Novel dengue virus NS2B/NS3 protease inhibitors

Abstract

Dengue fever is a severe, widespread, and neglected disease with more than 2 million diagnosed infections per year. The dengue virus NS2B/NS3 protease (PR) represents a prime target for rational drug design. At the moment, there are no clinical PR inhibitors (PIs) available. We have identified diaryl (thio)ethers as candidates for a novel class of PIs. Here, we report the selective and noncompetitive inhibition of the serotype 2 and 3 dengue virus PR in vitro and in cells by benzothiazole derivatives exhibiting 50% inhibitory concentrations (IC50s) in the low-micromolar range. Inhibition of replication of DENV serotypes 1 to 3 was specific, since all substances influenced neither hepatitis C virus (HCV) nor HIV-1 replication. Molecular docking suggests binding at a specific allosteric binding site. In addition to the in vitro assays, a cell-based PR assay was developed to test these substances in a replication-independent way. The new compounds inhibited the DENV PR with IC50s in the low-micromolar or submicromolar range in cells. Furthermore, these novel PIs inhibit viral replication at submicromolar concentrations.

FIG 1

Allosteric site of DENV-3 PR with the docked compounds 3, 6, and 8. The NS2B unit is shown in green and the NS3 unit in cyan. Ligands are rendered as sticks colored according to the Corey, Pauling, and Koltun (CPK) color scheme, with the exception of carbon atoms (compound 3 in white, compound 6 in yellow, and compound 8 in pink). Catalytic triad residues His51, Asp75, and Ser135 in the active site, which is behind the allosteric site, are represented as balls and sticks.

FIG 2

Predicted binding modes of compounds 3 and 8. The PIs were docked into DENV-3 PR by the LeadIT-FlexX program. The image was generated with PyMOL. (A) Surface view of the allosteric site with the docked compounds 3 and 8. Ligands are rendered as sticks colored according to the Corey, Pauling, and Koltun (CPK) color scheme, with the exception of carbon atoms (compound 3 in cyan and compound 8 in pink). (B) Binding mode of compound 8 showing the H bonds between two the hydroxyl groups and the amino acids Lys73, Lys74, and Asn152.

FIG 3

Docking results for PI 6 within the DENV-3 PR. (A) Three-dimensional interaction diagram of compound 6 with the amino acids Lys73, Thr120, Asn152, and Gln167. (B) Schematic view of all interactions (H bonds and hydrophobic interactions) of compound 6.

FIG 4

Inhibition of DENV-1 and DENV-2 replication. (A) Vero cells were preincubated with decreasing amounts of the compounds. DMSO served as a control. The cells were subsequently infected with DENV-2. Cell culture supernatants were collected and viruses titrated on Vero cells. All experiments were performed in triplicate assays. Error bars represent the standard deviation. Significances of differences in viral titers compared to the DMSO control were determined by the paired two-sample t test and are indicated by one (P < 0.05) or two (P < 0.01) asterisks above the bars. (B) Subtype specificity inhibition of replication by the PIs. Vero cells were preincubated with the compounds and infected with DENV-1 or DENV-2 (one genome copy [cp] per cell). Cell culture supernatants were collected, and viral genome amounts were determined by qRT-PCR. All experiments were performed in independent triplicate assays. Error bars represent the standard deviation.

FIG 5

Compounds 3 to 7 do not influence HIV-1. HEK 293T cells were transfected with the proviral HIV-1 plasmid pNL4-3 and subsequently incubated with the PIs. Viral titers were determined on TZM-bl indicator cells, which contain a LacZ gene controlled by the HIV-1 LTR promoter. All experiments were performed in triplicate assays. Error bars represent the standard deviation.

FIG 6

Components 1 and 3 to 7 do not influence HCV replication. (A) Scheme of the experimental approach. Huh7.5-Fluc cells were transfected with in vitro transcribed HCV RNA of the Renilla luciferase-encoding reporter virus JcR2a. (B) After 72 h, virus replication and cell viability were determined by measuring Renilla luciferase (Rluc) and firefly luciferase (Fluc) activity from cell lysates, respectively. AU, arbitrary fluorescence units. (C) Production of infectious particles was determined by infection of naive Huh7.5 cells with supernatants collected from transfected cells and quantification of Rluc activity after 72 h. Experiments were performed three times in triplicate (or sextuplicate in the case of DMSO-treated cells). The HCV protease inhibitor boceprevir was included as a positive control. A red dashed line indicates the limit of detection. All data represent the mean of three independent experiments, which were performed in triplicate assays. Error bars represent the standard deviation.

FIG 7

Compounds 1 and 3 to 7 inhibit DENV-2 PR in cells. (A) Scheme of the cleavage reporter construct. (B) Representative quantitative Western blotting analyses of reporter cleavage by the DENV-2 PR using a monoclonal anti-GFP antibody. Cells were transfected with the reporter vector alone (lane 1), with the PR expression vector alone, or with the reporter vector and the PR (lanes 3 to 11). PIs were added after transfection at four nontoxic concentrations (indicated above the panel). Substrate and products were visualized, and the inhibition was calculated as a ratio of fusion protein to fusion protein plus products. Expression of the DENV PR was monitored by Western blotting with monoclonal M2 antibodies. The positions of the molecular mass markers (kDa) are indicated.

FIG 8

Correlation of antiviral activity (expressed as log EC₅₀ [column 4 in Table 1]) and PR inhibition in cell culture (expressed as log IC₅₀ in the cell [column 5 in Table 1]). For the diagram, results for compounds 1, 3, 4, 5, 6, and 7 were taken.