日本脑炎病毒NS3蛋白酶活性检测及抑制剂高通量筛选方法的建立

日本脑炎病毒(Japanese encephalitis virus,JEV)是单股正链RNA病毒,全基因组仅含有一个开放阅读框,编码一条多聚蛋白前体,病毒编码的NS3蛋白酶在JEV多聚蛋白加工过程中起着重要作用,是重要的药物靶标。通过PCR扩增了NS2BH-NS3蛋白酶的编码区,构建了原核表达质粒并转化到大肠杆菌BL21(DE3),经IPTG诱导得到可溶性的NS3蛋白酶,用镍亲和层析方法进行了纯化。建立了基于荧光共振能量转移的NS3蛋白酶活性检测方法,并确定了最佳的反应条件,对113个化合物进行了筛选,发现其中两个化合物对JEV NS3蛋白酶具有一定的抑制活性。本研究为JEV NS3蛋白酶的活性研究及抑制剂筛选提供了一种操作方便、成本低廉的方法。     

Discovery of novel purine nucleoside derivatives as phosphodiesterase 2 (PDE2) inhibitors: Structure-based virtual screening,optimization and biological evaluation

Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders and pulmonary hypertension. Herein, we identified that clofarabine (4), an FDA-approved drug, displayed potential PDE2 inhibitory activity (IC₅₀?=?3.12?±?0.67?μM) by structure-based virtual screening and bioassay. Considering the potential therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the best compound 14e with a significant improvement of inhibitory potency (IC₅₀?=?0.32?±?0.04?μM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that 5′-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential compounds reported in this article and the valuable structure-activity relationships (SARs) might bring significant instruction for further development of potent PDE2 inhibitors.     

Analysis of the nucleoside triphosphatase, RNA triphosphatase, and unwinding activities of the helicase domain of dengue virus NS3 protein

The helicase domain of dengue virus NS3 protein (DENV NS3H) contains RNA-stimulated nucleoside triphosphatase (NTPase), ATPase/helicase, and RNA 5′-triphosphatase (RTPase) activities that are essential for viral RNA replication and capping. Here, we show that DENV NS3H unwinds 3′-tailed duplex with an RNA but not a DNA loading strand, and the helicase activity is poorly processive. The substrate of the divalent cation-dependent RTPase activity is not restricted to viral RNA 5′-terminus, a protruding 5′-terminus made the RNA 5′-triphosphate readily accessible to DENV NS3H. DENV NS3H preferentially binds RNA to DNA, and the functional interaction with RNA is sensitive to ionic strength.     

Structure-based virtual screening of essential Mycobacterium tuberculosis enzymes AspS and KatG for potential inhibitors

Mycobacterium tuberculosis (TB) is a leading global cause of disease-related death. Recent works have studied metabolic pathways of the mycobacterium, highlighting essential enzymes to target via competitive inhibition through computational molecular modeling to suppress the organism''s life cycle. We used the Protein Databank (PDB), the UniProt Knowledgebase and the iDock server in this study. In vitro toxicity screening and pharmacokinetic properties were assessed to determine potential ligand safety and drug properties. Our results have revealed five and nine potential ligands for the enzymes AspS and KatG respectively. The KatG active site has displayed binding affinities of -13.443 to -12.895 kcal/mol, while AspS ligands range from -6.580 to -6.490kcal/mol. The intermolecular forces responsible for the differing binding affinities of each enzyme are primarily Coulombic interactions for AspS, versus Coulombic and extensive hydrogen bonding interactions in KatG.     

Structure-based virtual screening for glycosyltransferase51

Glycosyltransferase is an essential and easily accessible drug target for antibiotic-resistance. The crystal structures of glycosyltransferase (GT₅₁) provide us with the chance to develop new antibiotics that interrupt a yet unexplored molecular target. Based on the crystal structure of GT₅₁, we have carried out computational screening of GT₅₁ in order to look for novel GT₅₁ inhibitors. The present study was accomplished by using advance docking and scoring methodology. It is the first example of virtual screening of GT₅₁ inhibitors. Two docking procedures (Surflex-Dock and FlexX-Pharm dockings) were applied and nine novel potential leads are proposed after thorough examination by a combination of methods.     

Structure-based virtual screening and biological evaluation of novel small-molecule BTK inhibitors

Bruton tyrosine kinase (BTK) is linked to multiple signalling pathways that regulate cellular survival, activation, and proliferation. A covalent BTK inhibitor has shown favourable outcomes for treating B cell malignant leukaemia. However, covalent inhibitors require a high reactive warhead that may contribute to unexpected toxicity, poor selectivity, or reduced effectiveness in solid tumours. Herein, we report the identification of a novel noncovalent BTK inhibitor. The binding interactions (i.e. interactions from known BTK inhibitors) for the BTK binding site were identified and incorporated into a structure-based virtual screening (SBVS). Top-rank compounds were selected and testing revealed a BTK inhibitor with >50% inhibition at 10 µM concentration. Examining analogues revealed further BTK inhibitors. When tested across solid tumour cell lines, one inhibitor showed favourable inhibitory activity, suggesting its potential for targeting BTK malignant tumours. This inhibitor could serve as a basis for developing an effective BTK inhibitor targeting solid cancers.     

Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNβ‐induced responses, for example, expression of IFN‐stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress‐induced‐phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca²⁺ levels while inhibiting nuclear translocalization of STAT1 and NFAT‐1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca²⁺/calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5‐mediated inhibition of IFNβ‐induced responses, for example, IFN‐sensitive response element driven luciferase, STAT1‐dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5‐expressing cells in response to IFNβ. Results prove Ca²⁺, calreticulin, and calcineurin involvement in STAT1‐mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.     

Structure-based virtual screening,synthesis and SAR of novel inhibitors of hepatitis C virus NS5B polymerase

Hepatitis C virus (HCV) NS5B polymerase is a key target for the development of therapeutic agents aimed at the treatment of HCV infections. Here we report on the identification of novel allosteric inhibitors of HCV NS5B through a combination of structure-based virtual screening, synthesis and structure–activity relationship (SAR) optimization approach. Virtual screening of 260,000 compounds from the ChemBridge database against the tetracyclic indole inhibitor binding pocket of NS5B (allosteric pocket-1, AP-1), sequentially down-sized the library by 4 orders of magnitude to yield 23 candidates. In vitro evaluation of the NS5B inhibitory activity of the in-silico selected compounds resulted in 17% hit rate, identifying two novel chemotypes. Of these, compound 3, bearing the rhodanine scaffold, proved amenable for productive SAR exploration and synthetic modification. As a result, 25 derivatives that exhibited IC₅₀ values ranging from 7.7 to 68.0 μM were developed. Docking analysis of lead compound 28 within the tetracyclic indole- and benzylidene-binding allosteric pockets (AP-1 and AP-3, respectively) of NS5B revealed topological similarities between these two pockets. Compound 28, a novel rhodanine analog with NS5B inhibitory potency in the low micromolar level range may be a promising lead for future development of more potent NS5B inhibitors.     

圣路易斯脑炎病毒NS2B-NS3蛋白酶的原核表达及其抑制剂的筛选

【目的】圣路易斯脑炎病毒(St. Louis encephalitis virus,SLEV)属于黄病毒科,是一种单股正链RNA病毒。黄病毒编码的非结构蛋白NS3在病毒复制以及多聚蛋白加工过程中起着重要作用,NS2B是其发挥作用的重要辅助因子。因此,NS2B-NS3蛋白酶复合物是抗病毒药物的重要靶标。本研究旨在构建SLEV NS2B-NS3蛋白酶的原核表达系统并建立其抑制剂的高通量筛选方法,从而发现其小分子抑制剂。【方法】通过PCR扩增SLEVNS2B-NS3蛋白的编码区,构建原核表达质粒;在大肠杆菌BL21(DE3)中,经异丙基硫代半乳糖苷(Isopropyl β-D-thiogalactoside)诱导得到可溶性的NS2B-NS3蛋白,并用镍亲和层析方法进行纯化;基于荧光共振能量转移(Fluorescence resonance energy transfer)技术检测NS2B-NS3蛋白酶活性,建立其抑制剂的高通量筛选平台。【结果】SLEV NS2B-NS3蛋白酶纯化程度高达95%以上,基于酶活测定的抑制剂筛选平台准确可行。对700多个上市药物进行筛选后,发现原花青素对SLEVNS2B-NS3蛋白酶具有明显的抑制活性。【结论】本研究为SLEVNS2B-NS3蛋白酶抑制剂提供了一种操作方便、高通量的筛选方法,并首次发现了原花青素具有抑制SLEV NS2B-NS3蛋白酶活性的功能,可以作为治疗SLEV感染的潜在靶向药物。     

Structure-based virtual screening approach to the discovery of phosphoinositide 3-kinase alpha inhibitors

Phosphoinositide 3-kinase alpha (PI3Kα) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. Herein we report a successful application of the structure-based virtual screening to identify the novel inhibitors of PI3Kα. These inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC₅₀ values ranging from 20 to 40 μM. Therefore, they deserve a consideration for further development by structure-activity relationship (SAR) studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of PI3Kα are addressed in detail.     

