Molecular Modeling-Based Evaluation of hTLR10 and Identification of Potential Ligands in Toll-Like Receptor Signaling

Toll-like receptors (TLRs) are pattern recognition receptors that recognize pathogens based on distinct molecular signatures. The human (h)TLR1, 2, 6 and 10 belong to the hTLR1 subfamilies, which are localized in the extracellular regions and activated in response to diverse ligand molecules. Due to the unavailability of the hTLR10 crystal structure, the understanding of its homo and heterodimerization with hTLR2 and hTLR1 and the ligand responsible for its activation is limited. To improve our understanding of the TLR10 receptor-ligand interaction, we used homology modeling to construct a three dimensional (3D) structure of hTLR10 and refined the model through molecular dynamics (MD) simulations. We utilized the optimized structures for the molecular docking in order to identify the potential site of interactions between the homo and heterodimer (hTLR10/2 and hTLR10/1). The docked complexes were then used for interaction with ligands (Pam₃CSK₄ and PamCysPamSK₄) using MOE-Dock and ASEDock. Our docking studies have shown the binding orientations of hTLR10 heterodimer to be similar with other TLR2 family members. However, the binding orientation of hTLR10 homodimer is different from the heterodimer due to the presence of negative charged surfaces at the LRR11-14, thereby providing a specific cavity for ligand binding. Moreover, the multiple protein-ligand docking approach revealed that Pam₃CSK₄ might be the ligand for the hTLR10/2 complex and PamCysPamSK_4, a di-acylated peptide, might activate hTLR10/1 hetero and hTLR10 homodimer. Therefore, the current modeled complexes can be a useful tool for further experimental studies on TLR biology.     

Toll-Like Receptor 7 Agonists: Chemical Feature Based Pharmacophore Identification and Molecular Docking Studies

Chemical feature based pharmacophore models were generated for Toll-like receptors 7 (TLR7) agonists using HypoGen algorithm, which is implemented in the Discovery Studio software. Several methods tools used in validation of pharmacophore model were presented. The first hypothesis Hypo1 was considered to be the best pharmacophore model, which consists of four features: one hydrogen bond acceptor, one hydrogen bond donor, and two hydrophobic features. In addition, homology modeling and molecular docking studies were employed to probe the intermolecular interactions between TLR7 and its agonists. The results further confirmed the reliability of the pharmacophore model. The obtained pharmacophore model (Hypo1) was then employed as a query to screen the Traditional Chinese Medicine Database (TCMD) for other potential lead compounds. One hit was identified as a potent TLR7 agonist, which has antiviral activity against hepatitis virus in vitro. Therefore, our current work provides confidence for the utility of the selected chemical feature based pharmacophore model to design novel TLR7 agonists with desired biological activity.     

Neolignans from mezilaurus itauba

The wood bark of Mezilaurus itauba afforded in addition to seven known neolignans, three new compounds rel-(7R,8R,1′S,3′S)-Δ^5′,8′-5′-methoxy-3,4-methylenedioxy-1′,2′,3′,4′-tetrahydro-2′,4′-dioxo-7.3′,8.1′-neolignan, rel-(7S,8S,1′S, 2′S, 3′R, 4′S)-Δ^8′-2′,4′-dihydroxy-3,4-methylenedioxy-1′,2′,3′,4′,5′,6′-hexahydro-5′-oxo-7.3′,8.1′-neolignan and rel-(7S,8S)-Δ^8′-6′-hydroxy 5′-methoxy-3,4-methylenedioxy-7·O·2′,8.3′-neolignan. The latter compound has been detected previously in Aniba terminalis. The structures were elucidated by spectroscopic methods and comparison with related compounds.     

