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

Highlights

• •Compound 3, 8, and 9 were identified to inhibit the ZIKV NS2B-NS3 protease activity of ZIKV.
• •Compound 3, 8, and 9 inhibit ZIKV NS2B-NS3 protease activity in a non-competitive mode.
• •Compound 8 significantly delayed lethality and illness symptoms in a ZIKV-infected suckling mouse model.
• •Compound 8 is worth further investigation for use as a potential future therapeutic agent against ZIKV infection.

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.

1. Introduction

Zika virus (ZIKV) is a positive single-stranded RNA Flavivirus that belongs to the Flaviviridae family. It is mainly transmitted through mosquito bites (Ferraris et al., 2019). According to statistics from the World Health Organization (WHO), 0.4 to 1.5 million cases of ZIKV infection were reported across several continents including America, Africa and Europe from 2015 to 2017 during a ZIKV outbreak that started in Brazil (Ferraris et al., 2019). The ZIKV genome encodes a polyprotein that can be processed by cellular or viral proteases into capsid protein (C), precursor protein (prM), an envelope protein (E), and non-structural proteins: NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 (Dikhit et al., 2016; Agrelli et al., 2019). ZIKV can target C-type lectin receptor on host cells through its N-glycosylated E protein (Agrelli et al., 2019), enter host cells by triggering clathrin-mediated endocytosis (Dai et al., 2016; Pandian and Sugiyama, 2012) and infect several types of target cells such as fibroblasts (Chen et al., 2016), neural progenitor cells (Dang et al., 2016; Tang et al., 2016) or placental trophoblasts (Saad et al., 2017); thereby, causing some severe symptoms. Brasil and colleagues indicated that a remarkable 42% of fetuses born to ZIKV-infected pregnant women were found to have gross abnormality upon brain imaging, such as cerebral calcifications or microcephaly (Ferraris et al., 2019; Tang et al., 2016). Cao-Lormeau et al. further suggested that ZIKV infection is highly associated with the development of Guillain-Barré Syndrome (GBS) (Cao-Lormeau et al., 2016), which is an autoimmune disorder that is associated with abnormal inflammation and targets peripheral nerves causing muscle weakness or even paralysis (Eichhorst et al., 2016). In a case-control study performed in French Polynesia, 41 of 42 patients (98%) with GBS had anti-ZIKV immunoglobulin M (IgM) or IgG as compared with 35 of 98 (35%) of hospitalized controls (Cao-Lormeau et al., 2016; Broutet et al., 2016). Based on the above, ZIKV infection has become an emerging threat to public health and an important healthcare issue that needs to be addressed.

Unfortunately, there is currently no vaccine or treatment available for ZIKV infection and supportive care is the major approach for ZIKV-infected patient therapy (Ferraris et al., 2019). Developing a safe and effective vaccine for preventing ZIKV infection may potentially protect over 2 billion people living in endemic areas (Bernatchez et al., 2020). However, according to a WHO project list, there are only eight projects still ongoing for ZIKV vaccine development and so far, two of them have advanced to phase II trials (Bernatchez et al., 2020; Born and Curtis, 2013). During the research and development of anti-ZIKV drugs, several molecules have been demonstrated to be inhibitors that interfere in different stages of the life cycle of ZIKV. For example, Rausch suggested that nanchangmycin can efficiently block ZIKV uptake and further infection by inhibiting clathrin-mediated endocytosis in cell infection assay (Rausch et al., 2017). Tabata and colleagues indicated that duramycin, a cyclic 19-amino acid peptide that specifically binds phosphatidylethanolamine (PE) in enveloped virions, significantly blocks ZIKV infection (> 80%) in primary placental cells in the dose range of 0.2 to 1 μM (Tabata et al., 2016). Chan et al. also demonstrated that bromocriptine can inhibit the proteolytic activity of ZIKV NS2B-NS3 protease by interacting with several active residues in its proteolytic cavity with an IC₅₀ of 21.6 μM and exhibit nearly 100% ZIKV-mediated plaque reduction in plaque reduction assay (Chan et al., 2017). Nevertheless, these drug candidates have not been tested in animals and the therapeutic efficacy and toxicity of these drugs in vivo still need to be studied for further clinical application. The development of an effective and safe treatment for patients suffering from ZIKV infection is an urgent need.

