Exploration of enzyme-ligand interactions in CYP2D6 & 3A4 homology models and crystal structures using a novel computational approach

New crystal structures of human CYP2D6 and CYP3A4 have recently been reported, and in this study, we wanted to compare them with previously used homology models with respect to predictions of site of metabolism and ligand-enzyme interactions. The data set consisted of a family of synthetic opioid analgesics with the aim to cover both CYP2D6 and CYP3A4, as most of these compounds are metabolized by both isoforms. The program MetaSite was used for the site of metabolism predictions, and the results were validated by experimental assessment of the major metabolites formed with recombinant CYP450s. This was made on a selection of 14 compounds in the data set. The prediction rates for MetaSite were 79-100% except for the CYP3A4 homology model, which picked the correct site in half of the cases. Despite differences in orientation of some important amino acids in the active sites, the MetaSite-predicted sites were the same for the different structures, with the exception of the CYP3A4 homology model. Further exploration of interactions with ligands was done by docking substrates/inhibitors in the different structures with the docking program GLUE. To address the challenge in interpreting patterns of enzyme-ligand interactions for the large number of different docking poses, a new computational tool to handle the results from the dockings was developed, in which the output highlights the relative importance of amino acids in CYP450-substrate/inhibitor interactions. The method is based on calculations of the interaction energies for each pose with the surrounding amino acids. For the CYP3A4 structures, this method was compared with consensus principal component analysis (CPCA), a commonly used method for structural comparison to evaluate the usefulness of the new method. The results from the two methods were comparable with each other, and the highlighted amino acids resemble those that were identified to have a different orientation in the compared structures. The new method has clear advantages over CPCA in that it is far simpler to interpret and there is no need for protein alignment. The methodology enables structural comparison but also gives insights on important amino acid substrate/inhibitor interactions and can therefore be very useful when suggesting modifications of new chemical entities to improve their metabolic profiles.     

人类2-氨基3-羧基粘康酸6-半醛脱羧酶(ACMSD)与底物及抑制剂作用模型的理论研究

利用同源模建和分子动力学模拟方法构建了人类2-氨基3-羧基粘康酸6-半醛脱羧酶(hACMSD)的三维结构, 并利用Profile-3D和Procheck等方法评估了模型的可靠性. 在此基础上, 用分子对接程序(Affinity), 将其底物2-氨基3-羧基粘康酸6-半醛(ACMS)和抑制剂喹啉酸(QA)分别与hACMSD进行对接, 获得了复合物结构的理论模型. 通过配体与受体之间相互作用能和结构分析给出了底物和抑制剂的具体结合方式, 明确了hACMSD与底物和抑制剂结合时起重要作用的氨基酸残基.     

Structural and thermodynamic studies on cation-Pi interactions in lectin-ligand complexes: high-affinity galectin-3 inhibitors through fine-tuning of an arginine-arene interaction

The high-resolution X-ray crystal structures of the carbohydrate recognition domain of human galectin-3 were solved in complex with N-acetyllactosamine (LacNAc) and the high-affinity inhibitor, methyl 2-acetamido-2-deoxy-4-O-(3-deoxy-3-[4-methoxy-2,3,5,6-tetrafluorobenzamido]-beta-D-galactopyranose)-beta-D-glucopyranoside, to gain insight into the basis for the affinity-enhancing effect of the 4-methoxy-2,3,5,6-tetrafluorobenzamido moiety. The structures show that the side chain of Arg144 stacks against the aromatic moiety of the inhibitor, an interaction made possible by a reorientation of the side chain relative to that seen in the LacNAc complex. Based on these structures, synthesis of second generation LacNAc derivatives carrying aromatic amides at 3'-C, followed by screening with a novel fluorescence polarization assay, has led to the identification of inhibitors with further enhanced affinity for galectin-3 (K(d) > or = 320 nM). The thermodynamic parameters describing the binding of the galectin-3 C-terminal to selected inhibitors were determined by isothermal titration calorimetry and showed that the affinity enhancements were due to favorable enthalpic contributions. These enhancements could be rationalized by the combined effects of the inhibitor aromatic structure on a cation-Pi interaction and of direct interactions between the aromatic substituents and the protein. The results demonstrate that protein-ligand interactions can be significantly enhanced by the fine-tuning of arginine-arene interactions.     

