Inhibitors Of NS2B-NS3 Protease Research Articles

The GA-Hecate Peptide inhibits the ZIKV Replicative Cycle in Different Steps and can Inhibit the Flavivirus NS2B-NS3 Protease after Cell Infection.

Peptide drugs are advantageous because they are subject to rational design and exhibit highly diverse structures and broad biological activities. The NS2B-NS3 protein is a particularly promising flavivirus therapeutic target, with extensive research on the development of inhibitors as therapeutic candidates, and was used as a model in this work to determine the mechanism by which GA-Hecate inhibits ZIKV replication. The present study aimed to evaluate the potential of GA-Hecate, a new antiviral developed by our group, against the Brazilian Zika virus and to evaluate the mechanism of action of this compound on the flavivirus NS2B-NS3 protein. Solid-phase peptide Synthesis, High-Performance Liquid Chromatography, and Mass Spectrometry were used to obtain, purify, and characterize the synthesized compound. Real-time and enzymatic assays were used to determine the antiviral potential of GA-Hecate against ZIKV. The RT-qPCR results showed that GA-Hecate decreased the number of ZIKV RNA copies in the virucidal, pre-treatment, and post-entry assays, with 5- to 6-fold fewer RNA copies at the higher nontoxic concentration in Vero cells (HNTC: 10 μM) than in the control cells. Enzymatic and kinetic assays indicated that GA-Hecate acts as a competitive ZIKV NS2B-NS3 protease inhibitor with an IC50 of 32 nM and has activity against the yellow fever virus protease. The results highlight the antiviral potential of the GA-Hecate bioconjugate and open the door for the development of new antivirals.

Synthesis and structural characterization of new macrocyclic inhibitors of the Zika virus NS2B-NS3 protease.

Three new series of macrocyclic active site-directed inhibitors of the Zika virus (ZIKV) NS2B-NS3 protease were synthesized. First, attempts were made to replace the basic P3 lysine residue of our previously described inhibitors with uncharged and more hydrophobic residues. This provided numerous compounds with inhibition constants between 30 and 50 nM. A stronger reduction of the inhibitory potency was observed when the P2 lysine was replaced by neutral residues, all of these inhibitors possess Ki values >1 µM. However, it is possible to replace the P2 lysine with the less basic 3-aminomethylphenylalanine, which provides a similarly potent inhibitor of the ZIKV protease (Ki = 2.69 nM). Crystal structure investigations showed that the P2 benzylamine structure forms comparable interactions with the protease as lysine. Twelve additional structures of these inhibitors in complex with the protease were determined, which explain many, but not all, SAR data obtained in this study. All individual modifications in the P2 or P3 position resulted in inhibitors with low antiviral efficacy in cell culture. Therefore, a third inhibitor series with combined modifications was synthesized; all of them contain a more hydrophobic d-cyclohexylalanine in the linker segment. At a concentration of 40 µM, two of these compounds possess similar antiviral potency as ribavirin at 100 µM. Due to their reliable crystallization in complex with the ZIKV protease, these cyclic compounds are very well suited for a rational structure-based development of improved inhibitors.

Molecular Docking and Pharmacokinetics Analysis of Phytochemicals from Piper caninum as Dengue NS2B-NS3 Protease Inhibitors

Dengue fever affects 390 million people each year. Currently, there is no specific medicine to treat this disease. Thus, the search for potential NS2B-NS3 protease inhibitors has attracted increasing research interest. The dengue protease NS2B-NS3 was used as a molecular target because of its vital function in viral replication. Piper caninum belongs to the Piperaceae family, found inhabited in Malaysia and Indonesia. This plant possesses various phytochemicals with various health benefits. However, the anti-dengue activity of this plant is yet to be discovered. Therefore, the objective of this research is to evaluate the inhibitory activity of phytochemicals from P. caninum against NS2B-NS3 using in silico experimentation. Molecular docking using AutoDock Vina was utilized to identify the binding interaction of phytochemicals on NS2B-NS3. SwissADME and ProTox-II web servers were used to analyse the ADMET (absorption, distribution, metabolism, excretion, and toxicity) of the phytochemicals. Results showed that cepharadione A, bornyl caffeate, and (+)-bornyl p-coumarate had comparable molecular interaction with the reference compound, curcumin. Analysis of in silico pharmacokinetics properties revealed that these phytochemicals have good pharmacokinetics profiles and excellent drug-ability, which obeyed Lipinski’s Rule of Five. This study shows the potential inhibitory activity of the phytochemicals against NS2B-NS3 for a lead in the development of dengue inhibitors

