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

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  • This article was downloaded by: [Northeastern University]On: 23 November 2014, At: 20:26Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK

    Journal of Biomolecular Structure and DynamicsPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tbsd20

    Benzodiazepine-Induced Hyperphagia: Developmentand Assessment of a 3D Pharmacophore ByComputational MethodsMarta Filizola a , Danni L. Harris a & Gilda H. Loew aa Molecular Research Institute , 2495 Old Middlefield Way, Mountain View , CA , 94043Published online: 15 May 2012.

    To cite this article: Marta Filizola , Danni L. Harris & Gilda H. Loew (2000) Benzodiazepine-Induced Hyperphagia:Development and Assessment of a 3D Pharmacophore By Computational Methods, Journal of Biomolecular Structure andDynamics, 17:5, 769-778, DOI: 10.1080/07391102.2000.10506566

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  • Benzodiazepine-Induced Hyperphagia: Development and Assessment of a

    3D Pharmacophore By Computational Methods

    http://www.adeninepress.com

    Abstract

    Benzodiazepine receptor (BDZR) ligands are structurally diverse compounds that bind tospecific binding sites on GABAA receptors and allosterically modulate the effect of GABAon chloride ion flux. The binding of BDZR ligands to this receptor system results in activi-ty at multiple behavioral endpoints, including anxiolytic, sedative, anticonvulsant, andhyperphagic effects. In the work presented here, a computational procedure developed in ourlaboratory has been used to obtain a 3D pharmacophore for ligand recognition of theGABAA/BDZRs initiating the hyperphagic response. To accomplish this goal, 17 structural-ly diverse compounds, previously assessed in our laboratory for activity at the hyperphagicendpoint, were used. The result is a four-component 3D pharmacophore. It consists of twoproton acceptor atoms, the centroid of an aromatic ring and the centroid of a hydrophobicmoiety in a common geometric arrangement in all compounds with activity at this endpoint.This 3D pharmacophore was then assessed and successfully validated using three differenttests. First, two BDZR ligands, which were included as negative controls in the set of sev-enteen 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 werefound to fit the pharmacophore. Finally, using the 3D pharmacophore developed in the pres-ent work to search 3D databases, over 50 classical benzodiazepines were found. Amongthem, were benzodiazepine ligands known to have an effect at the hyperphagic endpoint. Inaddition, the novel compounds also found in this search are promising therapeutic agentsthat could beneficially affect feeding behavior.

    Introduction

    The pharmacological effects of BDZR ligands are a consequence of their interac-tion with the pentameric chloride ion channel GABAA receptor located in the cen-tral nervous system (CNS). Cloning and sequencing have demonstrated the exis-tence of twenty-one different isoforms of the five GABAA receptor subunits,including 1-6, 1-4, 1-4, , 1-3, , and (1,2). Binding of BDZ ligands tothese different subunits or combinations of these subunits result in activity at mul-tiple behavioral endpoints, including hyperphagic, anxiolytic, sedative, hyperther-mic, and anticonvulsive effects.

    In recent experimental studies in our laboratory the effect caused by 21 structural-ly diverse BDZR ligands at these five behavioral endpoints, was determined. Theresults demonstrated behavioral heterogeneity for many of the compounds studied(3,4). Specifically, the same compound could have qualitatively different effects,i.e. be agonist, inverse agonist, antagonist, or have no effect, at different behavioralendpoints. Moreover, the pattern of heterogeneity was not the same for all com-pounds. For example, the subset of compounds that were agonists at the hyper-

    Journal of Biomolecular Structure &Dynamics, ISSN 0739-1102Volume 17, Issue Number 5, (2000)Adenine Press (2000)

    Marta Filizola * , Danni L. Harris,and Gilda H. LoewMolecular Research Institute,

    2495 Old Middlefield Way,

    Mountain View, CA 94043

    769

    *Phone: (650) 210-0310 Ext. 210;Fax: (650) 210-0318;E-mail: marta@purisima.molres.org

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  • phagic endpoint or had no effect at that endpoint was different from those that wereagonists or had no effect at the sedation endpoint. These results suggest that not allcombinations of subtypes leading to functional GABAA receptors are involved ineach behavioral endpoint.

    Results of a very recent study (5), provide dramatic new evidence for the conclu-sion drawn from the behavioral studies in our laboratory that not all combinationsof subtypes leading to functional GABAA receptors are involved in each behavioralendpoint. In this recent study, mice were engineered to contain a (H101R) mutantsubunit, by the technique of homologous recombination. Although these mice werenormal in the drug free state, diazepam failed to cause an effect on sedation andmemory, but retained anxiolytic activity. While a significant initial result, manyvery challenging investigations remain to be done in order to identify the specificfunctional GABAA/BDZRs initiating each behavioral response. However, thebehavioral heterogeneity observed in our laboratory for the 21 diverse BDZR lig-ands at five different endpoints suggests that the requirements for recognition of theGABA receptors initiating each endpoint are different.

    To further explore this possibility, 3D pharmacophores that contain the moleculardeterminants for recognition of receptors initiating anxiolysis, sedation, hyperphagia,hypothermia and anticonvulsant activity are being developed in our laboratory usingthe set of ligands for which assessment at each of these endpoints has been made.

