Synthesis, biological evaluation, and molecular modeling studies of methylene imidazole substituted biaryls as inhibitors of human 17alpha-hydroxylase-17,20-lyase (CYP17)--part II: Core rigidification and influence of substituents at the methylene bridge

Thirty-five novel substituted imidazolyl methylene biphenyls have been synthesized as CYP17 inhibitors for the potential treatment of prostate cancer. Their activities have been tested with recombinant human CYP17 expressed in Escherichia coli. Promising compounds were tested for selectivity against CYP11B1, CYP11B2, and hepatic CYP enzymes 3A4, 1A2, 2B6 and 2D6. The core rigidified compounds (30-35) were the most active ones, being much more potent than Ketoconazole and reaching the activity of Abiraterone. However, they were not very selective. Another rather potent and more selective inhibitor (compound 23, IC(50)=345 nM) was further examined in rats regarding plasma testosterone levels and pharmacokinetic properties. Compared to the reference Abiraterone, 23 was more active in vivo, showed a longer plasma half-life (10h) and a higher bioavailability. Using our CYP17 homology protein model, docking studies with selected compounds were performed to study possible interactions between inhibitors and amino acid residues of the active site.     

Dihydrobenzisoxazole-4-one compounds are novel selective inhibitors of aldosterone synthase (CYP11B2) with in vivo activity

6,7-Dihydro-5H-2,1-benzisoxazol-4-one analogs are potent inhibitors of aldosterone synthase (CYP11B2) with selectivity over the highly homologous enzyme cortisol synthase (CYP11B1). These compounds are unique among inhibitors of CYP11B2 in their lack of a strong-heme binding group such as a pyridine or imidazole. Poor metabolic stability in hepatocyte incubations was found to proceed via a reduction of the isoxazole ring. While the enzyme responsible for the reductive metabolism remains unknown, the rate of metabolism could be attenuated by the addition of polar functionality. The in vitro CYP11B2 potency and selectivity were confirmed in vivo in a cynomolgus monkey model by the inhibition of ACTH stimulated aldosterone production without impacting plasma cortisol concentrations.     

Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells

Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 enzyme catalyzing the last steps of aldosterone production in the adrenal cortex. A new pharmacological approach for the treatment of the aldosterone induced effects in congestive heart failure and all forms of hyperaldosteronism could be the use of CYP11B2 inhibitors. In search for such compounds, it was our goal to develop a cellular enzyme assay suitable for screening high numbers of compounds. An assay procedure for the evaluation of inhibitors using the human CYP11B2 expressed in fission yeast Schizosaccharomyces pombe was established and a series of 10 compounds was tested in this whole cellular system. Human 11beta-hydroxylase (CYP11B1), which catalyzes the production of glucocorticoids, shows more than 90% homology compared to human CYP11B2. As this enzyme should not be affected, strong inhibitors of CYP11B2 have to be tested for selectivity. For that purpose, an assay procedure with V79MZ cells that express human CYP11B1 and CYP11B2, respectively, was integrated into the evaluation process. Using these screening procedures a potent and rather selective non-steroidal inhibitor of human CYP11B2 was detected with an IC(50) value of 59nM. We also identified a very potent inhibitor of both enzymes showing a stronger inhibitory activity against the cortisol producing CYP11B1.     

The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors

Aldosterone plays a key role in salt and water homeostasis but is also involved in the development and progression of congestive heart failure and myocardial fibrosis. As a new pharmacological strategy for the treatment of these diseases, we propose the inhibition of the key enzyme of mineralcorticoid formation, CYP11B2 (aldosterone synthase). For studies of the effects of CYP11B2 inhibitors on the adrenal cortex, we selected the NCI-H295R cell line which expresses most of the key enzymes necessary for steroidogenesis. To evaluate this cell line as a test system for effects and side effects of CYP inhibitors, we established assays using radiolabeled substrates of CYP11B2 and CYP11B1 and subsequently tested a series of CYP11B2 inhibitors including the CYP19 inhibitor fadrozole. Fadrozole and compounds 6, 9 and 10 were more potent towards CYP11B2 compared to CYP11B1 with IC(50) values in the nanomolar range. To analyze their overall effect, the formation of steroids in the cell culture supernatant was monitored. All compounds led to a concentration-dependent reduction of the aldosterone secretion but also reduced the formation of cortisol and androgens. In conclusion, the H295R cell line is a suitable tool for the prediction of overall side effects of CYP11B2 inhibitors on steroidogenesis.     