Adsorption Activity and Molecular Dynamics Study on Anti-corrosion Mechanism of Q235 Steel

The correlation between inhibition efficiency and molecular structures of the inhibitor during hydrochloric acid corrosion of Q235 steel was studied by quantum chemical calculations and molecular dynamics(MD) simulation. The proton affinity(PA) calculations demonstrated that 2-(quinolin-2-yl)quinazolin-4(3H)-one inhibitor has the tendency to be protonated in hydrochloric acid, which was in good agreement with experimental observations. Besides, quantum chemical parameters revealed that the protonated corrosion inhibitor molecules were more easily adsorbed on Q235 steel surface and improved the corrosion resistance of steel. MD simulations were implemented to search for the adsorption behavior of this molecule on Fe (110) surface, which might be used as a convenient tool for estimating the interaction mechanism between inhibitor and iron surface.     

Comparative molecular field analysis (CoMFA) of new herbicidal sulfonylurea compounds

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The triterpenes 3β-lup-20(29)-en-3-ol and 3β-lup-20(29)-en-3-yl acetate and the carbohydrate 1,2,3,4,5,6-hexa-O-acetyl-dulcitol as photosynthesis light reactions inhibitors

Three compounds were isolated from Maytenus acanthophylla Reissek (Celastraceae): the pentacyclic triterpenes lup-20(29)-en-3β-ol (lupeol, 1) and 3β-lup-20(29)-en-3-yl acetate (2) and the carbohydrate 1,2,3,4,5,6-hexa-O-acetyldulcitol (3); lupeol was also isolated from Xylosma flexuosa. The compounds' structures were elucidated by spectroscopic and spectrometric analysis. Compound 1 acts as an energy transfer inhibitor, interacting with isolated CF? bound to thylakoid membrane, and dulcitol hexaacetate 3 behaves as a Hill reaction inhibitor and as an uncoupler, as determined by polarography. Chlorophyll a (Chl a) fluorescence induction kinetics from the minimum yield F? to the maximum yield F(M )provides information of the filling up from electrons coming from water to plastoquinone pool with reducing equivalents. In this paper we have examined the effects of compounds 1 and 3 on spinach leaf discs. Compound 1 induces the appearance of a K-band, which indicates that it inhibits the water splitting enzyme. In vivo assays measuring the fluorescence of chl a in P. ixocarpa leaves sprayed with compound 1, showed the appearance of the K-band and the PSII reaction centers was transformed to "heat sinks" or silent reaction centers unable to reduce Q(A). However, 3 also induced the appearance of a K band and a new band I appears in P. ixocarpa plants, therefore it inhibits at the water splitting enzyme complex and at the PQH? site on b?f complex. Compounds 1 and 3 did not affect chlorophyll a fluorescence of L. perenne plants.     

Synthesis and Conformational Analysis of 2′-Deoxy-2′-(3-methoxybenzamido)adenosine,a rational-designed inhibitor of trypanosomal glyceraldehyde phosphate dehydrogenase (GAPDH)

A series of 2′-benzamido-2′-deoxyadenosine analogues were synthesized in an effort to find new lead structures for the treatment of sleeping sickness. The 2′-deoxy-2′-(3-methoxybenzamido)adenosine ( 1h ) was proved to be a selective inhibitor of the parasite glyceraldehyde 3-phosphate dehydrogenase which confirms the modeling studies. The solution-state conformation of 2′-(thiophene-2-carboxamido) analogue 1d demonstrates a 2′-endo conformation, an orientation of the thiophene ring under the ribose moiety, and the base part occupying a ‘syn’/‘anti’ equilibrium.     

Diaporthichalasin, a novel CYP3A4 inhibitor from an endophytic Diaporthe sp.

A novel CYP3A4 inhibitor, diaporthichalasin, together with pycnidione were isolated from an endophytic fungus, Diaporthe sp. Their structures were elucidated on the basis of spectral data and the structure of diaporthichalasin was confirmed by X-ray crystallographic analysis. Diaporthichalasin exhibited significantly potent inhibition of CYP3A4 with an IC₅₀ value of 0.626 μM, while the IC₅₀ value of pycnidione was 465 μM.     

Solution-phase and solid-phase syntheses of enzyme inhibitor RK-682 and antibiotic agglomerins

The enzyme inhibitor RK-682 (5R)-(+)-1 was prepared in solution and on a solid support from (2R)-glycerates in five steps and ca. 40% overall yield. Key steps were a ring-closing tandem addition-Wittig alkenation reaction of the respective protected or immobilized glycerates with the ylide Ph(3)PCCO and the 3-acylation of the tetronic acids thus obtained with palmitic acid. A similar route extended by a mesylation-elimination sequence led to antibiotic agglomerins A-C 2 featuring 3-acyl-5-methylidenetetronic acid structures.     

