Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities

Abstract

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Keywords: DENV inhibitors; ICR-suckling mouse; NS3 protease; RdRp; synergy.

Figure 1.

Proposed binding for derivatives 4 (green) and 5 (magenta). Residues involved in interactions are reported as cyan stick. H-bonds are shown as yellow dot lines. Protease is reported as cartoon: grey for NS3 and red for NS2 subunits.

Scheme 1.

Synthesis of the final compounds 1–3. (a) TEA, reflux, 7 h. (b) 4-Cl- or 4-NO₂-C₆H₄SO₂Cl, pyridine, 60 °C, overnight. (c) LiOH, 50 °C, 15 min. (d) SnCl₂, 80 °C, 3 h. (e) 2,5-(OMe)₂-THF, 100 °C, 1 h. (f) benzoyl chloride, AlCl_3, closed vessel, 110 °C, 70 W, 4 min.

Scheme 2.

Synthesis of the final compounds 4 and 5. (a) 4-OH-3,5-OMe₂- or 3,5-OMe₂-C₆H₄COCl, AlCl₃, closed vessel, 110 °C, 70 W, 2 min. (b) TES, TFA, 48 h, rt.

Figure 2.

Inhibition of DENV RNA replication and RdRp activity by compounds 1–3. Panel A. Huh-7 cells were infected with DENV-2 at a multiplicity of infection (M.O.I) of 0.2 and followed by treatment with the DENV polymerase inhibitor for 3 days. The DENV RNA level was analysed by RT-qPCR with specific primer targeting viral NS5 gene, and relative viral RNA levels were normalised against cellular GADPH mRNA levels. Treatment of 50 μM 2′-C-methylcytidine (2CMC) direct against DENV RdRp served as positive control. 0.1% DMSO (Mock) served as negative control. Panel B. Huh-7 cells were transfected with pEG(MITA)SEAP and pcDNA-NS2B-GSG-NS3-Myc followed by incubation with each compound. The luciferase activity was analysed after 3 days treatment. Error bars denote the means ± SD of three independent experiments. *p < .05; **p < .01.

Figure. 3.

Inhibition of DENV RNA and NS3-protease activity by compounds 4 and 5. Panel A. Huh-7 cells were infected with DENV-2 at a multiplicity of infection (M.O.I) of 0.2 and followed by the treatment of each DENV protease inhibitors for 3 days. The DENV RNA level was analysed by RT-qPCR with specific primer targeting viral NS5 gene, and relative viral RNA levels were normalised against cellular GADPH mRNA levels. Treatment of 50 μM 2′-C-methylcytidine (2CMC) direct against DENV RdRp served as positive control. 0.1% DMSO (Mock) served as negative control. Panel B. Huh-7 cells were transfected with pEG(MITA)SEAP and pcDNA-NS2B-GSG-NS3-Myc followed by incubation of each compounds, and the luciferase activity was analysed after 3 days treatment. Error bars denote the means ± SD of three independent experiments. *p < .05; **p < .01.

Figure 4.

DENV RdRp inhibitor 3 protected ICR-suckling mice from DENV infection. 6-day-old ICR-suckling mice were intracerebrally injected with heat-inactive DENV (iDENV, n = 5) or active DENV (DENV, n = 4). Mice-receiving DENV were treated with 10 mg/kg of compound 3 (n = 5) at 1, 3, 5 dpi. Panel A, clinical scores; Panel B, body weight and Panel C, survival rate were recorded every day. Disease severity was scored as follow: 0: healthy, 1: slightly sick (reduced mobility), 2: inappetance, 3: weight loss and difficult to move, 4: paralysis, 5: death. Each group included 6 mice. Error bars denote the means ± SD.

Figure 5.

DENV protease inhibitor 4 protected ICR-suckling mice from DENV infection. Six-day-old ICR-suckling mice were intracerebrally injected with heat-inactive DENV (iDENV, n = 6) or active DENV (DENV, n = 6). Mice-receiving DENV were treated with 1 mg/kg of compound 4 (n = 6) at 1, 3, 5 dpi. Panel A, clinical scores, Panel B, body weight, and Panel C, survival rate were recorded every day. Disease severity was scored as follow: 0: healthy, 1: slightly sick (reduced mobility), 2: inappetance, 3: weight loss and difficult to move, 4: paralysis, 5: death. Each group included six mice. Error bars denote the means ± SD.

Figure 6.

Combination assay of RdRp (3) and NS3 protease (4) inhibitors. Huh-7 cells were infected with DENV-2 at a multiplicity of infection (M.O.I) of 0.2 and followed by treatment of 3 and 4 with indicated concentration for 3 days. The DENV RNA level was analysed by RT-qPCR with specific primer targeting viral NS5 gene, and relative viral RNA levels were normalised against cellular GADPH mRNA levels. Error bars denote the means ± SD of three independent experiments. *p < .05; **p < .01.