The development of a whole-cell based medium throughput screening system for the discovery of human aldosterone synthase (CYP11B2) inhibitors: old drugs disclose new applications for the therapy of congestive heart failure, myocardial fibrosis and hypertension

Cytochrome P450 enzymes play an important role in steroid hormone biosynthesis of the human adrenal gland, e.g., the production of cortisol and aldosterone. Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the salt and water homeostasis of the body and thus in the regulation of blood pressure, whereas cortisol is the most important glucocorticoid of the human body. CYP11B-dependent steroid hydroxylases are drug development targets, and since they are very closely related enzymes, the discovery of selective inhibitors has been subject to intense investigations for several years. Here we report the development of a whole-cell medium throughput screening technology for the discovery of CYP11B2 inhibitors. The new screening system displayed high reproducibility and was applied to investigate a library of pharmacologically active compounds. 1268 compounds were investigated during this study which revealed 5 selective inhibitors of CYP11B2 (after validation against CYP11B1). The new inhibitors of CYP11B2 are already existing drugs that could be used either in the treatment of hyperaldosteronism-related diseases or as lead compounds that could further be optimised to achieve safer and selective inhibitors of aldosterone synthase. Article from the Special issue on 'Targeted Inhibitors'.     

3-Pyridyl Substituted Aliphatic Cycles as CYP11B2 Inhibitors: Aromaticity Abolishment of the Core Significantly Increased Selectivity over CYP1A2

Aldosterone synthase (CYP11B2) is a promising therapeutic target for the treatment of cardiovascular diseases related to abnormally high aldosterone levels. On the basis of our previously identified lead compounds I–III, a series of 3-pyridinyl substituted aliphatic cycles were designed, synthesized and tested as CYP11B2 inhibitors. Aromaticity abolishment of the core was successfully applied to overcome the undesired CYP1A2 inhibition. This study resulted in a series of potent and selective CYP11B2 inhibitors, with compound 12 (IC₅₀ = 21 nM, SF = 50) as the most promising one, which shows no inhibition toward CYP1A2 at 2 µM. The design conception demonstrated in this study can be helpful in the optimization of CYP inhibitor drugs regarding CYP1A2 selectivity.     

Inhibitors of vitamin D hydroxylases: structure-activity relationships

Aiming at new drugs to efficiently treat diseases, in which either increased or decreased levels of active vitamin D are desirable, we have designed some 400 structurally different azole-type inhibitors and examined their capacity to selectively block vitamin D metabolism by CYP24 or synthesis by CYP27B, in human keratinocytes. Based on resulting data, we built pharmacophore models of the active sites using commercial software. The overlay of potent selective compounds indicated similar docking modes in the two-substrate pockets and allowed for identification of bioactive conformations. Superimposing these bioactive conformations with low energy conformers of 25(OH)D(3) suggested that the substrate-mimicked by strong inhibitors in size, shape and lipophilic character-binds to both enzymes in 6s-trans configuration. Pharmacophoric models implied a similar geometry of the substrate sites, nevertheless specific features of CYP24 and CYP27B could be defined. Bulky substituents in alpha-position to the azole caused selectivity for CYP24, whereas bulky substituents in beta-position could result in selectivity for CYP27B. Moreover, studies with small sterically restricted inhibitors revealed a probable location of the 3-OH-group of 25(OH)D(3) in CYP27B. In the absence of crystal structures, our inhibitors are valuable tools to model and understand the active sites of vitamin D hydroxylases, resulting in the design of powerful, selective therapeutics.     

