Development of NS2B-NS3 protease inhibitor that impairs Zika virus replication

Abstract

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes severe neurological disorders, such as microcephaly in fetuses. Most recently, an outbreak of ZIKV started in Brazil in 2015. To date, no therapeutic agents have been approved to treat ZIKV infection in the clinic. Here, we screened a small molecule inhibitor that can inhibit the function of ZIKV non-structural protein 2B (NS2B)-NS3 protease (ZIKV NS2B-NS3 protease), thereby interfering with viral replication and spread. First, we identified the half maximal inhibitory concentration (IC₅₀) of compound 3 (14.01 μM), 8 (6.85 μM), and 9 (14.2 μM) and confirmed that they are all non-competitive inhibitors. In addition, we have used the blind molecular docking method to simulate the inhibition area of three non-competitive inhibitors (compound 3, 8, and 9) with the ZIKV NS2B-NS3 protease. The results indicated that the four allosteric binding residues (Gln139, Trp148, Leu150, and Val220) could form hydrogen bonds or non-bonding interactions most frequently with the three compounds. The interaction might induce the reaction center conformation change of NS2B-NS3 protease to reduce catalyzed efficiency. The concentration of compounds required to reduce cell viability by 50% (CC₅₀), and the concentration of compounds required to inhibit virus-induced cytopathic effect by 50% (EC₅₀) of three potential compounds are >200 μM, 2.15 μM (compound 3), > 200 μM, 0.52 μM (compound 8) and 61.48 μM, 3.52 μM (compound 9), and Temoporfin are 61.05 μM, 2 μM, respectively. To select candidate compounds for further animal experiments, we analyzed the selectivity index (SI) of compound 3 (93.02), 8 (384.61), 9 (17.46), and Temoporfin (30.53, FDA-approved drug against cancer). Compound 8 has the highest SI value. Therefore, compound 8 was selected for verification in animal models. In vivo, compound 8 significantly delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in a ZIKV-infected suckling mouse model. We conclude that compound 8 is worth further investigation for use as a potential future therapeutic agent against ZIKV infection.

Keywords: Non-competitive; Small molecule inhibitor; ZIKV NS2B-NS3 protease; Zika virus (ZIKV).

Fig. 1

Screening of ZIKV NS2B-NS3 protease small molecule candidate inhibitors. (A) ZIKV NS2B-NS3 protease activity was determined by fluorescence quenching spectra and the fluorescence unit was converted into an activity percentage compared to ZIKV NS2B-NS3 protease alone. The solution first contained 5 μM ZIKV NS2B-NS3 protease; after adding 50 μM of compounds and 10 μM of fluorescence quencher substrate, it showed fluorescence emission at 460 nm at the excitation wavelength of 355 nm. (B) 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM of compound 1 to compound 11 or Temoporfin were mixed into 5 μM ZIKV NS2B-NS3 protease and 10 μM of fluorescence quencher substrate. To detect fluorescence emission at 460 nm at the excitation wavelength of 355 nm. Relative protease activity (%) = sample of different concentration / control *100%.

Fig. 2

Compounds inhibited ZIKV NS2B-NS3 protease activity in a non-competitive manner. ZIKV NS2B-NS3 protease (5 μM) was incubated with 75 μM (A) compound 3, (B) 8, (C) 9 and Bz-KKR-AMC (0 to 20 μM) in cleavage buffer. Lineweaver-Burk plots of kinetic analysis were used to determine the inhibition mechanism of compounds, which were calculated from a standard curve generated from an AMC-positive control solution. All three compounds inhibited ZIKV protease in a noncompetitive manner.

Fig. 3

The proposed inhibitor/ZIKV NS2B-NS3 protease complex structure. (A) Compound 3, (B) Compound 8 and (C) compound 9. The allosteric binding residues (residue no. Asp136, Lys138, Gln139, Trp148, Leu150, Aal152, Ala153, Trp154, Leu157, Thr183, Val211, Leu214 and Val220) of the protease is colored in yellow. The subtract reaction center residues (His116, Asp140 and Ser200) (6) are shown in red surface representation.

Fig. 4

Analysis of the CC₅₀ and EC₅₀ of compound 3, 8, and 9. (A) To add 200 μM serially diluted compound 3, 8, 9 and Temoporfin in Huh-7 cells for 48 h then analyze cell viability by ATPlite. Cell viability (%) = luminescence of sample/luminescence of control *100%. (B) Huh-7 cells were infected with ZIKV at MOI of 0.1 for 2 h, and the infected cells were treated with 6 μM compound 3, 8 and 9 at 24 h, 48 h and 72 h. The infected cells were treated with (C) compound 3, (D) 8 and (E) 9 at the indicated concentrations for 72 h. ZIKV RNA levels were analyzed by qRT-PCR, which were normalized to the RNA level of cellular GAPDH. The relative ZIKV RNA copies were relative to the mean of 0 h or DMF-treated Huh-7 cells. Error bars represent the mean ± SD; n.s., no significant difference; *, P < 0.05.

Fig. 5

Compound 8 delayed ZIKV-induced lethality and decreased monocyte infiltration. Six-day-old mice were intracerebrally injected with 104 pfu iZIKV or ZIKV and with compound 8 (1 mg/kg or 10 mg/kg) at day 1, 3, and 5. (A) Body weight, (B) clinical score and (C) survival rate were daily measured 6 day after ZIKV infection. All mice were sacrificed at 6 day after ZIKV infection. Brain tissue (0.1 g) was collected by RIAP buffer. (D) To evaluated for ZIKV infection level via stained NS1 and analysis monocyte infiltration level via stained Ly6C. (E) The level of NS2B was detected by WB. (F) The infiltration of monocytes was also quantification in the brain slices. Error bars represent the mean ± SD; *, P < 0.05.