Exploring the therapeutic potential of DV-B-120 as an inhibitor of dengue virus infection

Abstract

Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

Keywords: DV-B-120; NS2B-NS3 protease; competitive inhibitor; selectivity index.

Fig 1

Characterization of compounds targeting DENV NS2B-NS3 protease. Each compound includes the compound name, structural formula, and molecular weight description.

Fig 2

Fluorescence quenching spectra for evaluating the effect of small-molecule inhibitors on DENV 1–4 NS2B-NS3 protease activity. The activity percentage of the DENV NS2B-NS3 protease was compared to the protease alone. Initially, a solution containing 5 µM of the protease was prepared. Subsequently, 50 µM of the compound and 10 µM of a fluorescence quencher substrate were added, and the fluorescence was measured at 460 nm with an excitation wavelength of 355 nm. The analysis was performed for DENV-1 (A), DENV-2 (B), DENV-3 (C), and DENV-4 (D). Relative protease activity was calculated by dividing the sample fluorescence by the DMSO fluorescence (blank) and multiplying it by 100 (%). The error bars represent the mean ± SD, and statistical significance is indicated by *P < 0.05.

Fig 3

The IC₅₀ values of candidate DENV 1–4 NS2B-NS3 protease inhibitors were assessed. Compounds were combined with 5 µM of the DENV NS2B-NS3 protease and 10 µM of a fluorescence quencher substrate at concentrations ranging from 0.11 to 250 µM. Fluorescence emission was detected at 460 nm with an excitation wavelength of 355 nm. Relative protease activity was calculated as the sample concentration divided by the control concentration multiplied by 100 (%). The IC₅₀ value was determined using the formula IC₅₀ = lg-1[Xm-i(ΣP-0.5)], where Xm was the logarithm of the inhibitor maximum concentration, i was the logarithm of the concentration ratio for each concentration, ΣP was the sum of the growth inhibition rates for each group, and 0.5 was an empirical constant. The IC₅₀ value is the average of four repeated measurements.

Fig 4

The CC₅₀ and EC₅₀ values, virus titer, and NS2B-NS3 protease activity of DV-B-120 were analyzed. In (A), Huh-7 cells were incubated with serially diluted DV-B-120 or ARDP0006 (100 µM) for 48 hours, and cell viability was assessed using ATPlite. Cell viability percentage was calculated as the luminescence of the sample divided by the luminescence of the control multiplied by 100 (%). In (B), Huh-7 cells were infected with DENV 2–4 at an MOI of 0.1 for 2 hours, followed by treatment with 1 to 5 µM of DV-B-120 or ARDP0006 for 72 hours. DENV RNA levels were quantified using qRT-PCR and normalized to the RNA level of cellular GAPDH. The relative DENV RNA copies were compared to the mean of DMSO-treated Huh-7 cells. (C) Virus titer and (D) NS2B-NS3 protease activity after treatment were detected. The error bars represent the mean ± SD. Statistical significance is indicated by *P < 0.05.

Fig 5

The mechanism of noncompetitive inhibition of DENV-2 NS2B-NS3 protease activity by DV-B-120 is shown. The DENV-2 NS2B-NS3 protease (5 µM) was incubated with 25, 50, or 75 µM of DV-B-120 and fluorescence quencher substrate (0–20 µM) in a cleavage buffer. Kinetic analyses using Lineweaver–Burk plots were employed to determine DV-B-120’s mechanism of inhibition, which was calculated from a standard curve generated from an AMC-positive control solution. All three concentrations of DV-B-120 inhibited the DENV-2 NS2B-NS3 protease in a noncompetitive manner. 1/V represents the relationship between the reaction rate and substrate concentration and 1/S represents the affinity of the enzyme for the substrate.

Fig 6

DV-B-120 delayed DENV-2-induced mortality and reduced viral infection. On day 0, 6-day-old mice were tail-vein injected with 2.5 × 10⁵ PFU of heat-inactivated DENV (iDENV) or DENV, followed by ARDP0006 or DV-B-120 (1 or 10 mg/kg) on days 1, 3, and 5 post-DENV infection. (A) The survival rate, (B) body weight, and (C) clinical score were evaluated daily until day 6 post-DENV-2 infection. (D) The clinical scores were defined as follows: 0, healthy; 1, slight loss of weight; 2, slow motility; 3, asthenia and anorexia; 4, lethargy; and 5, death. (E) The brain tissue of mice sacrificed in each group was collected and subjected to immunohistochemical staining for NS1. The black arrow points to the obvious staining area. (F) The virus titer was measured in brain tissue collected from mice after sacrifice on day 6. The error bars represent the mean ± SD. Statistical significance is indicated by *P < 0.05.

Fig 7

DV-B-120 increased the RNA level of IFN and PKR in DENV-2 infections. On day 0, 6-day-old mice were tail-vein injected with 2.5 × 105 PFU of heat-inactivated DENV (iDENV) or DENV, followed by ARDP0006 (1 mg/kg or 10 mg/kg) or DV-B-120 (1 or 10 mg/kg) on days 1, 3, and 5 post-DENV-2 infection. The RNA levels of (A) IFN-alpha2, (B) IFN-alpha5, and (C) PKR in blood were monitored daily up to day 6 post-DENV-2 infection. The protein levels of (D) IFN-alpha2 and (E) IFN-alpha5 were also detected. The error bars represent the mean ± SD.