Evaluation of Phenol-Substituted Diphyllin Derivatives as Selective Antagonists for Ebola Virus Entry

Abstract

Ebola virus (EBOV) is an aggressive filoviral pathogen that can induce severe hemorrhagic fever in humans with up to 90% fatality rate. To date, there are no clinically effective small-molecule drugs for postexposure therapies to treat filoviral infections. EBOV cellular entry and infection involve uptake via macropinocytosis, navigation through the endocytic pathway, and pH-dependent escape into the cytoplasm. We report the inhibition of EBOV cell entry via selective inhibition of vacuolar (V)-ATPase by a new series of phenol-substituted derivatives of the natural product scaffold diphyllin. In cells challenged with Ebola virus, the diphyllin derivatives inhibit viral entry dependent upon structural variations to low nanomolar potencies. Mechanistically, the diphyllin derivatives had no effect on uptake and colocalization of viral particles with endocytic marker LAMP1 but directly modulated endosomal pH. The most potent effects were reversible exhibiting higher selectivity than bafilomycin or the parent diphyllin. Unlike general lysosomotrophic agents, the diphyllin derivatives showed no major disruptions of endocytic populations or morphology when examined with Rab5 and LAMP1 markers. The dilated vacuole phenotype induced by apilimod treatment or in constitutively active Rab5 mutant Q79L-expressing cells was both blocked and reversed by the diphyllin derivatives. The results are consistent with the action of the diphyllin scaffold as a selective pH-dependent viral entry block in late endosomes. Overall, the compounds show improved selectivity and minimal cytotoxicity relative to classical endosomal acidification blocking agents.

Keywords: V-ATPase inhibitor; diphyllin; endosome trafficking; macropinocytosis; viral entry inhibitor.

Figure 1

Natural-product-inspired V-ATPase inhibitors and/or other viral entry inhibitors.

Figure 2

Synthetic scheme for alkyl and amide inhibitors. (A) (i) ClCH₂CH₂CH₂R, K₂CO₃, dimethyl sulfoxide (DMSO), 3–16 h, 100 °C. (B) (ii) BrCH₂COOEt, K₂CO₃, DMSO, 100 °C, 2 h; (iii) 0.2 M NaOH H₂O:IPA (1:4), rt, 16 h; and (iv) R₂NH, triethylamine (TEA), HCTU, N,N-dimethylformamide (DMF), 3 h, MW (irradiation), 100 °C.

Figure 3

Time addition measurements of compound action and activity in primary human macrophages. (A) Time of addition of compounds indicates mechanism of action at early steps in virus replication. HeLa cells were pretreated for 1 h and then infected with wild-type EBOV or treated 2, 6, or 18 post infections. Cells were dosed with 1.5 (0.16 μM), 1.1 (2 μM), or 2 μM hydroxychloroquine. Infection efficiency was normalized to untreated controls, and one-way analysis of variance (ANOVA) was used to determine significance by P = <0.05 (*) between time points and the untreated controls. (B) EBOV infection in primary human macrophages. Primary human macrophages were infected with live GFP-EBOV after pretreatment with compounds for 1 h. Compounds were not removed before infection. Infected cells were fixed, stained, and imaged with epifluorescent microscopy, and infection efficiency was quantified by counting infected cells and normalizing to the total cell count in CellProfiler software. Prism 8 software was used to generate a graph, fit curves, and calculate IC₅₀ values.

Figure 4

Effect of diphyllin and derivatives on VLP cell binding. (A) Representative images of the Ebola GFP-VLP (green) internalization assay in HeLa cells pretreated with compounds and stained with a cell mask (yellow dotted line) 2 h post-VLP addition. Compounds were dosed at the following concentrations: EIPA at 50 μM, NH₄Cl at 100 mM, diphyllin (1.1) at 500 nM, 1.2–1.3 at 100 nM, 1.5 at 10 nM, 2.1 at 250 nM, 2.2–2.3 at 100 nM, 2.4 at 500 nM, and 2.6 at 250 nM. (B) Quantification of GFP-VLP internalization into cells using CellProfiler software. Statistics represent one-way ANOVA with multiple comparisons to no drug + VLP control.

Figure 5

Impact of diphyllin and derivatives on VLP trafficking to lysosomal compartments. (A) Representative images of pretreated HeLa cells infected with GFP-VLPs (green). Two hour post addition, cells were fixed, stained for LAMP1 (red), and imaged with epifluorescent imaging. Compounds were dosed at the following concentrations: NH₄Cl at 100 mM, diphyllin (1.1) at 500 nM, 1.2–1.3 at 100 nM, 1.5 at 10 nM, 2.1 at 250 nM, 2.2–2.3 at 100 nM, 2.4 at 500 nM, and 2.6 at 250 nM. (B) Quantification of the colocalization between GFP-VLPs and LAMP1+ vesicles. Statistics represent a one-way ANOVA with multiple comparisons to the no drug control.