In this study, we screened 11 potential compounds that exhibited efficient inhibition of ZIKV NS2B-NS3 protease by using a ZIKV NS2B-NS3 enzyme-based fluorescence peptide cleavage assay. Then, we determined the inhibition constant from the enzyme compounds of kinetics by these candid linear fitting action models and defined the interaction of enzyme and compounds affect pharmacokinetic pocket by molecular docking. A virus infection assay was further performed to confirm the inhibition activity of anti-ZIKV compounds to ZIKV replication. Finally, we evaluated the therapeutic efficacy of anti-ZIKV compounds to ZIKV-induced mortality in an ICR-suckling mouse model after ZIKV infection. It is expected that the anti-ZIKV compounds can efficiently inhibit the activity of NS2B-NS3 protease of ZIKV, and interfere with the process of ZIKV replication in vivo, thereby preventing the severe symptoms caused by ZIKV infection and blocking the spread of ZIKV.

2. Materials and methods

2.1. Plasmid construction, expression and purification of ZIKV NS2B-NS3 protease

The DNA sequences of NS2B and NS3 were amplified by polymerase chain reaction (PCR) from ZIKV cDNA, and the ZIKV NS2B (residues 46–99) and NS3 protease (residues 1–187) (Xiong et al., 2022), linked by native protease polyprotein cleavage sequence (Gly4SerGly4 or GGGGSGGGG), was cloned into the pET42b vector. The pET42b-NS2B-NS3 protease plasmid was expressed in BL21 (DE3) E. coli (Invitrogen) cultured in Luria bertani (LB) broth containing 0.05 mg/mL kanamycin (Calbiochem, Merck KGaA, Darmstadt, Germany) at 37 °C. When the culture absorbance at 600 nm reached 0.4, protein expression was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO, USA) at 25 °C for 8 h. The transformed BL21 cells were harvested, sonicated using a sonicator and the Histidine (8 X His) tagged NS2B-NS3 protease was purified by immobilized metal ion affinity chromatography.

2.2. Cells, bacteria, ZIKV strain, animals and reagents

Huh-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% non-essential amino acids and 1% antibiotic-antimycotic within 5% CO₂ supplement at 37 °C. BL-21 (DE3) Escherichia coli (E. coli) was purchased from Invitrogen (Calsbad, CA, USA). ZIKV (MR-766) was obtained from the Centers of Disease Control, Department of Health, Taiwan. ICR mice were purchased from BioLasco Taiwan. Animal experiments were carried out in accordance with institutional guidelines and reviewed and approved by the Animal Care and Use Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (IACUC: 108026). RIPA lysis buffer was purchased from Merck (Kenilworth, NJ, USA).

2.3. SDS page and western blot

The processed of the purified ZIKV NS2B-NS3 protease was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with coomassie blue staining. Using specific antibodies against mouse anti-His-Tag mouse mAb (AD1.1.10, sc-53073, 1:1000; Santa Cruz, Dallas, Texas, U.S.), and peroxidase-conjugated secondary antibody (Santa Cruz, Dallas, Texas, U.S.) to detect purified ZIKV NS2B-NS3 protease in Sup Fig. 1. Huh-7 cells were seeded into 24-well plate at a density of 4.5 × 10⁴ cells per well. After incubation for 16 h, Huh-7 cells were infected with ZIKV at a multiplicity of infection (MOI) of 0.1 for 2 h. The infected-Huh-7 cells were treated with compounds at the indicated concentrations for 72 h. Cell lysates were extracted with RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 2% SDS and 1% NP-40). The insoluble protein was removed by centrifugation, and the concentration of solute protein was measured by Bio-Rad protein assay kit (Hercules, CA, USA). The SDS-PAGE was used to separate different molecular size proteins, and the proteins were transferred to PVDF membranes as previous described. The levels of the proteins of interest were determined with specific antibodies against ZIKV NS2B or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10000; GeneTex, CA, USA). After washing, peroxidase-conjugated secondary antibody (Santa Cruz, Dallas, Texas, U.S.) was incubated in each indicated well. After extended washing, the peroxidase activity was developed using chemiluminescent substrate (Thermo Fisher Scientific) and a luminescent image analyzer (Image Quant LAS4000, General Electric Company, Boston, Massachusetts, U.S.) for detection of specific bands.

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%.

