Benzodiazepine-Induced Hyperphagia: Development and Assessment of a 3D Pharmacophore By Computational Methods

Abstract

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 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 GABAA/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.     

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 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.     

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.     

Molecular modelling study on human histamine H1 receptor and its applications in virtual lead identification for designing novel inverse agonists

Human histamine H1 receptor (HHR1) is one of the G protein-coupled receptors (GPCRs) known for their constitutive activation in the absence of agonist binding. Inverse agonists are the compounds that inhibit this constitutive activity of GPCRs. HHR1 is involved in allergic reactions and is also known to be constitutively active. An updated quantitative pharmacophore model, Hypo1, has been developed using a diverse set of known HHR1 inverse agonists employing the HypoGen algorithm as implemented in Accelrys Discovery Studio 2.1. Hypo1 comprised four pharmacophore features (each one of hydrogen bond acceptor, hydrophobic, ring aromatic and positive ionisable group) along with a high correlation value of 0.944. This pharmacophore model was validated using an external test set containing 25 diverse inverse agonists and CatScramble method. Three chemical databases were screened for novel chemical scaffolds using Hypo1 as a query, to be utilised in drug design. The 3D structure of HHR1 has been constructed using human β2 adrenergic receptor. Molecular docking studies were performed with the database hit compounds using GOLD 4.1 program. The combination of all results led us to identify novel compounds to be deployed in designing new generation HHR1 inverse agonists.     

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.     

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.     

A review of the molecular conformations of melatonin ligands at the melatonin receptor

This review examines the 1992-2000 literature on studies of the molecular conformations of melatonin ligands at the melatonin receptor. In order to investigate quantitative structure-affinity relationships between different chemical classes of melatonergic ligands binding to the melatonin GPCR, CoMFA has been applied to extended sets of compounds, to obtain 3D-QSAR agonist/antagonist models. The results of several authors have suggested that the active conformation of the C-3 aminoethyl side chain of melatonin and related compounds is in a folded form, orthogonal to the aromatic ring. Positive steric potentials were found in the C-2 region, surrounding the C-5 methoxy group and near the N -acyl group of the side chain, while substituents in positions C-6 and C-7 cause a decrease in affinity. Negative steric regions were found between indole N-1 and C-2. Receptor binding affinities have been predicted for a range of structurally diverse compounds for the sheep brain melatonin receptor considering steric, electrostatic and lipophilic fields.     

Complementarity of delta opioid ligand pharmacophore and receptor models

The elaboration of a pharmacophore model for the delta opioid receptor selective ligand JOM-13 (Tyr-c[D-Cys-Phe-D-Pen]OH) and the parallel, independent development of a structural model of the delta receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr(1) residue and in the Phe(3) side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained delta receptor ligands. Our model of the delta opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other delta ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the "alternate" allowed conformer, identified from analysis of the ligands but "disfavored" because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other delta ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ.     

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'.     

Design and synthesis of trivalent ligands targeting opioid,cholecystokinin, and melanocortin receptors for the treatment of pain

It has been known that co-administration of morphine with either cholecystokinin (CCK) receptor or melanocortin (MC) receptor antagonists enhance morphine’s analgesic efficacy by reducing serious side effects such as tolerance and addiction.1, 2, 3, 4 Considering these synergistic effects, we have designed trivalent ligands in which all three different pharmacophores for opioid, CCK, and MC receptors are combined in such a way as to conserve their own topographical pharmacophore structures. These ligands, excluding the cyclic compound, were synthesized by solid phase synthesis using Rink-amide resin under microwave assistance in very high yields. These trivalent ligands bind to their respective receptors well demonstrating that the topographical pharmacophore structures for the three receptors were retained for receptor binding. Ligand 10 was a lead compound to show the best biological activities at all three receptors.     

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.     