Figure 6

Effect of diphyllin and derivatives on the morphology and population of Rab5+ early endocytic vesicles. (A) Confocal imaging of GFP-Rab5-expressing HEK293 cells treated with drugs for 3 h and stained with Hoechst 33342 nuclear stain. Compounds were dosed at the following concentrations: bafilomycin at 10 nM, diphyllin (1.1) at 500 nM, 1.2–1.3 at 100 nM, 1.5 at 10 nM, 2.1 at 250 nM, 2.2–2.3 at 100 nM, 2.4 at 500 nM, and 2.6 at 250 nM. (B) Quantification of Rab5+ punctate vesicle diameters. Rab5+ vesicles were binned based on their appearance as either punctate or dilated structures. Diameters of vesicles were measured with ImageJ and recorded in Prism 9 software. (C) Quantification of Rab5+ vesicle population. Using ImageJ software, the number of vesicles (punctate and dilated) were counted per cell and analyzed by nested one-way ANOVA with multiple comparisons to DMSO.

Figure 7

Effect of diphyllin and derivatives on the morphology and population of LAMP1+ lysosomal vesicles. (A) Confocal imaging of mCherry-LAMP1-expressing HEK293 cells treated with drugs for 3 h and stained with Hoechst 33342 nuclear stain. Compounds were dosed at the following concentrations: bafilomycin at 10 nM, apilimod at 200 nM, diphyllin (1.1) at 500 nM, 1.2–1.3 at 100 nM, 1.5 at 10 nM, 2.1 at 250 nM, 2.2–2.3 at 100 nM, 2.4 at 500 nM, and 2.6 at 250 nM. (B) Quantification of LAMP1+ lysosome size. LAMP1+ vesicles were binned based on their appearance as either punctate or dilated structures. Diameters of vesicles were measured with ImageJ and recorded in Prism 9 software. (C) Quantification of LAMP1+ vesicle population. Using ImageJ software, the number of vesicles (punctate and dilated) was counted per cell. Statistics reflect a one-way nested ANOVA run with multiple comparisons to DMSO.

Figure 8

Blockage and reversal of apilimod-induced LAMP1 lysosomal coalescence phenotype by diphyllin and derivatives. (A) Representative images of mCherry-LAMP1-expressing HEK293 cells treated with 500 nM diphyllin and 100 nM derivatives (1.2–1.5) for 1 h followed by a 1 h 200 nM apilimod treatment. (B) Representative images of mCherry-LAMP1-expressing cells treated with apilimod for 1 h followed by a 1 h treatment with diphyllin and derivatives. (C) Quantification of the number of dilated mCherry+ vesicles >1 μM in size when mCherry-LAMP1-expressing HEK293 cells were treated with diphyllin and derivatives for 1 h followed by a 1 h apilimod treatment. (D) Quantification of the number of dilated mCherry+ vesicles >1 μM in size when mCherry-LAMP1-expressing HEK293 cells were treated with apilimod for 1 h followed by a 1 h treatment with diphyllin and derivatives. Statistics represent a one-way ANOVA with multiple comparisons to DMSO.

Figure 9

Reversibility of diphyllin and derivatives on endocytic pH. (A) HEK293 cells were treated with compounds for 3 h. Bafilomycin was dosed at 10 nM, diphyllin (1.1) at 500 nM, 1.2–1.4 at 100 nM, and 1.5 at 10 nM. Subsequently, cells were washed with phosphate-buffered saline and either loaded with acridine orange and read for the immediate post washout response or incubated in Opti-MEM media for an additional 24 h before loading and reading the 24 h post washout response. (B) Diphyllin and derivatives tested on the dual uptake assay. HEK293 cells pretreated with compounds for 1 h were loaded with a pH-sensitive probe (pHRodo Green Dextran 10 kDa) and a pH-insensitive probe (TMR-Dextran 10 kDa). Bafilomycin was dosed at 10 nM, diphyllin (1.1) at 500 nM, NH₄Cl at 40 mM, and all diphyllin analogues (1.2–1.5) at 100 nM. Twenty minutes after loading with the dyes, cells were stained with nuclear stain and imaged with live-cell confocal microscopy. CellProfiler software was used to identify stained vesicular structures and calculate the ratio of fluorescence intensities of the pH-sensitive dye to pH-insensitive dye. Structures were binned into small and large bins based on their size. Statistics represent a one-way ANOVA with multiple comparisons to the DMSO control for each bin.