Potential inhibitors isolated from Curcuma aeruginosa against dengue virus type 2 (DENV-2) NS2B-NS3 protease activity

Dengue virus (DENV) infection remains a formidable public health concern, causing significant fatalities worldwide. The unavailability of specific anti-DENV therapeutics and poor response towards dengue vaccine have impeded efforts to prevent and control this tropical disease, thereby, there is a pressing need for effective alternative therapeutics. Curcuma species including Curcuma aeruginosa have a long history of traditional use for the prevention and treatment of various ailments, including viral infections. While it is evident that several compounds isolated from these plants have antiviral activity against various viruses, their specific effects against DENV have not been thoroughly explored. The study aimed to investigate the potential of major compounds isolated from C. aeruginosa (demethoxycurcumin, curcumenol, furanodienone germacrone and zederone) in inhibiting DENV-2 NS2B-NS3 protease activity both in vitro and in silico. The results demonstrated that demethoxycurcumin (at 100 μM), furanodienone and germacrone (both at 25 μM) exhibited inhibitory effects on NS2B-NS3 protease activity comparable to aprotinin, a potent NS2B-NS3 protease inhibitor, albeit to a lesser extent. Importantly, these inhibitory concentrations did not adversely affect the viability of normal fibroblast cell line, L-929 cells, suggesting that the compounds’ effects were specific to the protease inhibition. Further in silico molecular docking analyses indicated that demethoxycurcumin, furanodienone and germacrone displayed significant binding affinities with both DENV E protein and DENV NS2B-NS3 protease, corroborating their inhibitory activity observed in the protease inhibition assay. These promising findings collectively suggest that demethoxycurcumin, furanodienone and germacrone from C. aeruginosa merit further development as distinctive inhibitors for treating DENV infection.

Synthesis and biological activity of imidazole phenazine derivatives as potential inhibitors for NS2B-NS3 dengue protease

A series of new imidazole-phenazine derivatives were synthesized via a two-step process. The condensation of 2,3-diaminophenazine and benzaldehyde derivatives proceeds with intermediate formation of an aniline Schiff base, which undergoes subsequent cyclodehydrogenation in situ. The structures of the synthesized compounds were characterized by 1D and 2D NMR, FTIR and HRMS. A total of thirteen imidazole phenazine derivatives were synthesized and validated for their inhibitory activity as anti-dengue agents by an in vitro DENV2 NS2B-NS3 protease assay using a fluorogenic Boc-Gly-Arg-Arg-AMC substrate. Two para-substituted imidazole phenazines, 3e and 3k, were found to be promising lead molecules for novel NS2B-NS3 protease inhibitors with IC50 of 54.8 μM and 71.9 μM, respectively, compared to quercetin as a control (IC50 104.8 μM). The in silico study was performed using AutoDock Vina to identify the binding energy and conformation of 3e and 3k with the active site of the DENV2 NS2B-NS3 protease Wichapong model. The results indicate better binding properties of 3e and 3k with calculated binding energies of −8.5 and −8.4 kcal mol−1, respectively, compared to the binding energy of quercetin of −7.2 kcal mol−1, which corroborates well with the experimental observations.

