Identification of a small-molecule entry inhibitor for filoviruses

Abstract

Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC(50)s) of 10 μM and 12 μM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.

FIG. 1.

Specificity of inhibition by compound 7. (A) Comparison of antiviral activity and cell toxicity of compound 7. GFP-ZEBOV was incubated with Vero E6 cells at an MOI of 5 for 1 h in the presence or absence of compound 7 in a dose-dependent manner as described in Materials and Methods. The blue diamonds represent anti-GFP-EBOV activity, while the red squares represent cytotoxicity. (B) The inhibitory effect of compound 7 on virus infectivity was investigated using HIV/MARV-GP pseudotype and infectious cell culture-grown MARV as described in Materials and Methods. Three independent experiments were performed to determine the effect of compound 7 on virus infectivity, and standard deviations are indicated. (C) Retroviral pseudotypes of LASV (HIV/LASV-GP), LCMV (HIV/LCMV-GP), and MACV (HIV/MACV-GP) were added to 293T cells, and influenza H5 [HIV/HA(H5)] pseudotype virus was added to A549 cells, in the presence of the indicated concentrations of compound 7. Pseudotype infection was assessed after 72 h by luciferase reporter assay. Three independent experiments were performed to determine the effect of compound 7 on virus infectivity, and standard deviations are indicated.

FIG. 2.

Compound 7 binds to EBOV-GP and inhibits virus infection. (A) Schematic diagram of a “single-cycle time-of-addition experiment” with HIV/EBOV-GP to determine the stage of virus entry blocked by compound 7. This experiment was designed to characterize the mechanism of action of the antiviral compounds. (B) A single-cycle time-of-addition experiment was done with HIV/EBOV-GP to determine the stage of EBOV entry blocked by compound 7. 293T cells were infected with 100 μl of p24-normalized HIV/EBOV-GP. Compound 7 was added and left for 1 h before infection (−1 h), for 1 h during adsorption (0 h), and for 1 h after infection (+1 h). Infected monolayers were washed with PBS and incubated for 72 h. Inhibition of HIV/EBOV-GP pseudotype infection was detected as a reduced luciferase signal. Error bars indicate standard deviations. (C) A Microcon Ultracell YM3 centrifugal device (Millipore) was used to study the binding of compound 7 to HIV/EBOV-GP and HIV/VSV-G pseudotype viruses. Pseudotype viruses (or a no-virus buffer control) were incubated with 25 μM compound 7 at room temperature for 60 min, and the virus-compound mixture was then loaded onto the Microcon Ultracell YM3 centrifugal device. After centrifugation, the flowthrough was analyzed for unbound compound by high-pressure liquid chromatography. The percentage of bound compound was calculated based on the peak area compared to that of a no-virus control. Error bars indicate standard deviations.

FIG. 3.

Computational studies locate a hydrophobic pocket at or near the GP1-GP2 interface in the GP prefusion crystal structure. (A) The three proposed binding sites S1 to S3, calculated with the Schrödinger Sitemap program, are shown in colored grids. Hydrophobic, ligand donor, and ligand acceptor maps are shown in yellow, blue, and red, respectively. EBOV-GP1 is shown in yellow and red ribbons, and GP2 is displayed in blue ribbons. (B) Magnified view of the S2 binding site circled in panel A. The putative binding site S2 was identified using Schrödinger SiteMap. Selected residues which are proposed to make contacts within the binding site are displayed as stick models. (C) The amino acid residues in the EBOV-GP1 sequence that are important for viral entry are shown. The charged residues are underlined, while hydrophobic residues are shown as gray shaded regions. The arrows indicate the positions of mutations in EBOV-GP1 of the HIV/EBOV-GP mutant pseudotype viruses utilized in this study.

FIG. 4.

Effect of compound 7 on the infectivity of HIV/EBOV-GP mutants. 293T cells were infected with mutant or wild-type HIV/EBOV-GP pseudotype virus in the presence of increasing concentrations of compound 7. Inhibition of HIV/EBOV-GP pseudotype infection was detected as a reduced luciferase signal. Error bars indicate standard deviations. (A) Effect of compound 7 on the infectivities of R64K, N69A, L70A, D78A, and K84A HIV/EBOV-GP mutants. (B) Effect of compound 7 on the infectivities of R172A, L184A, I185A, L186A, K190A/K191A, and Y225A HIV/EBOV-GP mutants.

FIG. 5.

Analysis of analogs of compound 7. (A) Summary of preliminary SAR results for benzodiazepine analogs of HIV/EBOV-GP compound 7. (B) Comparison of antiviral activity and cell toxicity of compound 12, as described in Materials and Methods. The blue diamonds represent anti-GFP-ZEBOV activity, while the red squares represent cytotoxicity. (C) Effect of compound 12 on the infectivities of N69A, L70A, L184A, I185A, L186A, and K190A/K191A HIV/EBOV-GP mutants. 293 T cells were infected with HIV/EBOV-GP pseudotype virus in the presence of increasing concentrations of compound 12. Inhibition of HIV/EBOV-GP infection was detected as a reduced luciferase signal. Error bars indicate standard deviations.