2.4. Activity assay of ZIKV NS2B-NS3 protease and cell viability

The activity analysis of ZIKV NS2B-NS3 protease was performed in a 96-well plate. Each reaction contains purified ZIKV NS2B-NS3 protease, compounds and Boc-Lys-Lys-Arg (KKR)-AMC substrate (BaChem, Bubendorf BL, Switzerland). Compounds at indicated concentrations were incubated with 5 μM ZIKV NS2B-NS3 protease in cleavage buffer (Phoo et al., 2018) at 37 °C for 30 min, and then subsequently incubated with 10 μM Boc-KKR-AMC for 1 h. The fluorescence of released AMC was determined by FL3–22 SpectroFluorometer (Horiba Jobin Yvon, Bucharest-Magurele, Romania) with an excitation of 355 nm and emission of 455 nm. To determine the inhibition mechanism of each compound against ZIKV NS2B-NS3 protease, 5 μM ZIKV NS2B-NS3 protease was incubated with each compound at a concentration of 75 μM in cleavage buffer for 30 mins at 37 °C, and then the Boc-KKR-AMC at concentrations ranging from 0 to 20 μM were added for another 1 h at 37 °C. The fluorescent signal was detected as mentioned above and the inhibition mechanistic model of each compound was calculated by using the Lineweaver-Burk plot.

2.5. Blind molecular docking and structure characterization of compound 3, 8, or 9/ZIKV NS2B-NS3 protease complex

Compound 3, 8 and 9 were optimized using SYBYL software. In order to check whether the three compounds could bind with the allosteric binding residues (residue no. Asp136, Lys138, Gln139, Trp148, Leu150, Ala152, Ala153, Trp154, Leu157, Thr183, Val211, Leu214 and Val220) of the protease (22) without any prior knowledge of the target pocket, the quickvina-w software was chosen for this study. The quickvina-w is a faster, accurate and the most suitable tool for “blind docking”, (not limited in search space size or number of residues)(23). Then the three compounds were docked into the whole surface of the ZIKV NS2B-NS3 protease (PDB ID: 5T1V). For every compound, 100 possible conformations were generated and then scored by the autodock-vina score (24). The binding modes of the best autodock-vina score were selected for subsequent binding modes analysis with the ligplot+ software (25).

2.6. CC₅₀ and quantification of and the intracellular RNA level of ZIKV

In CC₅₀ of compounds testing, 10³ Huh-7 cells were seeded in 96 well dishes overnight and the compound at the specified concentration was treated in the cell for 48 h. Then the cell viability was detected by ATPlite kit (510-17281, Blossom Biotechnologies). In order to analyze intracellular levels of viral RNA, 4.5 × 10⁴ Huh-7 cells were seeded into a 24-well plate. After incubation for 16 h, Huh-7 cells were infected with ZIKV at a multiplicity of infection (MOI) of 0.1 for 2 h, then the infection medium was replaced with fresh medium or compounds at the indicated concentrations, and cells were cultured for specified time. Cells were harvested and the viral RNA was extracted from cells using the Total RNA Miniprep Purification Kit (GeneMark Biolab, Taiwan) following the manufacturer's instruction. The same amount of RNA of each sample was reverse transcribed to cDNA by M-MLV reverse transcriptase (Promega, USA). The level of ZIKV RNA replication was determined by real-time quantitative PCR (RT-qPCR) with specific primers: a forward primer, 5′-aagcaaaaggtagccgcgcc- 3′; reverse primer, 5′-tgtcccagccagcagtgtca-3′ The cellular GAPDH RNA level of each sample was used to normalize ZIKV RNA levels (Faye et al., 2013). Applied Biosystems 7500 Software version 2.0.5 generated CT and DCT values, which were used to calculate DDCT and fold change, 2^DDCT (Thermo Fisher, Waltham, Massachusetts, U.S.).

2.7. Evaluation of anti-ZIKV-induced lethality in an ICR suckling mouse model

The procedure using the ZIKV-infected ICR suckling mice model was as described in a previous study (Wu et al., 2018). Briefly, 6-day-old ICR sucking mice were randomly divided into four groups (n = 6): group 1 were intracerebrally (i.c.) injected with 10⁴ plaque forming unit (pfu) of 60 °C heat-inactivated ZIKV (iZIKV); group 2 were i.c. injected with 10⁴ pfu of ZIKV and saline (ZIKV); group 3 were i.c. injected with 10⁴ pfu of ZIKV and 1 mg/kg of compound 8 (ZIKV + 1 mg/kg compound 8); group 4 were i.c. injected with 10⁴ pfu of ZIKV and 10 mg/kg of compound 8 (ZIKV + 10 mg/kg compound 8). The mice inoculated with iZIKV served as a mock control. The clinical score, body weight, and survival rate were measured every day after ZIKV infection (dpi). Mice were treated with compound 8 at day 1, 3, and 5 dpi. 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. The mice were sacrificed by CO₂ asphyxiation at day 6, and 0.1 g of brain tissue was collected by RIPA buffer to analyze the expression level of ZIKV NS2B protein. The remaining brain tissue was subjected to histopathology analysis.