A Combined Approach for Predicting Binding Affinity of Zika Virus NS2B-NS3 Protease Inhibitors Using Semi-Empirical Methods and Molecular Docking Algorithms

Zika virus (ZIKV) is a mosquito-borne virus that has emerged as a major public health concern due to its association with severe neurological disorders. In recent years, there has been increasing interest in exploring natural products as potential therapeutics for ZIKV infection. This study aimed to predict the binding affinity of natural compounds to the ZIKV NS2B-NS3 protease (NS2B-NS3pro) using multiple molecular docking algorithms and semiempirical quantum mechanical methods. Our results demonstrate that semiempirical methods can improve the accuracy of molecular docking studies for natural compounds. In this particular case, the PM7 (Parametric Method Number 7) method showed a significant improvement in the coefficient of determination (R2 = 0.85). We expect this combined approach to aid in the development of natural product-based therapies for ZIKV infection and to highlight the importance of continued research in this field.

ZIKV Inhibitors Based on Pyrazolo[3,4-d]pyridazine-7-one Core: Rational Design, In Vitro Evaluation, and Theoretical Studies.

The Zika virus (ZIKV) is believed to cause birth defects, and no anti-ZIKV drugs have been approved by medical organizations to date. Starting from antimicrobial lead compounds with a pyrazolo[3,4-d]pyridazine-7-one scaffold, we synthesized 16 derivatives and screened their ability to interfere with ZIKV infection utilizing a cell-based phenotypic assay. Of these, five compounds showed significant inhibition of ZIKV with a selective index value greater than 4.6. In particular, compound 9b showed the best anti-ZIKV activity with a selectivity index of 22.4 (half-maximal effective concentration = 25.6 μM and 50% cytotoxic concentration = 572.4 μM). Through the brine shrimp lethality bioassay, 9b, 10b, 12, 17a, and 19a showed median lethal dose values in a range of 87.2-100.3 μg/mL. Compound 9b was also targeted to the NS2B-NS3 protease of ZIKV using molecular docking protocols, in which it acted as a noncompetitive inhibitor and strongly bound to five key amino acids (His51, Asp75, Ser135, Ala132, Tyr161). Utilizing the pharmacophore model of 9b, the top 20 hits were identified as prospective inhibitors of NS2B-NS3 protease, and six of them were confirmed for their stability with the protease via redocking and molecular dynamics simulations.

Evaluating Known Zika Virus NS2B-NS3 Protease Inhibitor Scaffolds via In Silico Screening and Biochemical Assays.

The NS2B-NS3 protease (NS2B-NS3pro) is regarded as an interesting molecular target for drug design, discovery, and development because of its essential role in the Zika virus (ZIKV) cycle. Although no NS2B-NS3pro inhibitors have reached clinical trials, the employment of drug-like scaffolds can facilitate the screening process for new compounds. In this study, we performed a combination of ligand-based and structure-based in silico methods targeting two known non-peptide small-molecule scaffolds with micromolar inhibitory activity against ZIKV NS2B-NS3pro by a virtual screening (VS) of promising compounds. Based on these two scaffolds, we selected 13 compounds from an initial library of 509 compounds from ZINC15's similarity search. These compounds exhibited structural modifications that are distinct from previously known compounds yet keep pertinent features for binding. Despite promising outcomes from molecular docking and initial enzymatic assays against NS2B-NS3pro, confirmatory assays with a counter-screening enzyme revealed an artifactual inhibition of the assessed compounds. However, we report two compounds, 9 and 11, that exhibited antiviral properties at a concentration of 50 μM in cellular-based assays. Overall, this study provides valuable insights into the ongoing research on anti-ZIKV compounds to facilitate and improve the development of new inhibitors.