Hierarchical virtual screening of the dual MMP-2/HDAC-6 inhibitors from natural products based on pharmacophore models and molecular docking

The dual-target inhibitors tend to improve the response rate in treating tumors, comparing with the single-target inhibitors. Matrix metalloproteinase-2 (MMP-2) and histone deacetylase-6 (HDAC-6) are attractive targets for cancer therapy. In this study, the hierarchical virtual screening of dual MMP-2/HDAC-6 inhibitors from natural products is investigated. The pharmacophore model of MMP-2 inhibitors is built based on ligands, but the pharmacophore model of HDAC-6 inhibitors is built based on the experimental crystal structures of multiple receptor–ligand complexes. The reliability of these two pharmacophore models is validated subsequently. The hierarchical virtual screening, combining these two different pharmacophore models of MMP-2 and HDAC-6 inhibitors with molecular docking, is carried out to identify the dual MMP-2/HDAC-6 inhibitors from a database of natural products. The four potential dual MMP-2/HDAC-6 inhibitors of natural products, STOCK1 N-46177, STOCK1 N-52245, STOCK1 N-55477, and STOCK1 N-69706, are found. The studies of binding modes show that the screened four natural products can simultaneously well bind with the MMP-2 and HDAC-6 active sites by different kinds of interactions, to inhibit the MMP-2 and HDAC-6 activities. In addition, the ADMET properties of screened four natural products are assessed. These found dual MMP-2/HDAC-6 inhibitors of natural products could serve as the lead compounds for designing the new dual MMP-2/HDAC-6 inhibitors having higher biological activities by carrying out structural modifications and optimizations in the future studies.     

Flavonoids from Gutierrezia texana var. texana

Eleven flavonoids, including two new compounds, were isolated from Gutierrezia texana: the structures of the new compounds are 5,7,2′,5′-tetrahydroxy-3,4′-dimethoxyflavone and 5′-acetoxy-5,7,2′-trihydroxy-3,4′-dimethoxyflavone. The nine known compounds are 5,4′,5′-trihydroxy-3,6,7,8-tetramethoxyflavone, 5,7,2′,5′-tetrahydroxy-3,6,4′-trimethoxyflavone, 5,7,3′-trihydroxy-3,4′-dimethoxyflavone, 5,7,3′,4′-tetrahydroxyflavone, 3,5,7,3′,4′- pentahydroxyflavone, 5,7,3′,4′-tetrahydroxy-3-methoxyflavone, 5,6,7,3′,4′,-pentahydroxy-3-methoxyflavone, 5,7,3′,4′-tetrahydroxy-3-methoxyflavone 7-O-glucoside and 3,5,7,3′,4′-pentahydroxyflavanone.     

Pharmacoinformatics analysis to identify inhibitors of Mtb-ASADH

Aspartate-semialdehyde dehydrogenase (ASADH; EC 1.2.1.11) is a key enzyme in the biosynthesis of essential amino acids in prokaryotes and fungi, inhibition of ASADH leads to the development of novel antitubercular agents. In the present work, a combined structure and ligand-based pharmacophore modeling, molecular docking, and molecular dynamics (MD) approaches were employed to identify potent inhibitors of mycobacterium tuberculosis (Mtb)-ASADH. The structure-based pharmacophore hypothesis consists of three hydrogen bond acceptor (HBA), two negatively ionizable, and one positively ionizable center, while ligand-based pharmacophore consists of additional one HBA and one hydrogen bond donor features. The validated pharmacophore models were used to screen the chemical databases (ZINC and NCI). The screened hits were subjected to ADME and toxicity filters, and subsequently to the molecular docking analysis. Best-docked 25 compounds carry the characteristics of highly electronegative functional groups (–COOH and –NO₂) on both sides and exhibited the H-bonding interactions with highly conserved residues Arg99, Arg249, and His256. For further validation of docking results, MD simulation studies were carried out on two representative compounds NSC51108 and ZINC04203124. Both the compounds remain bound to the key active residues of Mtb-ASADH during the MD simulations. These identified hits can be further used for lead optimization and in the design more potent inhibitors against Mtb-ASADH.     