Modulation of enzymatic activity of dengue virus nonstructural protein NS3 nucleoside triphosphatase/helicase by poly(U)

The nonstructural protein 3 (NS3) appears to be the most promising target for anti-flavivirus therapy because of its multiple enzymatic activities that are indispensable for virus replication. NS3 of dengue virus type 2 (DEN2) is composed of two domains, a serine protease in the N-terminal domain (NS3pro) and RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase at the C-terminus (NS3h). NS3 plays an important role in viral replication and the coordinated regulation of all the catalytic activities in the full-length NS3 protein. In this study, a plasmid harboring the NS3 helicase domain (NS3h) was constructed by PCR. The 56.5 kDa NS3h protein was purified by metal-chelate affinity chromatography followed by renaturation, mediated by artificial chaperone-assisted refolding, which yielded the active helicase. NTPase activity was assayed with Malachite Green. The NTPase activity in the presence of poly(U) showed a higher turnover number (k _cat) and a lower K _m value than without poly(U). The activity increased approximately fourfold in the presence of polynucleotides. This indicates that NTPase activity of dengue NS3 can be stimulated by polynucleotides. A helicase assay based on internal fluorescence quenching was conducted using short internally quenched DNA oligonucleotides as substrates. Significant fluorescence signaling increase was observed in the absence of polynucleotides such as poly(U). No unwinding activity was observed with addition of poly(U). The approach we describe here is useful for the further characterization of substrate specificity and for the design of high-throughput assays aimed at discovery of inhibitors against NS3 NTPase/helicase activities.     

The discovery of novel HDAC3 inhibitors via virtual screening and in vitro bioassay

Histone deacetylase 3 (HDAC3) is a potential target for the treatment of human diseases such as cancers, diabetes, chronic inflammation and neurodegenerative diseases. Previously, we proposed a virtual screening (VS) pipeline named “Hypo1_FRED_SAHA-3” for the discovery of HDAC3 inhibitors (HDAC3Is) and had thoroughly validated it by theoretical calculations. In this study, we attempted to explore its practical utility in a large-scale VS campaign. To this end, we used the VS pipeline to hierarchically screen the Specs chemical library. In order to facilitate compound cherry-picking, we then developed a knowledge-based pose filter (PF) by using our in-house quantitative structure activity relationship- (QSAR-) modelling approach and coupled it with FRED and Autodock Vina. Afterward, we purchased and tested 11 diverse compounds for their HDAC3 inhibitory activity in vitro. The bioassay has identified compound 2 (Specs ID: AN-979/41971160) as a HDAC3I (IC₅₀?=?6.1?μM), which proved the efficacy of our workflow. As a medicinal chemistry study, we performed a follow-up substructure search and identified two more hit compounds of the same chemical type, i.e. 2–1 (AQ-390/42122119, IC₅₀?=?1.3?μM) and 2–2 (AN-329/43450111, IC₅₀?=?12.5?μM). Based on the chemical structures and activities, we have demonstrated the essential role of the capping group in maintaining the activity for this class of HDAC3Is. In addition, we tested the hit compounds for their in vitro activities on other HDACs, including HDAC1, HDAC2, HDAC8, HDAC4 and HDAC6. We have identified these compounds are HDAC1/2/3 selective inhibitors, of which compound 2 show the best selectivity profile. Taken together, the present study is an experimental validation and an update to our earlier VS strategy. The identified hits could be used as starting structures for the development of highly potent and selective HDAC3Is.     

Structure-based virtual screening for insect ecdysone receptor ligands using MM/PBSA

The ecdysone receptor (EcR) is an insect nuclear receptor that is activated by the molting hormone, 20-hydroxyecdysone. Because synthetic EcR ligands disrupt the normal growth of insects, they are attractive candidates for new insecticides. In this study, the Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method was used to predict the binding activity of EcR ligands. Validity analyses using 40 known EcR ligands showed that the binding activity was satisfactorily predicted when the ligand conformational free energy term was introduced. Subsequently, this MM/PBSA method was applied to structure-based hierarchical virtual screening, and 12 candidate compounds were selected from a database of 3.8 million compounds. Five of these compounds were active in a cell-based competitive binding assay. The most potent compound is a simple proline derivative with low micromolar binding activity, representing a valuable lead compound for further structural optimization.     

Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein

The NS3 protein of Japanese encephalitis virus (JEV) contains motifs typical of RNA helicase/NTPase but no RNA helicase activity has been reported for this protein. To identify and characterize the RNA helicase activity of JEV NS3, a truncated form of the protein with a His-tag was expressed in Escherichia coli and purified. The purified JEV NS3 protein showed an RNA helicase activity, which was dependent on divalent cations and ATP. An Asp-285-to-Ala substitution in motif II of the JEV NS3 protein abolished the ATPase and RNA helicase activities. These results indicate that the C-terminal 457 residues are sufficient to exhibit the RNA helicase activity of JEV NS3.     