Identification of Zika virus NS2B-NS3 protease and NS5 polymerase inhibitors by structure-based virtual screening of FDA-approved drugs

Zika virus (ZIKV) is a mosquito-borne human flavivirus responsible that causing emergency outbreaks in Brazil. ZIKV is suspected of causing Guillain-Barre syndrome in adults and microcephaly. The NS2B-NS3 protease and NS5 RNA-dependent RNA polymerase (RdRp), central to ZIKV multiplication, have been identified as attractive molecular targets for drugs. We performed a structure-based virtual screening of 2,659 FDA-approved small molecule drugs in the DrugBank database using AutoDock Vina in PyRx v0.8. Accordingly, 15 potential drugs were selected as ZIKV inhibitors because of their high values (binding affinity - binding energy) and we analyzed the molecular interactions between the active site amino acids and the compounds. Among these drugs, tamsulosin was found to interact most efficiently with NS2B/NS3 protease, as indicated by the lowest binding energy value (−8.27 kJ/mol), the highest binding affinity (−5.7 Kcal/mol), and formed H-bonds with amino acid residues TYRB130, SERB135, TYRB150. Furthermore, biotin was found to interact most efficiently with NS5 RdRp with a binding energy of −150.624 kJ/mol, a binding affinity of −5.6 Kcal/mol, and formed H-bonds with the amino acid residues ASPA665 and ASPA540. In vitro, in vivo, and clinical studies are needed to demonstrate anti-ZIKV safety and the efficacy of these FDA-approved drug candidates. Communicated by Ramaswamy H. Sarma

Boophilin D1, a Kunitz type protease inhibitor, as a source of inhibitors for the ZIKA virus NS2B-NS3 protease

Arboviruses are a global concern for a multitude of reasons, including their increased incidence and human mortality. Vectors associated with arboviruses include the mosquito Aedes sp., which is responsible for transmitting the Zika virus. Flaviviruses, like the Zika virus, present only one chymotrypsin-like serine protease (NS3) in their genome. Together with host enzymes, the NS2B co-factor NS3 protease complex are essential for the viral replication cycle by virus polyprotein processing. To search for Zika virus NS2B-NS3 protease (ZIKVPro) inhibitors, a phage display library was constructed using the Boophilin domain 1 (BoophD1), a thrombin inhibitor from the Kunitz family. A BoophilinD1 library mutated at positions P1–P4′ was constructed, presenting a titer of 2.9x106 (cfu), and screened utilizing purified ZIKVPro. The results demonstrated at the P1–P4’ positions the occurrence of 47% RALHA sequence (mut 12) and 11.8% RASWA sequence (mut14), SMRPT, or KALIP (wt) sequence. BoophD1-wt and mutants 12 and 14 were expressed and purified. The purified BoophD1 wt, mut 12 and 14, presented Ki values for ZIKVPro of 0.103, 0.116, and 0.101 μM, respectively. The BoophD1 mutant inhibitors inhibit the Dengue virus 2 protease (DENV2) with Ki values of 0.298, 0.271, and 0.379 μM, respectively. In conclusion, BoophD1 mut 12 and 14 selected for ZIKVPro demonstrated inhibitory activity like BoophD1-wt, suggesting that these are the strongest Zika inhibitors present in the BoophD1 mutated phage display library. Furthermore, BoophD1 mutants selected for ZIKVPro inhibit both Zika and Dengue 2 proteases making them potential pan-flavivirus inhibitors.

Identification of novel Zika virus NS3 protease inhibitors with different inhibition modes by integrative experimental and computational approaches

Zika virus (ZIKV) infection is associated with severe neurological disorders and congenital malformation. Despite efforts to eradicate the disease, there is still neither vaccine nor approved drugs to treat ZIKV infection. The NS2B-NS3 protease is a validated drug target since it is essential to polyprotein virus maturation. In the present study, we describe an experimental screening of 2,320 compounds from the chemical library of the Muséum National d’Histoire Naturelle of Paris on ZIKV NS2B-NS3 protease. A total of 96 hits were identified with 90% or more of inhibitory activity at 10 μM. Amongst the most active compounds, five were analyzed for their inhibitory mechanisms by kinetics assays and computational approaches such as molecular docking. 2-(3-methoxyphenoxy) benzoic acid (compound 945) show characteristics of a competitive inhibition (Ki = 0.49 μM) that was corroborated by its molecular docking at the active site of the NS2B-NS3 protease. Taxifolin (compound 2292) behaves as an allosteric inhibitor whereas 3,8,9-trihydroxy-2-methyl-1H-phenalen-1-one (compound 128), harmol (compound 368) and anthrapurpurin (compound 1499) show uncompetitive inhibitions. These new NS2B-NS3 protease inhibitors are valuable hits to further hit-to-lead optimization.