Hepatitis C virus NS3 protease requires its NS4A cofactor peptide for optimal binding of a boronic acid inhibitor as shown by NMR

NMR spectroscopy was used to characterize the hepatitis C virus (HCV) NS3 protease in a complex with the 24 residue peptide cofactor from NS4A and a boronic acid inhibitor, Ac-Asp-Glu-Val-Val-Pro-boroAlg-OH. Secondary-structure information, NOE constraints between protease and cofactor, and hydrogen-deuterium exchange rates revealed that the cofactor was an integral strand in the N-terminal beta-sheet of the complex as observed in X-ray crystal structures. Based upon chemical-shift perturbations, inhibitor-protein NOEs, and the protonation state of the catalytic histidine, the boronic acid inhibitor was bound in the substrate binding site as a transition state mimic. In the absence of cofactor, the inhibitor had a lower affinity for the protease. Although the inhibitor binds in the same location, differences were observed at the catalytic site of the protease.     

A ligand-based molecular modeling study on some matrix metalloproteinase-1 inhibitors using several 3D QSAR techniques

Some three-dimensional quantitative structure-activity relationship (3D-QSAR) models have been constructed using the comparative molecular field analysis (CoMFA) and comparative molecular similarity indices (CoMSIA) for a series of 84 proline-based plus 12 structurally more diversified nonproline matrix metalloproteinase inhibitors. The structures of these inhibitors were built from a structure template extracted from the crystal structure of stromelysin. The structures built were divided into the training and test sets for both the CoMFA and CoMSIA analyses for each being composed of 60 and 24 inhibitors, respectively. The structures in the training set were aligned using some alignment rules derived from the analysis of the Ligplot program on a recent crystal structure of ligand-collagenase-1 complex. Some stepwise CoMSIA's were performed on the aligned training set on which the best CoMFA result was obtained. The best CoMSIA model was identified from the stepwise results, and the corresponding pharmacophore features were used for the construction of a pharmacophore hypothesis by the Catalyst 4.9 program. The training set was extended to include 11 structurally more diversified and nonproline inhibitors. To construct a pharmacophore hypothesis, the conformation of 60 structurally aligned proline-based inhibitors was fixed, while that of the 11 structurally more diversified nonproline inhibitors was allowed to vary during the hypothesis construction process. It was found that the predicted activities by the top hypothesis constructed for both the training and test sets were as good in statistics as those predicted by the best CoMSIA model from which the hypothesis was derived. The top hypothesis was mapped onto the structures of several highly active inhibitors selected from both the training and test sets. The goodness of mapping on each inhibitor was found to be correlated well with the activity of each inhibitor.     

药效团检索设计新的HIV-1蛋白酶抑制剂

通过对自建的未开发化合物三维结构库进行药效团检索,得到了4个对HIV-1蛋白酶抑制活化的化合物,通过构象分析发现包含药效团的构象处于优势构象,而且4个结构都含有带两个邻位羟基的苯环和一个间位羰基的药效团以及公共子结构。通过计算发现它们的疏水参数都很小。在考虑满足包含药效团的结构特征和有适中的疏水参数两个因素的前提下,设计出了新的具有潜在抑制HIV-1蛋白酶活性的化合物。它们的结构都比检索得到的四个化合物更为简单,因此易于合成。     

Crystal structures of Salinosporamide A (NPI-0052) and B (NPI-0047) in complex with the 20S proteasome reveal important consequences of beta-lactone ring opening and a mechanism for irreversible binding

The crystal structures of the yeast 20S proteasome core particle (CP) in complex with Salinosporamides A (NPI-0052; 1) and B (4) were solved at <3 angstroms resolution. Each ligand is covalently bound to Thr1O(gamma) via an ester linkage to the carbonyl derived from the beta-lactone ring of the inhibitor. In the case of 1, nucleophilic addition to the beta-lactone ring is followed by addition of C-3O to the chloroethyl group, giving rise to a cyclic ether. The crystal structures were compared to that of the omuralide/CP structure solved previously, and the collective data provide new insights into the mechanism of inhibition and irreversible binding of 1. Upon opening of the beta-lactone ring, C-3O assumes the position occupied by a water molecule in the unligated enzyme and hinders deacylation of the enzyme-ligand complex. Furthermore, the resulting protonation state of Thr1NH2 deactivates the catalytic N-terminus.     