Design,synthesis and biological evaluation of amide-pyridine derivatives as novel dual-target (SE,CYP51) antifungal inhibitors

Based on the analysis of the squalene cyclooxygenase (SE) and 14α-demethylase (CYP51) inhibitors pharmacophore feature and the dual-target active sites, a series of compounds with amide-pyridine scaffolds have been designed and synthesized to treat the increasing incidence of drug-resistant fungal infections. In vitro evaluation showed that these compounds have a certain degree of antifungal activity. The most potent compounds 11a, 11b with MIC values in the range of 0.125–2 μg/ml had a broad-spectrum antifungal activity and exhibited excellent inhibitory activity against drug-resistant pathogenic fungi. Preliminary mechanism studies revealed that the compound 11b might play an antifungal role by inhibiting the activity of SE and CYP51. Notably compounds did not show the genotoxicity through plasmid binding assay. Finally, this study of molecular docking, ADME/T prediction and the construction of 3D QSAR model were performed. These results can point out the direction for further optimization of the lead compound.     

Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases.In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.     

Novel GSK-3beta inhibitors from sequential virtual screening

Glycogen synthase kinase-3 (GSK-3beta) has been emerging as a key therapeutic target for type-2 diabetics, Alzheimer's disease, cancer, and chronic inflammation. For the purpose of finding biologically active and novel compounds and providing new idea for drug-design, we performed virtual screening using commercially available database. Three-dimensional common feature pharmacophore model was developed by using HipHop program provided in Catalyst software and it was used as a query for screening database. Recursive partitioning (RP) model was developed as a filtering system, which was able to classify active and inactive compounds. Eventually, a sequential virtual screening procedure (SQSP) was conducted by applying the common feature pharmacophore and RP model in succession to discover novel potent GSK-3beta inhibitors. The final 56 hit compounds were carefully selected considering predicted docking mode in crystal structures. Subsequent enzyme assay for human GSK-3beta protein confirmed that three compounds of these hit compounds exhibit micromolar inhibitory activity. Here, we report novel hit compounds and their binding mode in the active site of GSK-3beta crystal structure.     

Combined structure- and ligand-based pharmacophore modeling and molecular dynamics simulation studies to identify selective inhibitors of MMP-8

Matrix metalloproteinase-8 (MMP-8) is the key mediator in initiating type I collagen degradation and is associated with rheumatoid arthritis. In the present study, a pharmacophore hypothesis was developed based on selective non zinc binding inhibitors of MMP-8. The pharmacophore hypothesis was refined manually and validated by observing structures and the interactions of MMP-8 inhibitors. The refined pharmacophore model was able to discriminate the non-zinc binding inhibitors of MMP-8 with respect to other inhibitors. Hence this study proposes a combined structure- and ligand-based pharmacophore model that is suitable for retrieving the novel inhibitors of MMP-8. The pharmacophore hypothesis AADRH was used as query for retrieving potential compounds from the Zinc database and hits were selected based on the catalytic selective amino acid residues of Arg 222, and Tyr 227. We identified six compounds as potent inhibitors and their selectivity profile were checked against different subtypes of MMPs using the cross-docking method. Molecular dynamics results indicated that ZINC 00673680 forms a stable interaction with the key amino acid residues and avoids the zinc atom with a distance of 5.49?Å. Our computational study might be useful for further development of selective MMP-8 inhibitors.     

CYP isoform specificity toward drug metabolism: analysis using common feature hypothesis

Three dimensional pharmacophoric maps were generated for each isoforms of CYP2C9, CYP2D6 and CYP3A4 separately using independent training sets consist of highly potent substrates (seven substrates for each isoform). HipHop module of CATALYST software was used in the generation of pharmacophore models. The best pharmacophore model was chosen out of the several models on the basis of (i) highest ranking score, (ii) better fit value among training set, (iii) capability to screen substrates from data set and (iv) efficiency to identify the isoform specificity. The individual pharmacophore models (CYP2C9-hypo1, CYP2D6-hypo1 and CYP3A4-hypo1) are characterized by the pharmacophoric features XZDH, RPZH and XYZHH for the CYP2C9, CYP2D6 and CYP3A4 respectively. Each of the chosen models was validated by using data sets of CYP substrates. This comparative study of CYP substrates demonstrates the importance of acidic character along with HBD and HBAl features for CYP2C9, basic character with ring aromatic features for CYP2D6 and hydrophobic features for CYP3A4. Acidity, basicity and hydrophobicity features arising from the functional groups of the substrates are also responsible for demonstrating CYP isoform specificity. Hence, these chemical features are incorporated in the decision tree along with pharmacophore maps. Finally, a decision tree based on chemical features and pharmacophore features was generated to identify the isoform specificity of novel query molecule toward the three isoforms.     