2.8. Histopathology

The method of histopathological observation followed the Ly6c antibody data sheet (ER-MP20, ab54223; Abcam, Cambridge, UK). In brief, the brain tissue was subjected to hematoxylin and eosin or IHC staining with Ly6c antibody to observe the ZIKV infection-induced injury or monocyte infiltration by a photomicroscope.

2.9. Statistical analysis

Data are presented as mean ± SD. The virologic results were analyzed via either the Student's t-test or one-way analysis of variance (ANOVA) to compare the statistical significance of the differences between the controls and samples. Statistical analysis was performed using GraphPad Prism v.6 and data were considered significant at a P value of less than 0.05.

2.10. Ethics statement

Animal experiments were carried out in accordance with institutional guidelines and reviewed and approved by the Animal Care and Use Committee of Kaohsiung Medical University, Kaohsiung, Taiwan (IACUC: 108026).

3. Results

3.1. Identification of the ZIKV NS2B-NS3 protease inhibitors

To identify the specific protease inhibitors of ZIKV NS2B-NS3, we first generated ZIKV NS2B-NS3 protease by connecting ZIKV NS2B (residues 46–99) and NS3 protease (residues 1–187) by a protease-resistant linker (Gly4SerGly4 or GGGGSGGGG), and cloned it to bacteria-expressing vector pET42b to form pET42b-ZIKV NS2B-NS3 protease expression vector. After large scale production of ZIKV NS2B-NS3 protease by BL21 (DE3) E. coli expression system and purification by immobilized metal ion affinity chromatography, we confirmed the molecular weight and purity of the purified ZIKV NS2B-NS3 protease by SDS-PAGE and WB (Sup fig 1.). We further identified ZIKV NS2B-NS3 protease inhibitors from the SPECS compound library by co-incubating 50 μM candidate compound 1 to 11 with 5 μM ZIKV NS2B-NS3 protease in cleavage buffer, and then subsequently incubating with 10 μM Boc-KKR-AMC fluorescent substrate. We also used temoporfin as a positive control for ZIKV NS2B-NS3 protease activity inhibition because temoporfin can inhibit the replication of a broad range of flaviviruses, including ZIKV, dengue virus (DENV), and so on, by interfering in protease function (Knyazhanskaya et al., 2021). As seen in (Fig. 1A),

Compounds 3, 8, and 9 had better protease activity inhibition efficacy than others with an inhibitor concentration of 50 μM. To increase the accuracy of compounds inhibiting ZIKV NS2B-NS3 protease. The compounds 1 to 11 and temoporfin of 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM were reacted with 5 μM NS2B-NS3 protease and 10 μM Boc-KKR-AMC fluorescent substrate. Compound 3, 8 and 9 were IC₅₀ of 14.01 μM, 6.85 μM, and 14.2 μM, respectively (Fig. 1B and Table 1). The inhibition of NS2B-NS3 protease activity by three compounds all exceeded temoporfin (IC₅₀ by 18.77 μM). Among them, Compound 8 has the strongest inhibitory efficiency.

Table 1.

IC₅₀ of compounds against ZIKV NS2B-NS3 protease.