Conformational analysis of tandospirone in aqueous solution: lead evolution of potent dopamine D4 receptor ligands

The significant contribution of folded conformation (2) of the anxiolytic tandospirone (1) in aqueous solution was verified by dynamic 1H NMR. A structurally rigid mimic of 2 was designed and synthesized to evaluate the implication of 2 towards neuroleptic receptor binding. The designed structures provided a new rigid scaffold for dopamine D4 ligands.     

6H,13H-Pyrazino[1,2-a;4,5-a']diindole analogs: probing the pharmacophore for allosteric ligands of muscarinic M2 receptors

A series of 6H,13H-pyrazino[1,2-a;4,5-a']diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M(2) receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [(3)H]N-methylscopolamine-occupied M(2) receptors (reflected by EC(50,diss)) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand-receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by K(i)) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M(2) receptors.     

In vitro and in vivo characterization of MPEP,an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article

Summary.  There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain. We have characterized novel subtype-selective mGlu₅ receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu₅ receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. Received July 2, 2001 Accepted August 6, 2001 Published online September 10, 2002     

A high-affinity fluorenone-based beta 2-adrenergic receptor antagonist with a photoactivatable pharmacophore

To develop molecules capable of directly probing the catechol binding region of the beta(2)-adrenergic receptor (beta(2)AR), novel benzophenone- and fluorenone-based beta(2)AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone+ ++ (iodoaminoflisopolol, IAmF), showed very high affinity for the beta(2)AR, inhibiting [(125)I]ICYP binding with an apparent K(i) of approximately 1 x 10(-)(9) M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon-carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous beta(2)AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta(2)AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the beta(2)AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([(125)I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [(125)I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta(2)AR in membranes (protectable by 10 microM alprenolol) was demonstrated. [(125)I]IAmF represents a new class of beta(2)AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the beta(2)AR ligand binding pocket.     

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     

3,4-Disubstituted indole acylsulfonamides: A novel series of potent and selective human EP3 receptor antagonists

A series of potent and selective EP₃ receptor antagonists are described. Utilizing a pharmacophore model developed for the EP₃ receptor, a series of 3,4-disubstituted indoles were shown to be high affinity ligands for this target. These compounds showed high selectivity over IP, FP and other EP receptors and are potent antagonists in functional assays.     

Identification of structural motifs in the E2 glycoprotein of Chikungunya involved in virus–host interaction

Chikungunya fever is one of the reemerging vector-borne diseases. It has become a major global health problem especially in the developing countries. There are no vaccines or specific antiviral drugs available to date. This study reports small molecule inhibitors of envelope glycoprotein 2 (E2 glycoprotein) which are predicted based on Chikungunya virus–host interactions. E2 glycoprotein of Chikungunya virus interacts at 216 residue of the host receptor protein which plays a vital role in initiating infection. Understanding the structural aspects of E2 glycoprotein is crucial to develop specific inhibitors to prevent the virus binding from host receptors. In silico method was adopted to predict the sequence motifs of envelope protein, as the method like yeast two hybrid system is laborious, time consuming, and costly. The E2 glycoprotein structure of the Indian isolate was modeled using two templates (2XFC and 3JOC) and then validated. The class III PDZ domain binding motif was found to be identified at 213–216 amino acids. The corresponding peptide structures which recognize the PDZ domain binding motif were identified by the literature search and were used for generating five point pharmacophore model (ADDDR) containing acceptor, donor and aromatic ring features. Databases such as Asinex, TosLab and Maybridge were searched for the matches for the predicted pharmacophore model. Two compounds were identified as lead molecules as their glide score is?>?5?kcal/mol. Since the pharmacophore model is developed based on Chikungunya virus–host interaction, it can be used for designing promising antiviral lead compounds for the treatment of Chikungunya fever.An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:21     

Monoclonal Antibodies with High Affinity for Spiroperidol

A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N-amino-phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to greater than 100 nM. The antigen binding sites of the anti-NH2SPD and anti-NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]-SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti-NH2SPD and four anti-NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2-selective antagonists of the benzamide class, or the agonist dopamine.