晶帽石斛的化学成分研究

该研究利用硅胶、凝胶、MCI、中压制备色谱(MPLC)和高效液相半制备色谱(semi-HPLC)等方法,对晶帽石斛(Dendrobium crystallinum)进行了化学成分研究。结果表明:提纯、分离共得到10个化合物,经波谱数据分析及与文献数据对照,分别鉴定为石斛酚(1),3,4'-二羟基-5-甲氧基联苄(2),3,4',5-三羟基-3-甲氧基联苄(3),二氢藜芦醇(4),安告佛醇(5),3',5,7-三羟基-4'-甲氧基黄烷酮(6),4',5,7-三羟基-6-甲氧基黄烷酮(7),丁香树脂醇(8),β-谷甾醇(9),β-胡萝卜苷(10)。其中,除化合物2和化合物10以外,其余8个化合物均在该种植物中首次发现。     

Novel virtual lead identification in the discovery of hematopoietic cell kinase (HCK) inhibitors: application of 3D QSAR and molecular dynamics simulation

High level of hematopoietic cell kinase (Hck) is associated with drug resistance in chronic myeloid leukemia. Additionally, Hck activity has also been connected with the pathogenesis of HIV-1 and chronic obstructive pulmonary disease. In this study, three-dimensional (3D) QSAR pharmacophore models were generated for Hck based on experimentally known inhibitors. A best pharmacophore model, Hypo1, was developed with high correlation coefficient (0.975), Low RMS deviation (0.60) and large cost difference (49.31), containing three ring aromatic and one hydrophobic aliphatic feature. It was further validated by the test set (r?=?0.96) and Fisher’s randomization method (95%). Hypo 1 was used as a 3D query for screening the chemical databases, and the hits were further screened by applying Lipinski’s rule of five and ADMET properties. Selected hit compounds were subjected to molecular docking to identify binding conformations in the active site. Finally, the appropriate binding modes of final hit compounds were revealed by molecular dynamics (MD) simulations and free energy calculation studies. Hence, we propose the final three hit compounds as virtual candidates for Hck inhibitors.     

Benzofuranoid Neolignans from Piper kadsura

In a continuing research for neolignans from Piper kadsura (Choisy) Ohwi, six benzofuranoid neolignans were isolated from the aerial part of the plant. Their structure determination were based on the spectroscopic analysis (UV, IR, MS, NMR and CD) and derivative synthesis. Three of the isolated compounds were identified as new structures: 7R, 8R, 1′S-△8′-3, 4-methylenedioxy-5′-methoxy-l′, 4′-dihydro-4′-oxo-7, 0, 2′, 8. l′-neolignan ( Ⅰ ), 7 R, 8 R, 1 ′ R- △8′ - 3,4- methylenedioxy- 1 ′- methoxy - 1′,6′- dihydro- 6′- oxo- 7.0.4′,8. 3′-neolignan (Ⅳ) and 7R, 8R, 1′S-△8′-3, 4-methylenedioxy-l′-methoxy-1′,6′-dihydro-6′-oxo-7.0.4′,8.3′-neolignan (Ⅴ). Known compounds among them are 7R, 8S,1′S-△8′-3, 4-methylenedioxy-5′-methoxy-1′, 4′-dihydro-4′-oxo-7. 0. 2′, 8. 1′-neolignan(Ⅱ), 7S, 8S, 1′R-△8′-3, 4, 5′-trimethoxy-1′, 4′-dihydro-4′-oxo-7.0. 2′, 8. 1′-neolignan (Ⅲ) and 75, 85, 1′S-△8′-3, 4, l′-trimethoxy-l′, 6′-dihydro-6′-oxo-7. 0. 4′, 8. 3′-neolignans (Ⅵ). All of them were isolated from the plant for the first time.     

Virtual screening of ABCC1 transporter nucleotidebinding domains as a therapeutic target in multidrug resistant cancer

ABCC1 is a member of the ATP-binding Cassette super family of transporters, actively effluxes xenobiotics from cells. Clinically, ABCC1 expression is linked to cancer multidrug resistance. Substrate efflux is energised by ATP binding and hydrolysis at the nucleotide-binding domains (NBDs) and inhibition of these events may help combat drug resistance. The aim of this study is to identify potential inhibitors of ABCC1 through virtual screening of National Cancer Institute (NCI) compounds. A threedimensional model of ABCC1 NBD2 was generated using MODELLER whilst the X-ray crystal structure of ABCC1 NBD1 was retrieved from the Protein Data Bank. A pharmacophore hypothesis was generated based on flavonoids known to bind at the NBDs using PHASE, and used to screen the NCI database. GLIDE was employed in molecular docking studies for all hit compounds identified by pharmacophore screening. The best potential inhibitors were identified as compounds possessing predicted binding affinities greater than ATP. Approximately 5% (13/265) of the hit compounds possessed lower docking scores than ATP in ABCC1 NBD1 (NSC93033, NSC662377, NSC319661, NSC333748, NSC683893, NSC226639, NSC94231, NSC55979, NSC169121, NSC166574, NSC73380, NSC127738, NSC115534), whereas approximately 7% (7/104) of docked NCI compounds were predicted to possess lower docking scores than ATP in ABCC1 NBD2 (NSC91789, NSC529483, NSC211168, NSC318214, NSC116519, NSC372332, NSC526974). Analyses of docking orientations revealed P-loop residues of each NBD and the aromatic amino acids Trp653 (NBD1) and Tyr1302 (NBD2) were key in interacting with high-affinity compounds. On the basis of docked orientation and docking score the compounds identified may be potential inhibitors of ABCC1 and require further pharmacological analysis.

