Benzodiazepine-induced hyperphagia: development and assessment of a 3D pharmacophore by computational methods

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABA(A) receptors and allosterically modulate the effect of GABA on chloride ion flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral endpoints, including anxiolytic, sedative, anticonvulsant, and hyperphagic effects. In the work presented here, a computational procedure developed in our laboratory has been used to obtain a 3D pharmacophore for ligand recognition of the GABA(A)/BDZRs initiating the hyperphagic response. To accomplish this goal, 17 structurally diverse compounds, previously assessed in our laboratory for activity at the hyperphagic endpoint, were used. The result is a four-component 3D pharmacophore. It consists of two proton acceptor atoms, the centroid of an aromatic ring and the centroid of a hydrophobic moiety in a common geometric arrangement in all compounds with activity at this endpoint. This 3D pharmacophore was then assessed and successfully validated using three different tests. First, two BDZR ligands, which were included as negative controls in the set of seventeen compounds used for the pharmacophore development, did not fit the pharmacophore. Second, some benzodiazepine ligands known to have activity at the hyperphagia endpoint, but not included in the pharmacophore development, were used as positive controls and were found to fit the pharmacophore. Finally, using the 3D pharmacophore developed in the present work to search 3D databases, over 50 classical benzodiazepines were found. Among them, were benzodiazepine ligands known to have an effect at the hyperphagic endpoint. In addition, the novel compounds also found in this search are promising therapeutic agents that could beneficially affect feeding behavior.     

Development and assessment of a 3D pharmacophore for ligand recognition of BDZR/GABAA receptors initiating the anxiolytic response

Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind to specific binding sites on GABAA receptors and allosterically modulate the effect of GABA on chloride flux. The binding of BDZR ligands to this receptor system results in activity at multiple behavioral end points including anxiolytic, sedative, hyperphagic, anticonvulsant and hyperthermic effects. In the work presented here, 17 structurally diverse BDZR ligands of the receptors initiating the anxiolytic response have been studied using a systematic computational procedure developed in our laboratory. Using this procedure, a five component 3D recognition pharmacophore was obtained consisting of two proton acceptors, a hydrophobic group, an aromatic electron accepting ring and a ring containing polar moieties, all found in a common geometric arrangement in the 15 compounds with an effect at the anxiolytic end point and absent in two control compounds. The 3D pharmacophore developed was validated by searching 3D databases and finding known BDZR ligands active at the anxiolytic end point, including 1,4-BDZ derivatives, imidazo BDZ and beta-carboline ligands.     

Development of a 3D pharmacophore for nonspecific ligand recognition of alpha1, alpha2, alpha3, alpha5, and alpha6 containing GABA(A)/benzodiazepine receptors

Transfected cells containing GABA(A) benzodiazepine receptors (BDZRs) have been utilized to systematically determine the affinity of ligands at alpha1, alpha2, alpha3, alpha5 and alpha6 subtypes in combination with beta2 and gamma2. All but a few of the ligands thus far studied have relatively high affinities for each of these alpha subtype receptors. Thus, these ligands must contain common stereochemical properties favorable for recognition by each of the subtype combinations. In the present work, such a common three-dimensional (3D) pharmacophore for recognition of alpha1, alpha2, alpha3, alpha5 and alpha6 containing GABA(A)/BDZRs types of receptors has been developed and assessed, using as a database receptor affinities measured in transfected cells for 27 diverse compounds. The 3D-recognition pharmacophore developed consists of three proton accepting groups, a hydrophobic group, and the centroid of an aromatic ring found in a common geometric arrangement in the 19 nonselective ligands used. Three tests were made to assess this pharmacophore: (i) Four low affinity compounds were used as negative controls, (ii) Four high affinity compounds, excluded from the pharmacophore development, were used as compounds for pharmacophore validation, (iii) The 3D pharmacophore was used to search 3D databases. The results of each of these types of assessments provided robust validation of the 3D pharmacophore. This 3D pharmacophore can now be used to discover novel nonselective ligands that could be activation selective at different behavioral end points. Additionally, it may serve as a guide in the design of more selective ligands, by determining if candidate ligands proposed for synthesis conform to this pharmacophore and selecting those that do not for further experimental assessment.     

