Quinoxaline-based inhibitors of Ebola and Marburg VP40 egress

Abstract

We prepared a series of quinoxalin-2-mercapto-acetyl-urea analogs and evaluated them for their ability to inhibit viral egress in our Marburg and Ebola VP40 VLP budding assays in HEK293T cells. We also evaluated selected compounds in our bimolecular complementation assay (BiMC) to detect and visualize a Marburg mVP40-Nedd4 interaction in live mammalian cells. Antiviral activity was assessed for selected compounds using a live recombinant vesicular stomatitis virus (VSV) (M40 virus) that expresses the EBOV VP40 PPxY L-domain. Finally selected compounds were evaluated in several ADME assays to have an early assessment of their drug properties. Our compounds had low nM potency in these assays (e.g., compounds 21, 24, 26, 39), and had good human liver microsome stability, as well as little or no inhibition of P450 3A4.

Keywords: Antiviral therapeutic; Budding; Ebola virus; L-domain; Marburg virus; Nedd4; PPxY; RNA viruses; Virus egress.

Figure 1

BiMC analysis of HEK293T cells co-expressing NYFP-Nedd4 and CYFP-mVP40 in the presence of DMSO alone, or 0.1 μM concentration of 21, 24 or 39. Representative images are shown where the total number of cells were quantified using NucBlue, and Green (YFP) indicates cells with a positive interaction between Nedd4 and mVP40. Scale bar, 200 μm. YFP-positive cells were quantified in triplicate using MetaMorph software and statistical analysis as described previously.

Figure 2

Marburg VP40 VLP budding assay. HEK293T cells were transfected with mVP40 plasmid in the presence of DMSO alone, or the indicated inhibitor (24 or 26) at the indicated concentrations. mVP40 was detected by Western blot in cell extracts and VLPs at 24 hours post-transfection. mVP40 was quantified using NIH Image-J software..

Figure 3

Marburg and Ebola VP40 VLP budding assays. HEK293T cells were transfected with either mVP40 or eVP40 plasmids in the presence of DMSO (0) alone, or budding inhibitor 21 at the indicated concentrations. mVP40 and eVP40 were detected by Western blot in cell extracts and VLPs at 24 hours post-transfection. mVP40 and eVP40 were quantified using NIH Image-J software. Bar graphs represent the average of three independent experiments.

Figure 4

Inhibition of Live VSV-M40 Virus Budding. HEK293T cells were infected (in triplicate) with VSV-M40 for 8 hours under the indicated conditions, and virion containing supernatants were harvested and quantified by plaque assay performed in triplicate. Virus titers are indicated as a percent relative to control. *** indicates a p values <0.001 as determined by a two-tailed student t-test. Western blots of infected cell extracts are shown for VSV M and actin, which demonstrate that treatment with the indicated concentrations of 21 and 39 for 8 hours did not affect viral or cellular protein levels compared to DMSO alone controls.

Scheme 1

General Preparation of Target Compounds

Scheme 2

Preparation of Certain Specific Target Compounds