Abbreviations

ABC - ATP-binding cassette, DHS - dehydrosilybin, MDR - multidrug resistance, NBD - nucleotide-binding domain, PDB - protein data bank.     

Exploration of Virtual Candidates for Human HMG-CoA Reductase Inhibitors Using Pharmacophore Modeling and Molecular Dynamics Simulations

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is a rate-controlling enzyme in the mevalonate pathway which involved in biosynthesis of cholesterol and other isoprenoids. This enzyme catalyzes the conversion of HMG-CoA to mevalonate and is regarded as a drug target to treat hypercholesterolemia. In this study, ten qualitative pharmacophore models were generated based on chemical features in active inhibitors of HMGR. The generated models were validated using a test set. In a validation process, the best hypothesis was selected based on the statistical parameters and used for virtual screening of chemical databases to find novel lead candidates. The screened compounds were sorted by applying drug-like properties. The compounds that satisfied all drug-like properties were used for molecular docking study to identify their binding conformations at active site of HMGR. The final hit compounds were selected based on docking score and binding orientation. The HMGR structures in complex with the hit compounds were subjected to 10 ns molecular dynamics simulations to refine the binding orientation as well as to check the stability of the hits. After simulation, binding modes including hydrogen bonding patterns and molecular interactions with the active site residues were analyzed. In conclusion, four hit compounds with new structural scaffold were suggested as novel and potent HMGR inhibitors.     

Neolignans from stem bark of ocotea veraguensis

The stem bark of Ocotea veraguensis has yielded nine neolignans of which five appear to be novel. The new neolignans, which were identified on the basis of spectral characteristics, are^* (7S,8R,1′S,2′S,3′R,4′S)-Δ^8′-2′,4′-dihydroxy-3,3′5′-trimethoxy-4,5-methylenedioxy-1′,2′,3′,4′-tetrahydro-7.3′,8.1′-neolignan, (7S,8R,1′S,3′S,4′S)-Δ^8′-4,4'-dihydroxy-3,3′,5′-trimethoxy-1′,2′,3′,4′-tetrahydro-2′-oxo-7.3′,8.1′-neolignan, (7S,8S,1′R)-Δ^8′-3′,5′-dimethoxy-3,4-methylenedioxy-1′,4′-dihydro-4′-oxo-7.0.2′,8.1′-neolignan, (7S,8S,1′R )-Δ^8′-1′-methoxy-3,4-methylenedioxy-1′,6′-dihydro-6′-oxo-7.0.4′,8.3′-neolignan and (7S,8S)-Δ^8′-2′,6′-dimethoxy-3,4-methylenedioxy-7.0.3′,8.4′,1′.0.7′-neolignan.     

Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9

Members of the Toll-like receptor family are essential components of the innate immune system. Herein we report the molecular cloning and characterization of three novel human Toll-like receptors (hTLRs) designated hTLR7, hTLR8, and hTLR9. Human TLR7-9, like the previously described members hTLR1-6 contain an ectodomain with multiple leucine-rich repeats (LRRs) and a cytoplasmic domain homologous to that of the human interleukin-1 (IL-1) receptor. When compared with hTLR1-6, the hTLR7-9 has a higher molecular weight largely as a result of a longer ectodomain. Phylogenetic analysis shows that hTLR7-9 belong to a new sub-family of the hTLRs. Analysis of mRNA expression at the tissue levels shows differential expression patterns; hTLR7 is predominantly expressed in lung, placenta and spleen, hTLR8 is more abundant in lung, peripheral blood leukocytes, and hTLR9 is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. The hTLR7 and hTLR8 genes are located on the sex chromosome X, hTLR9 gene is located on chromosome 3. Expression of constitutively active hTLR7-9 stimulates an NF-kappaB signaling pathway indirectly supporting the contention that these receptors are involved in cellular responses to stimuli, which activate innate immunity.     