Compounds	Structural formula	Molecular weight	IC50 (μM)a
(1) 4-((4,6-dioxo-1,3-diphenyl-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl)phenyl acetate		442.50	63.39
(2) 5-(3‑bromo-5‑methoxy-4-(naphthalen-1-ylmethoxy)benzylidene)−1,3-diphenyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione		649.56	72.54
(3) 5-(4-((2-chlorobenzyl)oxy)−3-iodo-5-methoxybenzylidene)−1,3-diphenyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione		680.94	14.01
(4) 5-(4‑hydroxy-3,5-diiodobenzylidene)−1,3-diphenyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione		652.24	> 100
(5) (E)−4-ethyl-N-(4-oxo-3-(phenylsulfonamido)naphthalen-1(4H)-ylidene)benzene sulfonamide		480.56	>100
(6) (E)−2,4,5-trimethyl-N-(4-oxo-3-(phenylsulfonamido) naphthalen-1(4H)-ylidene)benzenesulfonamide		494.58	>100
(7) (E)−4-methyl-N-(4-oxo-3-(phenylsulfonamido) naphthalen-1(4H)-ylidene)benzenesulfonamide		466.52	>100
(8) 5-(3‑bromo-4‑hydroxy-5-methoxybenzylidene)−2-thioxo-1,3-di-p-tolyldihydropyrimidine-4,6(1H,5H)-dione		537.43	6.85
(9) N-(3,4-dimethylphenyl)−2-(4-((4,6-dioxo-1,3-diphenyl-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl) phenoxy)acetamide		561.66	14.2
(10) (Z)−4-isopropyl-N-(4-oxo-3-(phenylsulfonamido) naphthalen-1(4H)-ylidene)benzenesulfonamide		494.58	>100
(11) (E)−2,4-dimethyl-N-(4-oxo-3-(phenylsulfonamido) naphthalen-1(4H)-ylidene)benzenesulfonamide		480.55	>100
Temoporfin		680.764	18.77

3.2. Characterization of the type of inhibition of ZIKV NS2B-NS3 protease afforded by compounds 3, 8 and 9

To investigate the type of inhibition of ZIKV NS2B-NS3 protease afforded by the inhibitors, detailed kinetic analyses were performed to determine the inhibition mechanisms of compound 3, 8, and 9. ZIKV NS2B-NS3 protease (5 μM) was incubated with compound 3, 8 and 9 at concentrations of 75 μM, respectively, and then subsequently incubated with different concentrations of Boc-KKR-AMC fluorescent substrate in a range of 0 to 20 μM in cleavage buffer. The inhibition mechanisms (competitive, uncompetitive, and non-competitive) were analyzed by using Lineweaver-Burk plots for each compound. The data showed that compound 3 (Fig. 2A), 8 (Fig. 2B) and 9 (Fig. 2C) can all significantly reduce the ZIKV NS2B-NS3 protease activity at the indicated concentrations and revealed a non-competitive inhibition model based on the different concentration plots of each compound intersection at the X-axis. The figures showed the trend lines of combined groups of compound 3, 8 and 9 were all higher than untreated protease and intersected at one point, showing that compound 3, 8, and 9 were all non-competitive inhibitors. We also calculate the Vmax value of untreated protease, compound 3, 8 and 9. It was 0.071 µmoles/min, 0.040 µmoles/min, 0.045 µmoles/min, and 0.043 µmoles/min. K_m value is 10.93 µM, 12.26 µM, 11.88 µM, 11.79 µM, from the above results, it could be found that compound 3, 8, 9 could reduce Vmax value, but the K_m value is slightly larger, which may be the effect of the compound on the conformational change of ZIKV NS2B-NS3 protease reduced the affinity of ZIKV NS2B-NS3 protease (Fig. 3). Finally, to understand the impact of compound 3, 8, and 9 on the turnover number of ZIKV NS2B-NS3 protease, K_cat of combinate ZIKV NS2B-NS3 protease with compounds was evaluated. K_cat of the value of untreated protease, compound 3, 8 and 9 were 0.0142/min, 0.008/min, 0.009/min and 0.0086/min, respectively. The results showed that the K_cat value of compound 3, 8 and 9 was lower than that of untreated protease and pointed out that compound 3, 8 and 9 could reduce the turnover number of ZIKV NS2B-NS3 protease.

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.

3.3. Molecular docking and binding modes analysis

Our blind docking results are shown in Fig. 3, and the autodock-vina scores of the compound 3, 8 and 9 are −8.5, −7.5 and −8.8 kcal/mol, respectively. Our binding modes analysis are shown in Supplementary Figure 2 to 4 and Table 1. Analyses of the binding mode of the allosteric binding residues of ZIKV NS2B-NS3 protease/compound 3 complex structures indicated that Gln139 can form hydrogen bonds in the complex structures, while Trp148, Leu150, leu214 and Val220 have non-bonding interactions (Sup fig. 2). Analyses of the binding mode of the allosteric binding residues of ZIKV NS2B-NS3 protease /compound 8 complex structures indicated that Gln139 can form hydrogen bonds in the complex structures (Sup fig. 3). Analyses of the binding mode of the allosteric binding residues of ZIKV NS2B-NS3 protease/compound 9 complex structures show that Gln139, Trp148, Leu150, Ala152, Thr183, Val211 and Val220 have non-bonding interactions (Sup fig. 3). While Trp148, Leu150 and Val220 have non-bonding interactions. The overall results of our simulations suggest that allosteric binding residues of ZIKV NS2B-NS3 protease could interact with the three inhibitors. Moreover, our simulations indicate that Gln139, Trp148, Leu150 and Val220 could form hydrogen bonds or the non-bonding interactions most frequently. Notably, the three non-competitive compound 3, 8 and 9 might interact with the allosteric binding residues and induce the reaction center conformation change of ZIKV NS2B-NS3 protease (27) (Tables 2 and 3).

