gene assay. The data shown are two independent experiments screening the same library. Luminescence is expressed as fold increase over background. A1–A10, B1–B10, C1–C10, D1–D10, E1–E10, F1–F10, and G1–G10 (black bars) represent mixtures of seven different compounds each. The samples labeled C (white bars) correspond to solvent only controls. 100 and 50% of mean control values are indicated as horizontal lines. The mixtures B1–B9 result in >50% inhibition of LASV pseudotype infection in both screens (underlined).
FIGURE 2.
FIGURE 2.
Candidate inhibitors for LASV entry from combinatorial chemical libraries. A, selected candidate single compounds obtained by HTS of combinatorial chemical libraries utilizing retroviral pseudotypes of LASV. B, IC50 for the compounds in A determined based on the dose-response characteristic for the neutralization of LASV pseudotype infection in permissive human A549 lung epithelial cells (supplemental Fig. S1). C, activity of candidate compounds against LASV pseudotypes in different cell lines. Pseudotypes of LASV or VSV were incubated with candidate compounds prior to infection of human epithelial cell lines HeLa and A549 and the primate fibroblast VeroE6. Infection was determined after 48 h by luciferase assay with luminescence expressed as fold increase over background (n = 3 + S.D.).
FIGURE 3.
FIGURE 3.
Activity of candidate compounds against pseudotypes of the South American HF viruses. Retroviral pseudotypes of the South American HF viruses GTOV, JUNV, and MACV, as well as VSV pseudotypes were pretreated with candidate compounds at 20 μm prior to infection of A549 cells and infection determined by luciferase assay as in Fig. 1B. Luminescence is expressed as fold increase over background (n = 3 + S.D.). Note the marked reduction in infection with pseudotypes of the South American HF viruses after exposure to candidate compounds 17C8 and 17C9.
FIGURE 4.
FIGURE 4.
Activity of compounds against live arenaviruses. A, infectious LASV was pretreated with candidate compounds 8C1 (20 μm), 17G8 (10 μm), and 17C8 (10 μm) or solvent control (Me2SO) and added to permissive VeroE6 cells. After 1 h of infection, the cells were washed and incubated for 16 h. The cells were fixed, and infection was detected by immunofluorescence detection of viral antigens as described under “Experimental Procedures.” B, blocking of infection of VeroE6 cells with LASV, JUNV, and MACV with candidate drugs as in A. The bars represent average numbers of infected cells in four independent visual fields. One representative example of several independent infections is shown. DMSO, dimethyl sulfoxide.
FIGURE 5.
FIGURE 5.
Compounds block pH-dependent membrane fusion. The candidate compounds 8C1 and 17C8 were tested for their ability to block cell-cell fusion mediated by the GPs of LASV and JUNV. The candidate compound 8C1 inhibited LASV GP-mediated fusion in a dose-dependent manner but was weak against JUNV GP-mediated fusion. However, 17C8 inhibited fusion mediated by both viral GPs. Fraction cell fusion is defined as the ratio of chemiluminescence between the indicated concentration of compound and an untreated cell control. The data represent two independent experiments per GP and drug or solvent control. DMSO, dimethyl sulfoxide.
FIGURE 6.
FIGURE 6.
Selective activity of compounds against arenaviruses. Retroviral pseudotypes of TACV, WWA, Ebola virus, and Amphotropic retrovirus, as well as recombinant measles virus expressing GFP and WSN influenza virus were incubated with the indicated concentrations of compounds and added to VeroE6 cells or Madrin-Darby canine kidney cells for influenza. Pseudotype infection was assessed after 48 h by luciferase reporter assay. To determine infection with recombinant measles virus, GFP-expressing cells were scored, and WSN infection was assessed by immunofluorescence staining for viral NP (for details see “Experimental Procedures”). Luminescence signals are given as fold increase over background (n = 3 + S.D.). For the quantification of measles virus and influenza infection, the total number of infected cell clusters was counted per well (n = 3 + S.D.).
FIGURE 7.
FIGURE 7.
Structure-activity relationships of candidate compounds. A, the structures of libraries 16 and 17. The library was synthesized and screened as single compounds containing single R1 (A1–A3), R2 (B1–B5), and R3 (C1–C10) substitutions. B, example of HTS of chemical libraries 16 and 17. LASV pseudotypes were preincubated with compounds (concentration, 50 μm) for 45 min and then added to monolayers of HeLa cells in 96-well plates. Infection was determined as in Fig. 1. Luminescence is expressed as fold increase over background. The samples labeled Me2SO (white bars) correspond to solvent only controls. The hits 16G8, 17C8, and 17C9 are indicated. Analysis of the results indicates that the key elements required for activity include the closely related 2-indole (C7), 2-benzofuran (C8), or 2-benzothiophene (C9) aryl substituent (R3) attached to the piperazinone core. These bicyclic aryl substituents proved more potent than the corresponding 2-pyrrole, 2-furan, 2-thiophene, or phenyl aryl substituents defining a clear pattern of activity. Similarly, although not as widely explored as R3 in the initial library, the most potent activity was observed with a phenethyl C6 substituent at position R1 (PhCH2CH2- > PhCH2-> CH2CH3), and the nature of the aryl substitutions pattern at N2 (R2) significantly modulate this activity.
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