Nootropic and anxiolytic properties of endogenous ligands of benzodiazepine receptors and their structural analogs

Experiments on mice and rats were made to study the nootropic and anxiolytic properties of endogenous ligands of benzodiazepine receptors of nicotinamide and inosin and of their new structural analogs--NMF and AZN. They were shown to have overt antihypoxic and anxiolytic effects. NMF and AZN given in 10-fold lower doses than endogenous benzodiazepine ligands appeared more active than these compounds and almost similar to diazepam as regards the activity. The data obtained point to the possibility of a purposeful search for new efficacious psychotropic and nootropic substances in the series of compounds structurally related to endogenous ligand of benzodiazepine receptors.     

The utilization of a unified pharmacophore query in the discovery of new antagonists of the adenosine receptor family

Pharmacophore queries from previously known potent selective A3 antagonists were generated by Chem-X. These queries were used to search a pharmacophore database of diverse compounds (CNS-Set). In vitro assays of 186 'hits' yielded over 30 active compounds, for four adenosine receptor subtypes. This search strategy may also be applicable to the discovery of new ligands via receptor homology data.     

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.     

Benzodiazepine receptor ligands. 8: synthesis and pharmacological evaluation of new pyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxide 3- and 8-disubstituted: high affinity ligands endowed with inverse-agonist pharmacological efficacy

The synthesis and the binding study of new 3-arylesters and 3-heteroarylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 8-substituted are reported. The nature of these substituents (in terms of lipophilic and electronic features) seems to influence the binding affinity. High-affinity ligands were studied in mice in vivo for their pharmacological effects, considering six potential benzodiazepine actions: anxiolytic-like effects, muscle relaxant effects, motor coordination, anticonvulsant action, spontaneous motor activity, and ethanol-potentiating action. Compounds 4d and 6d showed an inverse-agonist profile. These compounds were evaluated also for their binding at benzodiazepine site on GABAA receptor complex (GABAA/BzR complex) subtype to evaluate their subtype selectivity.     

A novel potential therapeutic avenue for autism: design, synthesis and pharmacophore generation of SSRIs with dual action

Autism symptoms are currently modulated by Selective Serotonin Reuptake Inhibitors (SSRIs). SSRIs slow onset of action limits their efficiency. The established synergistic activity of SSRIs and 5HT(1B/1D) autoreceptors antagonists motivated us to incorporate SSRIs and 5HT(1B/1D) antagonists in one 'hybrid' molecule. A library of virtual 'hybrid' molecules was designed using the tethering technique. A pharmacophore model was generated derived from 16 structurally diverse SSRIs (K(i)=0.013-5000 nM) and used as 3D query. Compounds with fit values (≥2) were chosen for synthesis and subsequent in vitro biological evaluation. Our pharmacophore model is a promising milestone to a class of SSRIs with dual action.     

Molecular determinants of non-specific recognition of delta, mu, and kappa opioid receptors

Identification of the molecular determinants of recognition common to all three opioid receptors embedded in a single three-dimensional (3D) non-specific recognition pharmacophore has been carried out. The working hypothesis that underlies the computational study reported here is that ligands that bind with significant affinity to all three cloned opioid receptors, delta, mu, and kappa, but with different combinations of activation and inhibition properties at these receptors, could be promising behaviorally selective analgesics with diminished side effects. The study presented here represents the first step towards the rational design of such therapeutic agents. The common 3D pharmacophore developed for recognition of delta, mu, and kappa opioid receptors was based on the receptor affinities determined for 23 different opioid ligands that display no specificity for any of the receptor subtypes. The pharmacophore centers identified are a protonated amine, two hydrophobic groups, and the centroid of an aromatic group in a geometric arrangement common to all 23, non-specific, opioid ligands studied. Using this three-dimensional pharmacophore as a query for searching 3D structural databases, novel compounds potentially involved in non-specific recognition of delta, mu, and kappa opioid receptors were retrieved. These compounds can be valuable candidates for novel behaviorally selective analgesics with diminished or no side effects, and thus with potential therapeutic usefulness.     