Homology modeling,docking and structure-based virtual screening for new inhibitor identification of Klebsiella pneumoniae heptosyltransferase-III

Abstract

Klebsiella pneumoniae (K. pneumoniae) is a Gram-negative opportunistic pathogen commonly associated with hospital-acquired infections that are often resistant even to antibiotics. Heptosyltransferase (HEP) belongs to the family of glycosyltransferase-B (GT-B) and plays an important in the synthesis of lipopolysaccharides (LPS) essential for the formation of bacterial cell membrane. HEP-III participates in the transfer of heptose sugar to the outer surface of bacteria to synthesize LPS. LPS truncation increases the bacterial sensitivity to hydrophobic antibiotics and detergents, making the HEP as a novel drug target. In the present study, we report the 3D homology model of K. pneumoniae HEP-III and its structure validation. Active site was identified based on similarities with known structures using Dali server, and structure-based pharmacophore model was developed for the active site substrate ADP. The generated pharmacophore model was used as a 3D search query for virtual screening of the ASINEX database. The hit compounds were further filtered based on fit value, molecular docking, docking scores, molecular dynamics (MD) simulations of HEP-III complexed with hit molecules, followed by binding free energy calculations using Molecular Mechanics-Poisson–Boltzmann Surface Area (MM-PBSA). The insights obtained in this work provide the rationale for design of novel inhibitors targeting K. pneumoniae HEP-III and the mechanistic aspects of their binding.

Communicated by Ramaswamy H. Sarma     

Discovery of novel 5α-reductase type II inhibitors by pharmacophore modelling,virtual screening,molecular docking and molecular dynamics simulations

Benign prostatic hyperplasia (BPH) is caused by augmented levels of androgen dihydrotestosterone (DHT) which is involved in the growth of the prostate in humans. 5α-Reductase type II (5αR2) is an intracellular enzyme that catalyses the formation of DHT from testosterone; hence, the inhibition of 5αR2 has emerged as one of the most promising strategies for the treatment of BPH. In this study, a computational approach that integrates ligand-based pharmacophore modelling, virtual screening, molecular docking and molecular dynamics (MD) simulations was adopted to discover novel 5αR2 inhibitors with less side effects. After validating by Fischer's randomisation and Güner–Henry test, the best quantitative pharmacophore model (Hypo1), consisting of two hydrogen-bond acceptors and three hydrophobic features, was subsequently used as a three-dimensional-query in virtual screening to identify potential hits from Maybridge and National Cancer Institute databases. These hits were further filtered by ADMET (absorption, distribution, metabolism, elimination and toxicology) and molecular docking experiments, and their binding stabilities were validated by 10-ns MD simulations. Finally, only one hit was identified as a potential lead based on higher predicted inhibitory activity to 5αR2 compared with the most active inhibitor (finasteride). Our results further suggest that this potential lead could easily be synthesised and has structural novelty, making it a promising candidate for treating BPH.     

Xanthones and Phenylpropanoids from the Whole Herb of Swertia pseudochinensis and Their Anti-Inflammatory Activity

An undescribed xanthone dimer, 1,3,5,8-tetrahydroxy-7-(1′,5′,8′-trihydroxy-3′-methoxy-2′-xanthonyl)xanthone ( 1 ) was separated together with eleven known compounds ( 2 – 12 ) from the dried whole herb of Swertia pseudochinensis. It was the first time that the compounds 8 – 12 were isolated from the Swertia genus. The structure of compound 1 was illuminated based on chemical evidence and spectral data analysis (UV, 1D and 2D-NMR, HR-ESI-MS). Moreover, the inhibitory effects of all compounds on NO production in LPS-induced RAW 264.7 cells were tested, compounds 8 , 9 , 10 , 11 and 12 showing significant inhibition. The IC₅₀ value of compound 12 was 3.05±1.10 μM. Using target screening and molecular docking, we hypothesized that compound 12 may bind neutrophil elastase to exert its anti-inflammatory effects.     