Table 2.

The binding modes of the compound 3, 8 and 9.

Compounds	Blind docking simulations
	Hydrogen bonds (residues)	Nonbonding interactions (residues)	Interact with the allosteric binding residues
3	Gln139	Trp134, Leu141, Trp148, Leu150, Ile188, Gly213, Leu214, Ans217, Gly218 and Val 220	Gln139, Trp148, Leu150, leu214 and Val220
8	Null	Gln139, Trp148, Leu150, Ala152, Thr183, Lys184, Asp185, Asp187, Ile188, Val211, Ile212, Gly213, Gly218 and Val 220	Gln139, Trp148, Leu150, Ala152, Thr183, Val211 and Val 220
9	Gln139	Trp134, Leu141, Pro147, Trp148, Leu150, Ile188, Ile212, Gly213 and Val 220	Gln139, Trp148, Leu150 and Val 220

Table 3.

Anti-ZIKV activity of compounds.

Compounds	IC50 (μM)a	CC50 (μM)b	EC50 (μM)c	SId
3	14.01	> 200	2.15	93.02
8	6.85	> 200	0.52	384.61
9	14.20	61.48	3.52	17.46
Temoporfin	18.77	61.05	2.00	30.53

^bCC₅₀ is defined as the concentration of compounds required to reduce cell viability by 50%.

^cEC₅₀ is defined as concentration of compounds required to inhibit virus-induced cytopathic effect by 50%.

^dSelectivity index (SI), the index to evaluate the compound has low cytotoxicity and inhibits ZIKV activity, SI = CC₅₀/EC_50.

3.4. Determination of the CC₅₀ and EC₅₀ of compound 3, 8 and 9

To confirm the effects of compounds 3, 8 and 9 on Huh-7 cells. The compounds were added to Huh-7 cells for 48 h and the cell activity was analyzed by ATP lite, the results showed that CC₅₀ of compound 3 (>200 μM) and compound 8 (>200 μM) are both higher than compound 9 (CC₅₀ = 61.48 μM) and Temoporfin (CC₅₀ = 61.05 μM) (Fig. 4A). To determine the inhibition ability of compound 3, 8, and 9 on ZIKV replication at different time. Huh-7 cells were infected with ZIKV at MOI of 0.1 for 2 h and further treated with 6 μM temoporfin, compound 3, 8, and 9 at 24 h, 48 h, 72 h. The ZIKV RNA level of cell pellets were analyzed by performing RT-qPCR. The data indicates that three compounds could reduce virus replication depending on time (Fig. 4B). To further verify the concentrations effect of compound 3, 8, and 9 on viral replication. To use 0.2 μM, 0.69 μM, 2 μM, 6 μM or 2 μM Temoporfin added to infected cells for 72 h. The ZIKV RNA level was analyzed by RT-qPCR. The data indicate that compound 3 (Fig. 3C), 8 (Fig. 3D), and 9 (Fig. 3E) could significantly inhibit ZIKV replication. The averages of their EC₅₀ were 2.15 μM, 0.52 μM, and 3.52 μM, respectively, and the EC₅₀ of compound 8 was the lowest. Based on the analysis of the above values, compound 8 was found to have the best values in IC₅₀, CC₅₀ and EC₅₀. In addition, the SI^c value of compound 8 was better than compound 3, compound 9 and Temoporfin. Therefore, compound 8 has the potential for animal testing.

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.