The benzodiazepine receptor ligands RO 5-4864 and RO 15-1788 do not block the inhibition of PAF-induced platelet aggregation seen with the hetrazepine WEB2086

The hypothesis was tested that the hetrazepine WEB 2086 acts as an inhibitor of PAF-induced platelet aggregation via interaction with the platelet benzodiazepine receptor(BDZR). WEB 2086 is a potent inhibitor of rabbit platelet aggregation and ATP secretion induced by 370 nM PAF. The two BDZR ligands RO 5-4864 and RO 15-1788 (7-96 microM) are inactive as PAF antagonists. When platelets were pretreated with either BDZR ligand, and then exposed to various concentrations of WEB 2086, there was no alteration of the dose-response relationship of the hetrazepine on PAF-induced aggregation, as reflected by threshold concentration, ED50, or maximum inhibition seen with WEB 2086. Pretreatment of platelets with the BDZR ligands also failed to block the inhibitory action of WEB 2086 on PAF-induced ATP release. The data are consistent with the notion that WEB 2086 acts as a PAF antagonist through its action at a specific PAF receptor, and is dissociated from, and independent of, interaction with the benzodiazepine receptor.     

Molecular modeling study on chemically diverse series of cyclooxygenase-2 selective inhibitors: generation of predictive pharmacophore model using Catalyst

Cyclooxygenase (COX) enzymes catalyse the biosynthesis of prostaglandins and thromboxane from arachidonic acid (AA). We summarize in this paper, the development of pharmacophores of a dataset of inhibitors for COX-2 by using the Catalyst/Hypogen module using six chemically diverse series of compounds. Training set consisting of 24 compounds was carefully selected. The activity spread of the training set molecules was from 0.1 to 10000 nM. The most predictive pharmacophore model (hypothesis 1), consisting of four features, namely, one hydrogen bond donor, one hydrogen bond acceptor, one hydrophobic aliphatic and one ring aromatic feature, had a correlation (r) of 0.954 and a root mean square deviation of 0.894. The entropy (configuration cost) value of the hypotheses was 16.79, within the allowed range. The difference between the null hypothesis and the fixed cost and between the null hypothesis and the total cost of the best hypothesis (hypothesis 1) was 88.37 and 78.51, respectively. The model was validated on a test set consisting of six different series of structurally diverse 22 compounds and performed well in classifying active and inactive molecules correctly. This validation approach provides confidence in the utility of the predictive pharmacophore model developed in this work as a 3D query tool in the virtual screening of drug like molecules to retrieve new chemical entities as potent COX-2 inhibitors. The model can also be used to predict the biological activities of compounds prior to their costly and time-consuming synthesis. Figure 3D Pharmacophore model generated using structurally diverse COX-2 inhibitors     

Discovery and functional evaluation of diverse novel human CB1 receptor ligands

Ligand-based virtual screening with a 3D pharmacophore led to the discovery of 30 novel, diverse and drug-like ligands of the human cannabinoid receptor 1 (hCB₁). The pharmacophore was validated with a hit rate of 16%, binding selectivity versus hCB₂, and expected functional profiles. The discovered compounds provide new tools for exploring cannabinoid pharmacology.     

基于微管蛋白和秋水仙碱结合位点化合物构建3D-药效团新模型及先导物虚拟筛选

基于作用于微管蛋白秋水仙碱结合位点的小分子抑制剂与生物靶标的复合晶体结构,采用分子模拟软件Discovery Studio 3.0的受体-配体药效团产生程序建立了系列3D药效团新模型（M1-M6）,并用20个已知微管抑制剂验证了其可靠性.用新药效团模型对约10000个化合物的数据库进行了虚拟筛选,发现了一些潜在先导物.据此合成的二芳烃胺类新化合物20在抑制人白血细胞K562的初步实验中显示出了明显的细胞形态变化和抑制活性,对多种人癌细胞A549,KB,KBvin和DU145均有较强的抑制活性（GI500.17～1.02μmol/L）,表明以此新构建的药效团模型进行理性设计和寻找新型抗癌先导物的方法具有一定的可行性.     