A structure-based design approach to advance the allyltyrosine-based series of HIV integrase inhibitors

As of mid-2017, only one structure of the human immunodeficiency virus (HIV) integrase core domain co-crystallised with an active site inhibitor was reported. In this structure (1QS4), integrase is complexed with a diketo-acid based strand-transfer inhibitor (INSTI). This structure has been a preferred platform for the structure-based design of INSTIs despite concerns relating to structural irregularities arising from crystallographic packing effects. A survey of the current pool of 297 reported integrase catalytic core structures indicated that the anatomy of the active site in the complex structure 1QS4 exhibits subtle variations relative to all other structures examined. Consequently, the 1QS4 structure was employed for docking studies. From the docking of twenty-seven allyltyrosine analogues, a 3-point inhibitor binding motif required for activity was established and successfully utilised in the development of a tripeptide displaying an EC₅₀ value of 10 ± 5 μM in HIV infected human T-cells. Additional docking of “in-house” compound libraries unearthed a methyl ester based nitrile derivative displaying an IC₅₀ value of 0.5 μM in a combined 3′-processing and strand-transfer assay.     

Comparing the quality and predictiveness between 3D QSAR models obtained from manual and automated alignment

A set of 113 flexible cyclic urea inhibitors of human immunodeficiency virus protease (HIV-1 PR) was used to compare the quality and predictive power of CoMFA and CoMSIA models for manually or automatically aligned inhibitor set. Inhibitors that were aligned automatically with molecular docking were in agreement with information obtained from existing X-ray structures. Both alignment methods produced statistically significant CoMFA and CoMSIA models, with the best q(2) value being 0.649 and the best predictive r(2) being 0.754. The manual alignment gave statistically higher values, whereas the automated alignment gave more robust models for predicting the activities of an external inhibitor set. Both models utilized similar amino acids in the HIV-1 PR active site, supporting the idea that hydrogen bonds form between an inhibitor and the backbone carbonyl oxygens of Gly48 and Gly48' and also the backbone NH group of Asp30, Gly48, Asp29', and Gly48' of the enzyme. These results suggest that an automated inhibitor alignment can yield predictive 3D QSAR models that are well comparable to manual methods. Thus, an automated alignment method in creating 3D QSAR models is encouragable when a well-characterized structure of the target protein is available.     

Potent active site-directed inhibition of steroid sulphatase by tricyclic coumarin-based sulphamates

BACKGROUND: There is now abundant evidence that inhibition of steroid sulphatase alone or in conjunction with inhibition of aromatase may enhance the response of postmenopausal patients with hormone-dependent breast cancer to this type of endocrine therapy. Additionally, sulphatase inhibition has been proposed to be of potential therapeutic benefit in the immune system and for neuro-degenerative diseases. After the finding that our first highly potent active site-directed steroid sulphatase inhibitor, oestrone-3-O-sulphamate (EMATE), was highly oestrogenic, we proposed non-steroidal coumarin sulphamates such as 4-methylcoumarin-7-O-sulphamate (COUMATE) as alternative non-steroidal steroid sulphatase inhibitors. In this work, we describe how tricyclic coumarin-based sulphamates have been developed which are even more potent than COUMATE, are non-oestrogenic and orally active. We also discuss potential mechanisms of action. RESULTS: 4-Ethyl- (4), 4-(n-propyl)- (6), 3-ethyl-4-methyl- (8), 4-methyl-3-(n-propyl)coumarin-7-O-sulphamate (11); the tricyclic derivatives 665COUMATE (13), 666COUMATE (15), 667COUMATE (17), 668COUMATE (20) and the tricyclic oxepin sulphamate (22) were synthesised. In a placental microsome preparation, all of these analogues were found to be more active than COUMATE in the inhibition of oestrone sulphatase, with the most potent inhibitor being 667COUMATE which has an IC(50) of 8 nM, some 3-fold lower than that for EMATE (25 nM). In addition, 667COUMATE was also found to inhibit DHEA-sulphatase some 25-fold more potently than EMATE in a placental microsome preparation. Like EMATE, 667COUMATE acts in a time- and concentration-dependent manner, suggesting that it is an active site-directed inhibitor. However, in contrast to EMATE, 667COUMATE has the important advantage of not being oestrogenic. In addition, we propose several diverse mechanisms of action for this active site-directed steroid sulphatase inhibitor in the light of recent publications on the crystal structures of human arylsulphatases A and B and the catalytic site topology for the hydrolysis of a sulphate ester. CONCLUSIONS: A highly potent non-steroidal, non-oestrogenic and irreversible steroid sulphatase inhibitor has been developed. Several mechanisms of action for an active site-directed steroid sulphatase inhibitor are proposed. With 667COUMATE now in pre-clinical development for clinical trial, this should allow the biological and/or clinical significance of steroid sulphatase inhibitors in the treatment of postmenopausal women with hormone-dependent breast cancer and other therapeutic indications to be fully evaluated.     