Identification of potential inhibitors for HCV NS5b of genotype 4a by combining dynamic simulation,protein–ligand interaction fingerprint, 3D pharmacophore,docking and 3D QSAR

Abstract

HCV NS5B polymerase has been one of the most attractive targets for developing new drugs for HCV infection and many drugs were successfully developed, but all of them were designed for targeting Hepatitis C Virus genotype 1 (HCV GT1). Hepatitis C virus genotype 4a (HCV GT4a) dominant in Egypt has paid less attention. Here, we describe our protocol of virtual screening in identification of novel potential potent inhibitors for HCV NS5B polymerase of GT4a using homology modeling, protein–ligand interaction fingerprint (PLIF), docking, pharmacophore, and 3D CoMFA quantitative structure activity relationship (QSAR). Firstly, a high-quality 3D model of HCV NS5B polymerase of GT4a was constructed using crystal structure of HCV NS5B polymerase of GT1 (PDB ID: 3hkw) as a template. Then, both the model and the template were simulated to compare conformational stability. PLIF was generated using five crystal structures of HCV NS5B (PDB ID: 4mia, 4mib, 4mk9, 4mka, and 4mkb), which revealed the most important residues and their interactions with the co-crystalized ligands. After that, a 3D pharmacophore model was developed from the generated PLIF data and then used as a screening filter for 17000328 drug-like zinc database compounds. 900 compounds passed the pharmacophore filter and entered the docking-based virtual screening stage. Finally, a 3D CoMFA QSAR was developed using 42 compounds as a training and 19 compounds as a test set. The 3D CoMFA QSAR was used to design and screen some potential inhibitors, these compounds were further evaluated by the docking stage. The highest ranked five hits from docking result (compounds (p1–p4) and compound q1) were selected for further analysis.

Communicated by Ramaswamy H. Sarma     

Pharmacophore-based discovery of 2-(phenylamino)aceto-hydrazides as potent eosinophil peroxidase (EPO) inhibitors

There is an increasing interest in developing novel eosinophil peroxidase (EPO) inhibitors, in order to provide new treatment strategies against chronic inflammatory and neurodegenerative diseases caused by eosinophilic disorder. Within this study, a ligand-based pharmacophore model for EPO inhibitors was generated and used for in silico screening of large 3?D molecular structure databases, containing more than 4 million compounds. Hits obtained were clustered and a total of 277 compounds were selected for biological assessment. A class of 2-(phenyl)amino-aceto-hydrazides with different substitution pattern on the aromatic ring was found to contain the most potent EPO inhibitors, exhibiting IC₅₀ values down to 10?nM. The generated pharmacophore model therefore, represents a valuable tool for the selection of compounds for biological testing. The compounds identified as potent EPO inhibitors will serve to initiate a hit to lead and lead optimisation program for the development of new therapeutics against eosinophilic disorders.     

Ligand-based and structure-based approaches in identifying ideal pharmacophore against c-Jun N-terminal kinase-3

Structure and ligand based pharmacophore modeling and docking studies carried out using diversified set of c-Jun N-terminal kinase-3 (JNK3) inhibitors are presented in this paper. Ligand based pharmacophore model (LBPM) was developed for 106 inhibitors of JNK3 using a training set of 21 compounds to reveal structural and chemical features necessary for these molecules to inhibit JNK3. Hypo1 consisted of two hydrogen bond acceptors (HBA), one hydrogen bond donor (HBD), and a hydrophobic (HY) feature with a correlation coefficient (r²) of 0.950. This pharmacophore model was validated using test set containing 85 inhibitors and had a good r² of 0.846. All the molecules were docked using Glide software and interestingly, all the docked conformations showed hydrogen bond interactions with important hinge region amino acids (Gln155 and Met149) and these interactions were compared with Hypo1 features. The results of ligand based pharmacophore model (LBPM) and docking studies are validated each other. The structure based pharmacophore model (SBPM) studies have identified additional features, two hydrogen bond donors and one hydrogen bond acceptor. The combination of these methodologies is useful in designing ideal pharmacophore which provides a powerful tool for the discovery of novel and selective JNK3 inhibitors.     

