Identification of 2,4-Diaminoquinazoline Derivative as a Potential Small-Molecule Inhibitor against Chikungunya and Ross River Viruses

Abstract

Alphaviruses are serious zoonotic threats responsible for significant morbidity, causing arthritis or encephalitis. So far, no licensed drugs or vaccines are available to combat alphaviral infections. About 300,000 chikungunya virus (CHIKV) infections have been reported in 2023, with more than 300 deaths, including reports of a few cases in the USA as well. The discovery and development of small-molecule drugs have been revolutionized over the last decade. Here, we employed a cell-based screening approach using a series of in-house small-molecule libraries to test for their ability to inhibit CHIKV replication. DCR 137, a quinazoline derivative, was found to be the most potent inhibitor of CHIKV replication in our screening assay. Both, the cytopathic effect, and immunofluorescence of infected cells were reduced in a dose-dependent manner with DCR 137 post-treatment. Most importantly, DCR 137 was more protective than the traditional ribavirin drug and reduced CHIKV plaque-forming units by several log units. CHIKV-E2 protein levels were also reduced in a dose-dependent manner. Further, DCR 137 was probed for its antiviral activity against another alphavirus, the Ross River virus, which revealed effective inhibition of viral replication. These results led to the identification of a potential quinazoline candidate for future optimization that might act as a pan-alphavirus inhibitor.

Keywords: 2,4-diaminoquinazoline derivative; Ross River virus; antiviral drug; chikungunya virus; drug screening; pan-alphavirus inhibitor.

Figure 1

(A) In vitro screening of anti-CHIKV compounds (using MNTD as indicated in Table 2) by measurement of cell viability as readout gave rise to nine SMs that inhibited CHIKV replication by preserving >50% cell survival rate at 72 hpi. (B) MS and (C) ¹H-NMR spectra of best shortlisted compound DCR 137.

Figure 2

Cytotoxicity assay and determination of MNTD of DCR 137 in Vero cells. Cells were treated with different concentrations of DCR 137. (A) Microscopic images showing morphology of treated Vero cells at 48 hpi. No significant cytotoxicity was observed at 312.5 µM DCR 137. Data shown here are representative of one of the three experimental repeats. (B) Maximum non-toxic dose (MNTD) of DCR 137 was calculated through cell viability MTT assay on Vero cells.

Figure 3

Mode of inhibition of DCR 137. (A) Schematic illustration of the time-of-addition/mode of inhibition experiment. Vero cells were infected with CHIKV at an MOI of 1, and treated with DCR 137 pre (−24 h), during (0–1 h) and post (2 h) infection. DMSO at 0.1% was added at the same time as a control. (B) Post-treatment mode showed significant viral inhibition after treatment with 300 µM DCR 137 compared to pre-treatment and simultaneous treatment. (C) Inhibition of CHIKV post-treatment with DCR 137 in a dose-dependent manner was assessed by MTT assay at 48 hpi to determine the percent viability after treatment. Data represent the mean ± SD of three independent experiments. The asterisk indicates statistical significance (*** p < 0.001).

Figure 4

Reduction in CPE and immunofluorescence assay. Vero cells were infected with CHIKV, followed by addition of different concentrations of DCR 137 post 2 h virus adsorption. (A) Microscopic images showing cytopathic effect at 48 hpi after respective treatment. (B) Immunofluorescence assay: Cells were observed at 36 hpi, green fluorescence indicates the virus load as assessed with anti-CHIKV E2 mAb and secondary antibody conjugated with FITC, whereas blue fluorescence indicates the nuclear staining with DAPI. Data shown here are representative of one of the three experimental repeats.

Figure 5

Plaque reduction assay, Western blot, and flow cytometry assay. (A) (i) Representative image of the plaques for the CHIKV load in cell supernatant at 48 hpi. (ii) CHIKV-infected cells were treated with 300 µM of DCR 137. Cell supernatant was collected at 24 hpi and 48 hpi. Virus titer was assessed by plaque assay. Plaques were visualized by staining with crystal violet. Titer was calculated considering the volume and dilution factor of the inoculum. Data represent the mean ± SD of three independent experiments. The asterisk indicates statistical significance (** p < 0.01, *** p < 0.001). (B) CHIKV E2 expression was analyzed by Western blot, which revealed significant inhibition of CHIKV in a dose-dependent manner after treatment. β-actin served as a loading control. (C) Analysis of reduction in CHIKV infectivity level after treatment with 300 µM DCR 137 by flow cytometry; filled histogram is control sample (control cell + DMSO). The mean fluorescence intensity (MFI) of CHIKV E2 expression in each group was plotted. Mean ± SD of MFI (*** p < 0.001) of three independent experiments is shown.

Figure 6

Antiviral activity of DCR137 against RRV. (A) Microscopic images showing cytopathic effect at 48 hpi after respective treatment. (B) Immunofluorescence assay: Cells were observed at 36 hpi, green fluorescence indicates the virus load as assessed with mouse anti-native RRV polyclonal antibodies and secondary antibodies conjugated with FITC; and blue fluorescence indicates the nuclear staining with DAPI at 20X. Data shown here are representative of one of the three experimental repeats. (C) Vero cells were infected with RRV, followed by addition of 300 µM DCR 137 post-2 h virus adsorption, MTT assay was performed 48 hpi to determine the percent viability after treatment. (D) (i) Representative image of the plaques for the RRV load in cell supernatant at 48 hpi. (ii) RRV-infected cells were treated with 300 µM of DCR 137. Cell supernatant was collected at 24 hpi and 48 hpi. Virus titer was assessed by plaque assay. Data represent the mean ± SD of three independent experiments. The asterisk indicates statistical significance (** p < 0.01, *** p < 0.001).