Synthesis of macrocyclic,potential protease inhibitors using a generic scaffold

A generic macrocyclic peptide structure 2 was designed as a potential inhibitor of a range of proteinases, by using as a basis for the design the known structures of a series of enzyme-inhibitor complexes. The macrocyclic nature of the target 2 was chosen so as to reduce the entropic advantage in the hydrolytic enzymatic step, and thereby to inhibit the function of the enzyme. The nature of the linking group was identified as a benzoxazole by molecular modeling, so as to preserve the recognized conformation of the peptide chain. The specificity of the potential inhibitor was tuned by variation of the P(1) group (by incorporating phenylalanine, aspartic acid, or lysine), to allow recognition by different enzyme classes. The targets were prepared from the bis-amino acid derivative 5, itself prepared using the Pd-catalyzed coupling of an organozinc reagent with the iodobenzothiazole 7 and subsequent macrocyclization of the open-chain derivatives 22-24 using HATU. None of the macrocylic compounds 25, 28-30, and 32 inhibited their target enzymes. NMR and MS studies on the interaction of macrocycle 29 and chymotrypsin established that compound 29 was in fact a substrate of the enzyme. This result indicated that while the design had been partially successful in identifying a compound that bound, the reduction in entropic advantage due to its macrocyclic nature was not sufficient to allow 29 to act as an inhibitor.     

Scaffold hopping in drug discovery using inductive logic programming

In chemoinformatics, searching for compounds which are structurally diverse and share a biological activity is called scaffold hopping. Scaffold hopping is important since it can be used to obtain alternative structures when the compound under development has unexpected side-effects. Pharmaceutical companies use scaffold hopping when they wish to circumvent prior patents for targets of interest. We propose a new method for scaffold hopping using inductive logic programming (ILP). ILP uses the observed spatial relationships between pharmacophore types in pretested active and inactive compounds and learns human-readable rules describing the diverse structures of active compounds. The ILP-based scaffold hopping method is compared to two previous algorithms (chemically advanced template search, CATS, and CATS3D) on 10 data sets with diverse scaffolds. The comparison shows that the ILP-based method is significantly better than random selection while the other two algorithms are not. In addition, the ILP-based method retrieves new active scaffolds which were not found by CATS and CATS3D. The results show that the ILP-based method is at least as good as the other methods in this study. ILP produces human-readable rules, which makes it possible to identify the three-dimensional features that lead to scaffold hopping. A minor variant of a rule learnt by ILP for scaffold hopping was subsequently found to cover an inhibitor identified by an independent study. This provides a successful result in a blind trial of the effectiveness of ILP to generate rules for scaffold hopping. We conclude that ILP provides a valuable new approach for scaffold hopping.     

Discovery of Anti-SARS Coronavirus Drug Based on Molecular Docking and Database Screening

The active site of 3CL proteinase (3CL^por) for coronavirus was identified by comparing the crystal structures of human and porcine coronavirus. The inhibitor of the main protein of rhinovirus (Ag7088) could bind with 3CL^pro of human coronavirus, then it was selected as the reference for molecular docking and database screening. The ligands from two databases were used to search potential lead structures with molecular docking. Several structures from natural products and ACD-SC databases were found to have lower binding free energy with 3CL^pro than that of Ag7088. These structures have similar hydrophobicity to Ag7088. They have complementary electrostatic potential and hydrogen bond aeceptor and donor with 3CL^pro, showing that the strategy of anti-SARS drug design based on molecular docking and database screening is feasible.     

Aromatic polyketide production in Cordyceps indigotica, an entomopathogenic fungus, induced by exposure to a histone deacetylase inhibitor

Cultivation of Cordyceps indigotica, an entomopathogenic fungus, in the presence of suberoyl bis-hydroxamic acid (an HDAC inhibitor) greatly activated its polyketide synthesis apparatus to afford six novel aromatic polyketides, indigotides C-F (1-4), 13-hydroxyindigotide A (5), and 8-O-methylindigotide B (6). The structures of these compounds were determined by NMR spectroscopic analyses. Among the compounds, indigotides C-E (1-3) possessed unprecedented dimeric polyketide frameworks possibly generated via a [4 + 2] cycloaddition or Michael type reaction.