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

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.

Identification of NS2B-NS3 Protease Inhibitors for Therapeutic Application in ZIKV Infection: A Pharmacophore-Based High-Throughput Virtual Screening and MD Simulations Approaches

Zika virus (ZIKV) pandemic and its implication in congenital malformations and severe neurological disorders had created serious threats to global health. ZIKV is a mosquito-borne flavivirus which spread rapidly and infect a large number of people in a shorter time-span. Due to the lack of effective therapeutics, this had become paramount urgency to discover effective drug molecules to encounter the viral infection. Various anti-ZIKV drug discovery efforts during the past several years had been unsuccessful to develop an effective cure. The NS2B-NS3 protein was reported as an attractive therapeutic target for inhibiting viral proliferation, due to its central role in viral replication and maturation of non-structural viral proteins. Therefore, the current in silico drug exploration aimed to identify the novel inhibitors of Zika NS2B-NS3 protease by implementing an e-pharmacophore-based high-throughput virtual screening. A 3D e-pharmacophore model was generated based on the five-featured (ADPRR) pharmacophore hypothesis. Subsequently, the predicted model is further subjected to the high-throughput virtual screening to reveal top hit molecules from the various small molecule databases. Initial hits were examined in terms of binding free energies and ADME properties to identify the candidate hit exhibiting a favourable pharmacokinetic profile. Eventually, molecular dynamic (MD) simulations studies were conducted to evaluate the binding stability of the hit molecule inside the receptor cavity. The findings of the in silico analysis manifested affirmative evidence for three hit molecules with -64.28, -55.15 and -50.16 kcal/mol binding free energies, as potent inhibitors of Zika NS2B-NS3 protease. Hence, these molecules holds the promising potential to serve as a prospective candidates to design effective drugs against ZIKV and related viral infections.

Computational study of nitro-benzylidene phenazine as dengue virus-2 NS2B-NS3 protease inhibitor.

According to the World Health Organisation (WHO), as of week 23 of 2022, there were more than 1,311 cases of dengue in Malaysia, with 13 deaths reported. Furthermore, there was an increase of 65.7% during the same period in 2021. Despite the increase in cumulative dengue incidence, there is no effective antiviral drug available for dengue treatment. This work aimed to evaluate several nitro-benzylidene phenazine compounds, especially those that contain 4-hydroxy-3,5-bis((2-(4-nitrophenyl)hydrazinylidene)-methyl)benzoate through pharmacophore queries selection method as potential dengue virus 2 (DENV2) NS2B-NS3 protease inhibitors. Herein, molecular docking was employed to correlate the energies of selected hits' free binding and their binding affinities. Pan assay interference compounds (PAINS) filter was also adopted to identify and assess the drug-likeness, toxicity, mutagenicity potentials, and pharmacokinetic profiles to select hit compounds that can be considered as lead DENV2 NS2B-NS3 protease inhibitors. Molecular dynamics assessment of two nitro-benzylidene phenazine derivatives bearing dinitro and hydroxy groups at the benzylidene ring showed their stability at the main binding pocket of DENV2 protease, where their MM-PBSA binding energies were between -22.53 and -17.01kcal/mol. This work reports those two nitro-benzylidene phenazine derivatives as hits with 52-55% efficiency as antiviral candidates. Therefore, further optimisation is required to minimise the lead compounds' toxicity and mutagenicity.

