Zika virus (ZIKV) is a single stranded positive stranded RNA virus that is mainly transmitted through mosquito vectors. The virus can cross the blood-brain barrier, causing some neurological complications and congenital Zika virus syndrome, and causing irreversible neurological damage. With the rapid spread of the virus worldwide, it has attracted high attention from the International Health Organization. However, there are currently no specific drugs on the market for Zika virus, so it is urgent to seek anti Zika virus drugs.
The main function of ZIKV NS3 protease is to cleave the polyprotein formed by the virus into independent functional proteins, which are essential for virus replication and therefore a potential target for antiviral drugs. This study conducted screening of antiviral inhibitors based on the activity of ZIKV NS2B-NS3 protease. The activity of the protease was detected in vitro using the Fluorescence Resonance Energy Transfer (FRET) method, and a high-throughput screening method was established based on this. We screened the compounds stored in our laboratory and obtained some compounds with good inhibitory effects on the protease through re screening. We selected a compound with relatively good inhibitory effects and conducted antiviral studies at the cellular and animal levels. The specific experimental results are as follows:
1. Soluble expression of ZIKV NS2B-NS3 protease and high-throughput screening of compound library. This study explored the expression vector, competent strain, induction temperature, and time, and finally obtained the optimal soluble expression conditions. A large amount of soluble ZIKV NS2B-NS3 protease was purified by nickel column affinity chromatography. Then, the FRET method was used to detect the activity of ZIKV NS2B-NS3 protease, and S/B=13.60 and Z ‘=0.51 were calculated, which met the requirements of high-throughput screening. Therefore, more than 4000 small molecule compound libraries stored in the laboratory were screened. Through initial screening and re screening, ten small molecule inhibitors with inhibition rates greater than 80% were obtained by removing false positive compounds with fluorescence and false positive compounds caused by sample addition errors. And the above ten compounds were subjected to in vitro enzyme activity inhibition experiments on DENV, JEV, and WNV NS3 proteases. According to the experiments, eight compounds showed good inhibitory effects on both DENV and JEV proteases, while only three compounds showed good inhibitory effects on WNV protease. Based on this experiment, we selected a compound CS-004/04003052 for further research. Obtain the following results:
2. Enzyme kinetics study of compound CS-004/04003052 and its specificity in inhibiting ZIKV protease. The compound was diluted at different concentrations and the reaction rate of protease was measured at different substrate concentrations. The reaction rate substrate concentration double reciprocal curve was plotted. According to the Mie equation, the inhibition constant Ki value of the compound was 0.1346 ± 0.0243 μ M/L. Furthermore, compared to the X-axis, it was found that the compound exhibited competitive inhibition with the substrate. The enzyme binding constant Kd value of the compound was determined to be 12.262 ± 5.549 μ M through Microscale thermophoresis Test (MST) analysis of data. Secondly, we conducted in vitro enzyme activity inhibition experiments on compound CS-004/04003052 on ZIKV, DENV, JEV NS3 protease, and PEDV NSP5 protease, and found that compound CS-004/04003052 only exhibited highly specific inhibitory effects on ZIKV NS3 protease.
3. Cell level antiviral activity detection: Compound CS-004/04003052 was diluted at a ratio of 200 μ M, 100 μ M, 50 μ M, 25 μ M, 12.5 μ M, 6.25 μ M, 3.125 μ M, and 1.59 μ M and added to Vero, BHK-21, and Hela cells, respectively. After 48 hours, Cell Titer Glo Luminescent Cell Viability Assay reagent was added, and an equal volume of detection reagent was added. The data was read and saved, analyzed, and CC50 was calculated. Combined with the measured IC50, it was found that the compound had very low toxicity to the three types of cells at higher concentrations. The analysis of the plaque test results showed that the compound can inhibit the titer of Zika virus; The results of indirect immunofluorescence assay and Western blot assay both indicate that the compound can inhibit the protein expression of Zika virus. At the same time, we also investigated whether the compound had an inhibitory effect on the replication of Japanese encephalitis virus at the cellular level. The results of the plaque assay and Western blot assay showed that the compound had no inhibitory effect on the replication of Japanese encephalitis virus at the cellular level.
4. Animal level antiviral effect: Compound CS-004/04003052 was injected into AG129 mice via tail vein to study its in vivo antiviral effect against Zika virus. The experimental results indicate that the compound in the virus group can delay the time of death of mice infected with Zika virus. And according to the analysis of HE section staining results, it was found that the pathological symptoms in the treated group were significantly improved compared to the virus group in the brain, testes, ovaries, and spleen tissues; According to the results of fluorescence quantitative PCR and plaque assay, the virus content in the drug group was significantly lower than that in the control group in the above tissues. Comprehensive analysis shows that compound CS-004/04003052 has inhibitory effects on the Zika virus at the animal level.