3.5. Evaluation of the antiviral activity of compound 8 in a ZIKV-infected ICR suckling mouse model

To investigate whether compound 8 exhibited protective efficacy against ZIKV infection in ICR sulking mice, 6-day-old suckling mice were intracerebrally injected with ZIKV at day 0 and simultaneously treated with normal saline, 1 mg/kg or 10 mg/kg of compound 8 at days 1, 3 and 5. Suckling mice injected with heat-inactive ZIKV (iZIKV) served as mock controls. The clinical scores and survival rates of ZIKV-infected mice administered with saline or compound 8 were measured daily for 6 days. Fig. 5 shows that the normal saline-treated ZIKV-infected suckling mice suffered severe sickness and even death within 3–6 dpi as compared with iZIKV-infected mice. As shown in Fig. 5A and B, the clinical score showed that ZIKV-infected suckling mice developed severe paralysis, anorexia, and asthenia and lost 28% body weight. In contrast, compound 8 -treated ZIKV-infected suckling mice showed a delay in the sickness induced by ZIKV infection and lost only 2% body weight. Treatment with compound 8 protected all ZIKV-infected suckling mice from lethal ZIKV infection as compared with the saline-treated control group (Fig. 5C). We collected the brain tissue of each mouse at 6 days post infection (dpi) and detected viral protein and monocytes by performing western blotting and immunohistochemistry (IHC) analysis. The results indicated that ZIKV NS1 (Fig. 5D, above) and NS2B protein level (Fig. 5E) were significantly decreased in the brain tissue of compound 8 treated ZIKV-infected suckling mice as compared with the saline-treated group, suggesting that compound 8 could efficiently inhibit ZIKV replication in ZIKV-infected suckling mice. The level of monocyte infiltration in brain tissue was further evaluated as the immune cell infiltration into the brain is associated with ZIKV-caused neurological disorders such as GBS. Fig. 5D, below and Fig. 5F indicate that Ly6C⁺ monocytes were significantly less accumulated in the adjacent blood vessels in brain tissue in compound 8 treated ZIKV-infected suckling mice as compared with the saline-treated group, indicating that the compound 8 treatment decreased the monocyte infiltration in the brain of ZIKV-infected suckling mice.

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.

4. Discussion

In this study, we successfully screened out potential compounds (i.e., compound 3, 8, and 9) that could efficiently inhibit ZIKV NS2B-NS3 protease activity from the SPECS compound library and defined its pharmacodynamic pocket. The three candidate compounds inhibited NS2B-NS3 protease activity in a noncompetitive manner and compound 8 exhibited the highest antiviral activity in viral replication assay, and the lowest cytotoxicity as compared with temoporfin, which is a clinical drug that possesses baviadirase proteases. As expected compound 8 also effectively delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in the ZIKV-infected suckling mouse model. We believe that compound 8 may serve as a potential anti-ZIKV agent for treating and preventing the pandemic outbreak of ZIKV infection

ZIKV NS2B-NS3 protease as the target of ZIKV inhibition, supplemented by the screening of small molecule database is a rapid and effective strategy for the development of small molecule virus inhibitors, because viral proteases play an important role in converting polyprotein precursors into mature viral proteins in various types of virus (Bortolotti et al., 2021; Blakeney et al., 2003). Protease-targeting inhibitors have been developed for treating infectious diseases such as human immunodeficiency virus (HIV) or hepatitis C virus (HCV) in the clinic (Van Dycke et al., 2021; Chang et al., 2019), and several preclinical studies have attempted to develop anti-ZIKV agents using a drug repurposing strategy (Knyazhanskaya et al., 2021); however, as yet no therapeutic agent has been approved for clinical use.

We found compounds of 11 from the SPECS compound library and test they inhibit ZIKV NS2B-NS3 protease potential by purifying ZIKV NS1-NS2B proteases for preliminary screening, and the results did show that compound 8 had the best inhibitory ability. Next, we used purified ZIKV NS1-NS2B proteases for IC₅₀ analysis of compounds. Compound 3, 8 and 9 could efficiently inhibit ZIKV NS2B-NS3 protease activity with IC₅₀ of 14.01, 6.85, and 14.2 μM, respectively. The inhibition efficacies of all these compounds are better than temoporfin (IC₅₀ by 18.77 μM; Fig. 1B). Among them, compound 8 showed the highest efficacy for inhibiting ZIKV NS2B-NS3 protease activity, significantly delayed ZIKV-induced lethality and illness symptoms, and decreased ZIKV-induced weight loss in the ZIKV-infected suckling mouse model (Fig. 5). We expect that compound 8 may be suitable to become a first-in-class anti-ZIKV small molecule drug for controlling ZIKV infection and pandemics in the future.