A Combination of Receptor-Based Pharmacophore Modeling & QM Techniques for Identification of Human Chymase Inhibitors

Inhibition of chymase is likely to divulge therapeutic ways for the treatment of cardiovascular diseases, and fibrotic disorders. To find novel and potent chymase inhibitors and to provide a new idea for drug design, we used both ligand-based and structure-based methods to perform the virtual screening(VS) of commercially available databases. Different pharmacophore models generated from various crystal structures of enzyme may depict diverse inhibitor binding modes. Therefore, multiple pharmacophore-based approach is applied in this study. X-ray crystallographic data of chymase in complex with different inhibitors were used to generate four structure–based pharmacophore models. One ligand–based pharmacophore model was also developed from experimentally known inhibitors. After successful validation, all pharmacophore models were employed in database screening to retrieve hits with novel chemical scaffolds. Drug-like hit compounds were subjected to molecular docking using GOLD and AutoDock. Finally four structurally diverse compounds with high GOLD score and binding affinity for several crystal structures of chymase were selected as final hits. Identification of final hits by three different pharmacophore models necessitates the use of multiple pharmacophore-based approach in VS process. Quantum mechanical calculation is also conducted for analysis of electrostatic characteristics of compounds which illustrates their significant role in driving the inhibitor to adopt a suitable bioactive conformation oriented in the active site of enzyme. In general, this study is used as example to illustrate how multiple pharmacophore approach can be useful in identifying structurally diverse hits which may bind to all possible bioactive conformations available in the active site of enzyme. The strategy used in the current study could be appropriate to design drugs for other enzymes as well.     

Pharmacophore mapping of a series of pyrrolopyrimidines,indolopyrimidines and their congeners as multidrug-resistance-associated protein (MRP1) modulators

Pharmacophore mapping studies were undertaken for a series of molecules belonging to pyrrolopyrimidines, indolopyrimidines and their congeners as multidrug resistance-associated protein (MRP1) modulators. A five-point pharmacophore with two hydrogen bond acceptors (A), one lipophilic/hydrophobic group (H), one positive ionic feature (P) and one aromatic ring (R) as pharmacophoric features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of r ² = 0.799 for training set molecules. The model generated showed excellent predictive power, with a correlation coefficient Q ² = 0.679 for an external test set of 20 molecules. The pharmacophore was further validated using four structurally diverse compounds with MRP1 modulatory activity. These compounds mapped well onto four of the five features of the pharmacophore. The pharmacophore proposed here was then utilised for the successful retrieval of active molecules with diverse chemotypes from database search. The geometry and features of pharmacophore are expected to be useful for the design of selective MRP1 inhibitors. Figure Alignment of multidrug resistance-associated protein (MRP1) inhibitors with the developed pharmacophore. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular modeling and QSAR analysis of the interaction of flavone derivatives with the benzodiazepine binding site of the GABA(A) receptor complex

A large number of structurally different classes of ligands, many of them sharing the main characteristics of the benzodiazepine (BDZ) nucleus, are active in the modulation of anxiety, sedation, convulsion, myorelaxation, hypnotic and amnesic states in mammals. These compounds have high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Since 1989 onwards our laboratories established that some natural flavonoids were ligands for the BDZ-bs which exhibit medium to high affinity in vitro and anxiolytic activity in vivo. Further research resulted in the production of synthetic flavonoid derivatives with increased biochemical and pharmacological activities. The currently accepted receptor/pharmacophore model of the BDZ-bs (Zhang, W.; Koeler, K. F.; Zhang, P.; Cook, J. M. Drug Des. Dev. 1995, 12, 193) accounts for the general requirements that should be met by this receptor for ligand recognition. In this paper we present a model pharmacophore which defines the characteristics for a ligand to be able to interact and bind to a flavone site, in the GABA(A) receptor. closely related to the BDZ-bs. A model of a flavone binding site has already been described (Dekermendjian, K.; Kahnberg, P.; Witt, M. R.; Sterner, O.; Nielsen, M.; Liljerfors, T. J. Med. Chem. 1999, 42, 4343). However, this alternative model is based only on graphic superposition techniques using as template a non-BDZ agonist. In this investigation all the natural and synthetic flavonoids found to be ligands for the BDZ-bs have been compared with the classical BDZ diazepam. A QSAR regression analysis of the parameters that describe the interaction demonstrates the relevance of the electronic effects for the ligand binding, and shows that they are associated with the negatively charged oxygen atom of the carbonyl group of the flavonoids and with the nature of the substituent in position 3'.     