Targeting the NTPase site of Zika virus NS3 helicase for inhibitor discovery

Abstract

The major threats linked to Zika virus (ZIKV) are microcephaly, Guillain-Barre syndrome, and the ability to transfer through sexual transmission. Despite these threats, Zika specific FDA approved drugs or vaccines are not available as of yet. Additionally, the involvement of pregnant women makes the drug screening process lengthy and complicated in terms of safety and minimum toxicity of the molecules. Since NS3 helicase of ZIKV performs the critical function of unwinding double-stranded RNA during replication, it is considered as a promising drug target to block ZIKV replication. In the present study, we have exploited the NTPase site of ZIKV NS3 helicase for screening potential inhibitor compounds by molecular docking, and molecular dynamics (MD) simulation approaches. NS3 helicase hydrolyzes the ATP to use its energy for unwinding RNA. We have chosen twenty natural compounds from ZINC library with known antiviral properties and a helicase focused library (HFL) of small molecules from Life Chemicals compounds. After going through docking, the top hit molecules from ZINC and HFL library were further analysed by MD simulations to find out stable binding poses. Finally, we have reported the molecules with potential of binding at NTPase pocket of ZIKV NS3 helicase, which could be further tested on virus through in vitro experiments to check their efficacy.

Communicated by Ramaswamy H. Sarma     

Partial nucleotide sequencing of the NS3/helicase region of hepatitis G virus to prove vertical transmission

To study non-parental transmission of hepatitis G virus and/or GB virus C (HGV/GBV-C), we sequenced and compared the NS3/helicase region of the virus for five HGV/GBV-C RNA-positive mothers and their 11 children who had experienced neither blood transfusion nor overt hepatitis and were negative for HBV, HCV and HIV, except in one mother coinfected with HCV. The nucleotide sequences of the familial HGV/GBV-C isolates showed high similarity of 99-100% (mean 99.8%, 100% at the deduced amino acid level) between mother and her child(ren) in each family. These findings strongly suggest the spontaneous occurrence of mother-to-child transmission of HGV/GBV-C as reported previously. They also suggest that nucleotide sequence analysis on the NS3/helicase region of HGV/GBV-C may be a useful tool to study HGV/GBV-C transmission.     

Structure-based virtual screening toward the discovery of novel inhibitors for impeding the protein-protein interaction between HIV-1 integrase and human lens epithelium-derived growth factor (LEDGF/p75)

HIV-1 integrase is a unique promising component of the viral replication cycle, catalyzing the integration of reverse transcribed viral cDNA into the host cell genome. Generally, IN activity requires both viral as well as a cellular co-factor in the processing replication cycle. Among them, the human lens epithelium-derived growth factor (LEDGF/p75) represented as promising cellular co-factor which supports the viral replication by tethering IN to the chromatin. Due to its major importance in the early steps of HIV replication, the interaction between IN and LEDGF/p75 has become a pleasing target for anti-HIV drug discovery. The present study involves the finding of novel inhibitor based on the information of dimeric CCD of IN in complex with known inhibitor, which were carried out by applying a structure-based virtual screening concept with molecular docking. Additionally, Free binding energy, ADME properties, PAINS analysis, Density Functional Theory, and Enrichment Calculations were performed on selected compounds for getting a best lead molecule. On the basis of these analyses, the current study proposes top 3 compounds: Enamine-Z742267384, Maybridge-HTS02400, and Specs-AE-848/37125099 with acceptable pharmacological properties and enhanced binding affinity to inhibit the interaction between IN and LEDGF/p75. Furthermore, Simulation studies were carried out on these molecules to expose their dynamics behavior and stability. We expect that the findings obtained here could be future therapeutic agents and may provide an outline for the experimental studies to stimulate the innovative strategy for research community.     

Structure-based design of a novel series of azetidine inhibitors of the hepatitis C virus NS3/4A serine protease

Structural homology between thrombin inhibitors and the early tetrapeptide HCV protease inhibitor led to the bioisosteric replacement of the P2 proline by a 2,4-disubstituted azetidine within the macrocyclic β-strand mimic. Molecular modeling guided the design of the series. This approach was validated by the excellent activity and selectivity in biochemical and cell based assays of this novel series and confirmed by the co-crystal structure of the inhibitor with the NS3/4A protein (PDB code: 4TYD).     

Structure-based virtual screening and biological evaluation of potent and selective ADAM12 inhibitors

We describe a series of potent and selective inhibitors of ADAM12 that were discovered using computational screening of a focused virtual library. The initial structure-based virtual screening selected 64 compounds from a 3D database of 67,062 molecules. Being evaluated by a cell-based ADAM12 activity assay, compounds 5, 11, 14, 16 were further identified as the potent and selective inhibitors of ADAM12 with low nanomolar IC₅₀ values. The mechanism underlying the potency and selectivity of a representative compound, 5, was investigated through molecular docking studies.