Novel Aporphine- and Proaporphine-Clerodane Hybrids Identified from the Barks of Taiwanese Polyalthia longifolia (Sonn.) Thwaites var. pendula with Strong Anti-DENV2 Activity.

Hybrid natural products produced via mixed biosynthetic pathways are unique and often surprise one with unexpected medicinal properties in addition to their fascinating structural complexity/diversity. In view of chemical structures, hybridization is a way of diversifying natural products usually through dimerization of two similar or dissimilar subcomponents through a C–C or N–C covalent linkage. Here, we report four structurally attractive diterpene–alkaloid conjugates polyalongarins A–D (1–4), clerodane-containing aporphine and proaporphine alkaloids, the first of its kind from the barks of Taiwanese Polyalthia longifolia (Sonn.) Thwaites var. pendula. In addition to conventional spectroscopic analysis, single crystal X-ray crystallography was employed to determine the chemical structures and stereo-configurations of 1. Compounds 1–4 were subsequently subjected to in vitro antiviral examination against DENV2 by evaluating the expression level of the NS2B protein in DENV2-infected Huh-7 cells. These compounds display encouraging anti-DENV2 activity with superb EC50 (2.8–6.4 μM) and CC50 values (50.4–200 μM). The inhibitory mechanism of 1–4 on NS2B was further explored drawing on in-silico molecular docking analysis. Based on calculated binding affinities and predicted interactions between the functional groups of 1–4 and the allosteric-site residues of the DENV2 NS2B-NS3 protease, our analysis concludes that the clerodane–aporphine/proaporphine-type hybrids are novel and effective DENV NS2B-NS3 protease inhibitors.

A Critical Observation on the Design and Development of Reported Peptide Inhibitors of DENV NS2B-NS3 Protease in the Last Two Decades.

Dengue is one of the neglected tropical diseases, which remains a reason for concern as cases seem to rise every year. The failure of the only dengue vaccine, Dengvaxia® , has made the problem more severe and humanity has no immediate respite from this global burden. Dengue virus (DENV) NS2B-NS3 protease is an attractive target partly due to its role in polyprotein processing. Also, since it is among the most conserved domains in the viral genome, it could produce a broad scope of opportunities toward antiviral drug discovery in general. This review has made a detailed analysis of each case of the design and development of peptide inhibitors against DENV NS2B-NS3 protease in the last two decades. Also, we have discussed the reasons attributed to their inhibitory activity, and wherever possible, we have highlighted the concerns raised, challenges met, and suggestions to improve the inhibitory activity. Thus, we attempt to take the readers through the designing and development of reported peptide inhibitors and gain insight from these developments, which could further contribute toward strategizing the designing and development of peptide inhibitors of DENV protease with improved properties in the coming future.

Structure-Based Optimization and Characterization of Macrocyclic Zika Virus NS2B-NS3 Protease Inhibitors.

Zika virus (ZIKV) is a human pathogenic arbovirus. So far, neither a specific treatment nor a vaccination against ZIKV infections has been approved. Starting from our previously described lead structure, a series of 29 new macrocyclic inhibitors of the Zika virus protease containing different linker motifs have been synthesized. By selecting hydrophobic d-amino acids as part of the linker, numerous inhibitors with Ki values < 5 nM were obtained. For 12 inhibitors, crystal structures in complex with the ZIKV protease up to 1.30 Å resolution were determined, which contribute to the understanding of the observed structure-activity relationship (SAR). In immunofluorescence assays, an antiviral effect was observed for compound 26 containing a d-homocyclohexylalanine residue in its linker segment. Due to its excellent selectivity profile and low cytotoxicity, this inhibitor scaffold could be a suitable starting point for the development of peptidic drugs against the Zika virus and related flaviviruses.