Development of an inhibitor that suppresses the function of ZIKV NS2B-NS3 protease of ZIKV may potentially be applied to other flaviviruses as the flavivirus proteases share a high level of sequence similarity (approximately 30 to 80%) (Kang et al., 2017). Therefore, it is very important to define the pharmacokinetic pocket of the inhibitor. In Li and colleagues’ study, they suggested that temoporfin, niclosamide, and nitazoxanide, which were screened from the NIH Chemical Genomic Center (NCGC) Pharmaceutical Collection library, can broadly inhibit NS2B-NS3 protease of flaviviruses at micromolar efficacy (Li et al., 2017). However, they lack definition of drug pockets, which limits compound optimization. To reduce the limitations of subsequent applications. In our study, we used molecular docking to define the common pharmacodynamic functional groups of compound no. 3, 8, 9 (Fig. 3). Compound 8 was also able to significantly inhibit ZIKV NS2B-NS3 protease activity (Fig. 1) and viral replication (Fig. 4) at the micromolar level and further delayed ZIKV-induced lethality (Fig. 5). We still need to verify the inhibition ability and efficacy of compound 8 to other flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and yellow fever virus (YFV), in vitro to evaluate the potential of compound 8 as a pan-flaviviral protease inhibitor. We also intend to investigate the selectivity, cellular activity and pharmacokinetics of compound 8 in vivo to assess its availability for clinical use.

The permeability of the placenta and safety of anti-viral drugs is important to treat pregnant females who have been infected by viruses that are transmitted through sexual and maternal-fetal modes. Previous studies reported that ZIKV can infect various types of cells in the human placenta, including endothelial cells, placental trophoblasts and fetal Hofbauer macrophages in the intervillous space (Tabata et al., 2016), thereby infecting the fetus and causing neurological disorders such as microcephaly in neonates, congenital malformations and even fetal demise (Russell, 2016; Wang et al., 2016). Although some FDA-approved drugs, such as sofosbuvir, azithromycin, niclosamide and chloroquine, are considered for use in the treatment of pregnant females, the therapeutic mechanism and efficacy of these repurposing drugs to ZIKV infection are still unknown. On the other hand, the permeability of anti-Zika viral drug to pass through blood-brain barrier (BBB) is also a critical issue that need to be considered for treating Zika virus-infected fetus. Our results have shown that compound 8 could significantly inhibit ZIKV NS2B-NS3 protease activity (Fig. 1) and viral replication (Fig. 4) in vitro and intracranially administrating compound 8 delays ZIKV-induced lethality in vivo (Fig. 5). Therefore, further study is necessary to analyze the availability of compound 8 in a pregnant AG129 mouse model and evaluate and confirm the BBB-transported efficiency for and evaluating the potential of compound 8 as a therapeutic option for ZIKV-infected pregnant patients in the future.

In conclusion, we found a potential compound 8 that can efficiently inhibit the activity of ZIKV NS2B-NS3 protease in a noncompetitive inhibition manner, decrease the viral replication in ZIKV-infected cells and thereby delay the ZIKV-induced lethality in a ZIKV-infected suckling mouse model. This viral protease inhibitor has the following advantages: (1) Compound 8 exhibits lower to ZIKV NS2B-NS3 protease inhibition than the clinical drug temoporfin. (2) Compound 8 inhibits ZIKV NS2B-NS3 protease in a noncompetitive inhibition manner, which means that compound 8 can interfere with the activity of ZIKV NS2B-NS3 protease no matter whether it interacts with the substrate or not, suggesting that compound 8 exerts a more comprehensive inhibitory effect on protease activity during ZIKV infection of the host. (3) Compound 8 significantly delayed ZIKV-induced lethality and illness symptoms and decreased ZIKV-induced weight loss in an animal model. Thus, we expect that after further investigation, compound 8 may be considered as potential therapeutic agent for ZIKV infection in the future.

CRediT authorship contribution statement

Wen-Wei Lin: Conceptualization, Data curation, Investigation, Methodology, Software, Writing – original draft. Yi-Jung Huang: Conceptualization, Data curation, Investigation, Methodology, Software, Writing – original draft. Yen-Tseng Wang: Formal analysis, Funding acquisition. Yun-Syuan Lin: Data curation, Supervision, Validation. Nonsikelelo Mazibuko: Data curation. Chien-Shu Chen: Supervision, Validation. Tian-Lu Cheng: Visualization. Chih-Shiang Chang: Writing – review & editing. Yu-Ling Leu: Supervision. Chiao-Yun Chen: Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Writing – original draft, Writing – review & editing. Chih-Hung Chuang: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Visualization, Writing – original draft, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no competing interests.

Data availability

• Data will be made available on request.