Two new dihydrobenzofuran-type neolignans from Breynia fruticosa

(7S,8R,7′S)-9,7′,9′-Trihydroxy-3,4-methylenedioxy-3′-methoxy [7-O-4′,8-5′] neolignan (1) and (7S,8R,7′S)-9,9′-dihydroxy-3,4-methylenedioxy-3′,7′-dimethoxy [7-O-4′,8-5′] neolignan (2), two new natural dihydrobenzofuran-type neolignans, along with 9,9′-dihydroxy-3,4-methylenedioxy-3′-methoxy [7-O-4′,8-5′] neolignan (3) and (-)-machicendiol (4), were isolated from the whole plants of Breynia fruticosa. The structures of 1 and 2, including the absolute configurations, were determined by spectroscopic methods and circular dichroism (CD) techniques. The absolute configuration of 4 was confirmed by calculations of the OR spectrum, together with OR and ECD spectra of its p-bromobenzoate ester (4a).     

Chemical constituents of Trilepisium madagascariense (Moraceae) and their antimicrobial activity

The chemical study of Trilepisium madagascariense has led to the isolation of two previously undescribed compounds, (+)-(2S)-7-hydroxy-3′,4′-dimethoxyflavan (trilepisflavan) and (E)-4-[3-(3,4-dihydroxyphenyl)prop-2-enoyloxy]-3-hydroxybenzoic acid (trilepisuimic acid), together with ten known compounds caffeic acid, catechin, erythrodiol-3-O-palmitate, 8-C-glucopyranosylapigenin, dihydrokaempferol, protocatechuic acid, 3′,7-dihydroxy-4′-methoxyflavan, isoliquiritigenin, luteolin and 1,3-dimethoxybenzene. Their structures were elucidated on the basis of spectroscopic evidence. Crude extracts, trilepisflavan, dihydrokaempferol and 8-C-glucopyranosylapigenin showed significant antimicrobial activity.     

Molecular Dynamics Simulations of Sonic Hedgehog-Receptor and Inhibitor Complexes and Their Applications for Potential Anticancer Agent Discovery

The sonic hedgehog (Shh) signaling pathway is necessary for a variety of development and differentiation during embryogenesis as well as maintenance and renascence of diverse adult tissues. However, an abnormal activation of the signaling pathway is related to various cancers. In this pathway, the Shh signaling transduction is facilitated by binding of Shh to its receptor protein, Ptch. In this study, we modeled the 3D structure of functionally important key loop peptides of Ptch based on homologous proteins. Using this loop model, the molecular interactions between the structural components present in the pseudo-active site of Shh and key residues of Ptch was investigated in atomic level through molecular dynamics (MD) simulations. For the purpose of developing inhibitor candidates of the Shh signaling pathway, the Shh pseudo-active site of this interface region was selected as a target to block the direct binding between Shh and Ptch. Two different structure-based pharmacophore models were generated considering the key loop of Ptch and known inhibitor-induced conformational changes of the Shh through MD simulations. Finally two hit compounds were retrieved through a series of virtual screening combined with molecular docking simulations and we propose two hit compounds as potential inhibitory lead candidates to block the Shh signaling pathway based on their strong interactions to receptor or inhibitor induced conformations of the Shh.     

Synthesis of metabolic intermediates of diethylstilbestrol

This report details the synthesis of 1) 3,4,4′-trihydroxy-α,α′-diethyl-trans-stilbene; 2) 3,4-bis-(p-hydroxyphenyl)-trans-3-hexenol; 3) 3,4-bis-(p-hydroxyphenyl)-2,4-cis,cis-hexadienol; 4) 3,4-bis-(3′-methoxy-4′-hydroxyphenyl)-trans-3-hexene; 5) 3,4-bis-(3′, 4′-dimethoxyphenyl)-trans-3-hexene. These compounds are suspected metabolites of diethylstilbestrol.