Identification of Montelukast as flavivirus NS2B-NS3 protease inhibitor by inverse virtual screening and experimental validation

Flavivirus, such as Dengue Virus (DENV) and Zika virus (ZIKV), infects millions of people and cause the death of thousands of people every year. Despite many efforts, there is no approved anti-flaviviral treatment available. In particular, some antiflavivirus compounds were investigated the cellular activities of DENV and ZIKV, but lacking the exploration of specific target enzyme, thereby resulting in the hindrance of structure-based drug design. One example is Montlukast, which was found to inhibit the replicon replication in DENV and ZIKV infected cells, with EC50 values as 1.03 μM (DENV) and 1.14 μM (ZIKV), while the underlying mechanism remains unclear. In our study, the inhibitory mechanisms of Montelukast against the replicon replication of DENV and ZIKV infected cells were studied by using in silico approaches including inverse virtual screening (IVS), molecular dynamics (MD) simulations and binding free energy calculation, and validated through in vitro protease assay, confirming Montelukast could bind to NS2B-NS3 proteases of DENV and ZIKV as a competitive inhibitor (IC50 for DENV: 25.65 μM, for ZIKV: 15.57 μM). Moreover, Montelukast has no potential off-target effect on NS2B-NS3 protease from thrombin and trypsin inhibitory assay. Overall, Montelukast may be used as a potential candidate to block NS2B-NS3 protease as well as lead for structural modification.

Identification of a small compound that specifically inhibits Zika virus in vitro and in vivo by targeting the NS2B-NS3 protease

Zika virus (ZIKV) has rapid become a global threat, but no ZIKV-specific vaccines or drugs are currently available. In this study, inhibitors of ZIKV NS2B-NS3 protease were screened from a library containing 4,452 compound fragments. One of the compounds, 6-bromo-1,2-naphthalenedione, exhibited high specific inhibition against ZIKV NS2B-NS3 protease, but had no inhibitory effects against other viral proteases. A microscale thermophoresis (MST) assay confirmed that the compound bound to ZIKV NS2B-NS3 protein with a binding constant (Kd) of 12.26 μM. Indirect immunofluorescence assays, Western blots, and plaque assays indicated that the compound inhibited virus replication in cells. Virus titer was reduced by more than 75% when the compound was present at 1 μM. A time-of-addition assay showed that inhibition occurred at the virus replication stage, but not at the adsorption or invasion stages. The half cytotoxicity concentration (CC50) of the compound on HeLa, Vero, and BHK-21 cells were 445.44 μM, 123.87 μM, and 123.64 μM, respectively. In vivo tests using infected AG129 mice demonstrated that treatment with the compound reduced mortality by up to 60%. Mice treated with the compound showed a reduction in histopathological lesions in brain, testis, and ovary. Viral RNA, IL-1β, and IL-6 mRNA levels decreased significantly in these tissues. In summary, this study has identified a small compound with high and specific inhibitory effects on ZIKV. The compound can be used as a therapeutic agent and is also an ideal starting point for drug optimization.

SAR evolution towards potent C-terminal carboxamide peptide inhibitors of Zika virus NS2B-NS3 protease

Zika virus (ZIKV) is a member of the Flaviviridae family that can cause neurological disorders and congenital malformations. The NS2B-NS3 viral serine protease is an attractive target for the development of new antiviral agents against ZIKV. We report here a SAR study on a series of substrate-like linear tripeptides that inhibit in a non-covalent manner the NS2B-NS3 protease. Optimization of the residues at positions P1, P2, P3 and of the N-terminal and C-terminal portions of the tripeptide allowed the identification of inhibitors with sub-micromolar potency with phenylglycine as arginine-mimicking group and benzylamide as C-terminal fragment. Further SAR exploration and application of these structural changes to a series of peptides having a 4-substituted phenylglycine residue at the P1 position led to potent compounds showing double digit nanomolar inhibition of the Zika protease (IC50 = 30 nM) with high selectivity against trypsin-like proteases and the proteases of other flavivirus, such as Dengue 2 virus (DEN2V) and West Nile virus (WNV).