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. 2021 Jul 16;65(8):e0234920.
doi: 10.1128/AAC.02349-20. Epub 2021 Jul 16.

A Novel Series of Indole Alkaloid Derivatives Inhibit Dengue and Zika Virus Infection by Interference with the Viral Replication Complex

Antonios Fikatas  1 , Peter Vervaeke  1 , Eef Meyen  1 , Nuria Llor  2 , Sergi Ordeix  2 , Ine Boonen  3 , Magda Bletsa  3 , Liana Eleni Kafetzopoulou  3 , Philippe Lemey  3 , Mercedes Amat  2 , Christophe Pannecouque #  1 , Dominique Schols #  1
Affiliations

A Novel Series of Indole Alkaloid Derivatives Inhibit Dengue and Zika Virus Infection by Interference with the Viral Replication Complex

Antonios Fikatas et al. Antimicrob Agents Chemother. .

Abstract

Here, we identified a novel class of compounds which demonstrated good antiviral activity against dengue and Zika virus infection. These derivatives constitute intermediates in the synthesis of indole (ervatamine-silicine) alkaloids and share a tetracyclic structure, with an indole and a piperidine fused to a seven-membered carbocyclic ring. Structure-activity relationship studies indicated the importance of substituent at position C-6 and especially the presence of a benzyl ester for the activity and cytotoxicity of the molecules. In addition, the stereochemistry at C-7 and C-8, as well as the presence of an oxazolidine ring, influenced the potency of the compounds. Mechanism of action studies with two analogues of this family (compounds 22 and trans-14) showed that this class of molecules can suppress viral infection during the later stages of the replication cycle (RNA replication/assembly). Moreover, a cell-dependent antiviral profile of the compounds against several Zika strains was observed, possibly implying the involvement of a cellular factor(s) in the activity of the molecules. Sequencing of compound-resistant Zika mutants revealed a single nonsynonymous amino acid mutation (aspartic acid to histidine) at the beginning of the predicted transmembrane domain 1 of NS4B protein, which plays a vital role in the formation of the viral replication complex. To conclude, our study provides detailed information on a new class of NS4B-associated inhibitors and strengthens the importance of identifying host-virus interactions in order to tackle flavivirus infections.

Keywords: NS4B protein; SAR studies; Zika virus; cross resistance; dengue virus; indole alkaloids; mechanisms of action.

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Figures

FIG 1
FIG 1
Structure of indole alkaloid derivatives. Chemical groups and sites crucial for the enhancement of antiviral activity are marked with circles and arrows.
FIG 2
FIG 2
Detection of ZIKV envelope protein in the presence of compound 22 using immunofluorescence staining in A549 and Vero cells. Both A549 and Vero cells were exposed to the ZIKV MR766 strain at an MOI of 0.2 and incubated with several concentrations of compound 22. Fluorescence images were acquired 72 h postinfection. (A) Cell control; (B) virus control; (C to F) 0.4 μM, 2 μM, 10 μM, and 50 μM compound 22.
FIG 3
FIG 3
Compounds interfere with the viral RNA replication/assembly. (A) Treatment of Vero cells with a 9 μΜ concentration of compounds 22 (red) and trans-14 (blue) shows inhibition of DENV infection even at later stages (8 h to 12 h p.i.), as assessed by qRT-PCR of cell lysates 20 h p.i. The viral polymerase inhibitor 7-deaza-2′-C-methyladenosine (7DMA) (black) presents a similar profile, as it suppresses the infection at 12 h p.i. On the other hand, the entry inhibitor dextran sulfate (DS) (green) inhibits virus replication at the early stages (−2 h to 0 h). Horizontal red lines represent virus controls. (B) Compounds 22 and trans-14 were incubated with BHK/DENV cells at different concentrations. A decrease in the luciferase activity was observed for both compounds, as well as the viral inhibitor 7DMA (blue), as assessed by luminescence. These results indicate an interaction with the nonstructural proteins or the replication complex. Incubation of compounds 22 and trans-14 shows no cellular cytotoxicity at the tested concentrations (red). All data are averages from two independent experiments.
FIG 4
FIG 4
Compounds do not interact with the intact viral particles. (A) Compound 22 at 50μΜ, 10μΜ, and 2μΜ, as well as the positive control epigallocatechin gallate (EGCG) (1 mg/ml), was mixed with DENV2 stock and passed through a filter. Incubation of compound 22-treated samples on BHK cells resulted in no inhibition of viral infection in each tested condition (formation of viral plaques). Inoculation of cells with EGCG-treated sample demonstrated full inhibition of viral infection. (B) Compound 22 T 10 μΜ was mixed with DENV2 viral stock and further diluted 100 times to infect BHK cells. No suppression of viral infection was noticed in the compound-treated sample, as assessed by the plaque assay. Both positive (virus-infected cells) and negative (noninfected, DMSO-treated cells) controls were included in the assays.
FIG 5
FIG 5
Sequencing analysis of the viruses used in the cross-resistance assay. (A) NS4B membrane topology and alignment of amino acid sequence at the N-terminal site of the protein in compound 22- and trans-14-resistant virus mutants compared to virus control. An aspartic acid-to-histidine (D31H) nonsynonymous mutation is located at the pTMD1 domain and marked in orange. Passage 16 (P16) of each virus strain was used in the assay. (B) Antiviral activity of several derivatives against the viruses used above (wild-type ZIKV MR766 and compound 22- and trans-14-resistant virus mutants) was evaluated by qRT-PCR in the supernatant of A549 cells at 72 h p.i. Fold shifts in EC50 are indicated in parentheses, while major findings on the activity of some analogues against the trans-14-resistant mutant and wild-type virus are highlighted in yellow.
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