Discovery of novel inhibitors of 11beta-hydroxysteroid dehydrogenase type 1 by docking and pharmacophore modeling

11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is a potential target for treatment of diabetes and metabolic syndrome. Docking and pharmacophore modeling have been used to discover novel inhibitors of 11beta-HSD1. Several compounds, with large structural diversity and good potency against 11beta-HSD1, have been found and their potency was determined by the enzyme assay. New scaffolds of 11beta-HSD1 inhibitors are also reported.     

Identification of Putative Plant-Based ALR-2 Inhibitors to Treat Diabetic Peripheral Neuropathy

Diabetic peripheral neuropathy (DPN) is a common diabetes complication (DM). Aldose reductase -2 (ALR-2) is an oxidoreductase enzyme that is most extensively studied therapeutic target for diabetes-related complications that can be inhibited by epalrestat, which has severe adverse effects; hence the discovery of potent natural inhibitors is desired. In response, a pharmacophore model based on the properties of eplarestat was generated. The specified pharmacophore model searched the NuBBE_DB database of natural compounds for prospective lead candidates. To assess the drug-likeness and ADMET profile of the compounds, a series of in silico filtering procedures were applied. The compounds were then put through molecular docking and interaction analysis. In comparison to the reference drug, four compounds showed increased binding affinity and demonstrated critical residue interactions with greater stability and specificity. As a result, we have identified four potent inhibitors: ZINC000002895847, ZINC000002566593, ZINC000012447255, and ZINC000065074786, that could be used as pharmacological niches to develop novel ALR-2 inhibitors.     

Structure-based and shape-complemented pharmacophore modeling for the discovery of novel checkpoint kinase 1 inhibitors

Checkpoint kinase 1 (Chk1), a member of the serine/threonine kinase family, is an attractive therapeutic target for anticancer combination therapy. A structure-based modeling approach complemented with shape components was pursued to develop a reliable pharmacophore model for ATP-competitive Chk1 inhibitors. Common chemical features of the pharmacophore model were derived by clustering multiple structure-based pharmacophore features from different Chk1-ligand complexes in comparable binding modes. The final model consisted of one hydrogen bond acceptor (HBA), one hydrogen bond donor (HBD), two hydrophobic (HY) features, several excluded volumes and shape constraints. In the validation study, this feature-shape query yielded an enrichment factor of 9.196 and performed fairly well at distinguishing active from inactive compounds, suggesting that the pharmacophore model can serve as a reliable tool for virtual screening to facilitate the discovery of novel Chk1 inhibitors. Besides, these pharmacophore features were assumed to be essential for Chk1 inhibitors, which might be useful for the identification of potential Chk1 inhibitors.     

Insights from ligand and structure based methods in virtual screening of selective Ni-peptide deformylase inhibitors

In recent years, there has been a growing interest in developing bacterial peptide deformylase (PDF) inhibitors as novel antibiotics. The purpose of the study is to generate a three-dimensional (3D) pharmacophore model by using diverse PDF inhibitors which is useful for designing of potential antibiotics. Twenty one structurally diverse compounds were considered for the generation of quantitative pharmacophore model using HypoGen of Catalyst, further model was validated using 78 compounds. Pharmacophore model demonstrated the importance of two acceptors, one donor and one hydrophobic feature toward the biological activity. The inhibitors were also docked into the binding site of PDF to comprehend the structural insights of the active site. Combination of ligand and structure based methods were used to find the potential antibiotics.     

结合分子相似性、药效团和分子对接筛选新的HIV-1蛋白酶抑制剂

结合分子相似性、药效团和分子对接建立兼顾计算效率和预测准确度的HIV-1蛋白酶抑制剂筛选方法。首先通过对现有HIV-1蛋白酶抑制剂分子进行相似性分析,选取代表性的HIV-1蛋白酶抑制剂作为模板分子,构建和优化药效团模型,并从1万个化合物中优先筛选出500个化合物。而后采用分子对接方法进一步考察化合物与HIV-1蛋白酶结合情况,得到4个新的活性候选化合物,并进行其结合自由能计算和抗突变性分析。结果表明新候选化合物ST025723和HIV-1蛋白酶表现出较好的相互作用和抗突变性,具有深入研究的价值,同时也证明分子相似性、药效团和分子对接相结合能够快速有效地发现新颖活性候选化合物。