Discovery of potent inhibitors for interleukin-2-inducible T-cell kinase: structure-based virtual screening and molecular dynamics simulation approaches

In our study, a structure-based virtual screening study was conducted to identify potent ITK inhibitors, as ITK is considered to play an important role in the treatment of inflammatory diseases. We developed a structure-based pharmacophore model using the crystal structure (PDB ID: 3MJ2) of ITK complexed with BMS-50944. The most predictive model, SB-Hypo1, consisted of six features: three hydrogen-bond acceptors (HBA), one hydrogen-bond donor (HBD), one ring aromatic (RA), and one hydrophobic (HY). The statistical significance of SB-Hypo1 was validated using wide range of test set molecules and a decoy set. The resulting well-validated model could then be confidently used as a 3D query to screen for drug-like molecules in a database, in order to retrieve new chemical scaffolds that may be potent ITK inhibitors. The hits retrieved from this search were filtered based on the maximum fit value, drug-likeness, and ADMET properties, and the hits that were retained were used in a molecular docking study to find the binding mode and molecular interactions with crucial residues at the active site of the protein. These hits were then fed into a molecular dynamics simulation to study the flexibility of the activation loop of ITK upon ligand binding. This combination of methodologies is a valuable tool for identifying structurally diverse molecules with desired biological activities, and for designing new classes of selective ITK inhibitors.

3D QSAR studies on T-type calcium channel blockers using CoMFA and CoMSIA

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on a series of isoxazolyl compounds as a potent T-type calcium channel blockers. A set of 24 structurally similar compounds served to establish the model. Four different conformations of the most active compound were used as template structures for the alignment, three of which were obtained from Catalyst pharmacophore modeling and one by using SYBYL random search option. All CoMFA and CoMSIA models gave cross-validated r(2) (q(2)) value of more than 0.5 and conventional r(2) value of more than 0.85. The predictive ability of the models was validated by an external test set of 10 compounds, which gave satisfactory pred r(2) values ranging from 0.577 to 0.866 for all models. Best predictions were obtained with CoMFA std model of Conformer no: 3 alignment (q(2)=0.756, r(2)=0.963), giving predictive r(2) value of 0.866 for the test set. CoMFA and CoMSIA contour maps were used to analyze the structural features of the ligands accounting for the activity in terms of positively contributing physicochemical properties: steric, electrostatic, hydrophobic and hydrogen bonding fields.     

Identification of selective MMP-9 inhibitors through multiple e-pharmacophore,ligand-based pharmacophore,molecular docking,and density functional theory approaches

Matrix metalloproteinase-9 (MMP-9) is a significant target for the development of drugs for the treatment of arthritis, CNS disorders, and cancer metastasis. The structure-based and ligand-based methods were used for the virtual screening (VS) of database compounds to obtain potent and selective MMP-9 inhibitors. Experimentally known MMP-9 inhibitors were used to grow up ligand-based three pharmacophore models utilizing Schrodinger suite. The X-ray crystallographic structures of MMP-9 with different inhibitors were used to develop five energy-optimized structure-based (e-pharmacophore) models. All developed pharmacophores were validated and applied to screen the Zinc database. Pharmacophore matched compounds were subjected to molecular docking to retrieve hits with novel scaffolds. The molecules with diverse structures, high docking scores and low binding energies for various crystal structures of MMP-9, were selected as final hits. The Induced fit docking (IFD) analysis provided significant information about the driving of inhibitor to approve a suitable bioactive conformational position in the active site of protein. Since charge transfer reaction occurs during receptor–ligand interaction, therefore, electronic features of hits (ligands) are interesting parameters to explain the binding interactions. Density functional theory (DFT) at B3LYP/6-31G* level was utilized to explore electronic features of hits. The docking study of hits using AutoDock was helpful to establish the binding interactions. The study illustrates that the combined pharmacophore approach is advantageous to identify diverse hits which have better binding affinity to the active site of the enzyme for all possible bioactive conformations. The approach used in the study is worthy to design drugs for other targets.