    In very recent work, 3D pharmacophores for recognition of receptors initiatinganxiolysis (6) and sedation (7) have been developed and validated in our laborato-ry using these structurally diverse ligands and were found to be qualitatively dif-ferent. These results provide further evidence for the hypothesis that different sub-sets of functional GABA receptors initiate different behavioral effects. In addition,the finding of these different 3D pharmacophores should be useful in the discoveryof behaviorally selective BDZR/GABAA ligands that separate anxiolytic fromsedative activity.

    In the present work, this effort has been extended to the development of a 3D phar-macophore for recognition of receptors initiating the hyperphagia behavioral end-point. This result should be useful since, in addition to the widespread clinical useof benzodiazepine receptor agonists as anxiolytics and sedatives, it is well knownthat they can also induce hyperphagia (8,9) in many mammalian species. Wise andDawson first proposed this possible effect of BDZR agonists almost thirty yearsago (10). Further elaboration of this behavior was provided by Cooper and col-leagues, whose studies supported the hypothesis that benzodiazepine agonistsincrease food intake by selective enhancement of taste-related palatability (8,11).

    At present, it is well established that a wide variety of structurally diverse knownBDZR ligands produce stimulation of the appetite, which is blocked by the admin-istration of either antagonists or inverse-agonists. Specifically, compounds such asflunitrazepam (3), diazepam (12,13), chlordiazepoxide (11,14), clonazepam(15,16), midazolam (17,18), CGS 9896 (19), Ro 16-6028 (20), Ro 17-1812 (21,22), Ro 23-0364 (23), ZK 93423 (22), and ZK 91296 (22) are known to increasefood consumption. By contrast, BDZR ligands such as the pyrazoloquinoline CGS-8216 (19) and the imidazobenzodiazepine Ro 15-4513 (24) are inverse agonists atthis endpoint, decreasing feeding. Finally, the effects of both agonists and inverseagonists are blocked by the administration of the BDZR antagonist Ro 15-1788(flumazenil) (3,25).

    The 3D pharmacophore for recognition of BDZR/GABAA receptors initiating ben-zodiazepine-induced hyperphagia reported here has been developed using seven-teen benzodiazepine ligands from diverse chemical families, whose activity at thisendpoint was assessed in our laboratory. Among them, were eleven agonists, three

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  • antagonists, one inverse agonist, and two compounds with no effect at the hyper-phagia endpoint.

    A novel computational procedure embodied in the computer program MOLMOD,developed in our laboratory (6), was used to identify the molecular determinantsresponsible of recognition of BDZR/GABAA receptors initiating hyperphagia. Thisprogram allows identification of a common spatial arrangement of candidate chem-ical moieties in each agonist, antagonist, or inverse agonist at a specific behavioralendpoint that is not satisfied by compounds having no effect at that endpoint. Thesystematic procedures used to generate 3D pharmacophores can, in principal, beused to examine both receptor recognition and activation requirements. No a pri-ori assumption of a bioactive conformation of any of the ligands needs be made, notemplate is required and both structurally diverse and conformationally flexibleligands can be included in the compounds used for development of the pharma-cophore of interest.

    The 3D pharmacophore developed for recognition of benzodiazepine receptors ini-tiating hyperphagia was successfully validated by three distinct types of assess-ment. First, the two BDZR ligands, that had no activity at this endpoint were usedas negative controls and found not to fit the pharmacophore. Second, some benzo-diazepine ligands known as agonists, antagonists, or inverse agonists at the hyper-phagia endpoint and excluded from the pharmacophore development were used aspositive controls and were found to fit the pharmacophore. Finally, the 3D phar-macophore was used to search 3D databases and BDZR ligands known to have aneffect at the hyperphagia endpoint were extracted. Thus, the remaining compoundsdetermined from such searches could be promising therapeutic agents for the con-trol of feeding behavior.

    Methods

    The MSI/Quanta package (MSI-Quanta. Biosym/MSI, San Diego, CA) was used toconstruct initial structures of the seventeen benzodiazepine ligands used in thiswork for the development of a 3D pharmacophore for recognition ofBDZR/GABAA receptors initiating hyperphagia. These initial structures were thenenergy minimized using the Quanta/CHARMm force field. The minimization pro-cedure consisted of using a dielectric constant of 80, no cutoff, and 200 steps ofsteepest descent followed by 2000-3000 steps of conjugate gradient method.Convergence was achieved when the root mean square deviation (rmsd) changeswere less than 0.01.

    A set of energy minimized unique conformations within 3 kcal/mol with respect tothe lowest energy conformation have already been determined for each of the sev-enteen compounds in a previous study in our laboratory (6). These were used asinput to the 3D pharmacophore generating program MOLMOD (6).

    Briefly, the method selected for exploring the conformational space of all seven-teen compounds depended on the number of rotatable bonds in each of the diversemolecules. Despite the use of the same Quanta/CHARMm force field, a nestedrotation method was applied for the BDZR ligands with 3-4 significant rotatablebonds while a hybrid genetic algorithm (GA)/minimization procedure (CCEMD,Sandia, CA) was used for more flexible ligands. Briefly, the use of nested rotationmethod consisted of employing increments of 30 degrees for each rotatable bondand energy minimizing the resulting conformations. In the case of the hybridGA/minimization procedure, three separate steps were performed for each GA run.Specifically, the three steps consisted of: i) generating an initial population of lowenergy conformers using a genetic algorithm step; ii) clustering of the generatedpopulation into families of unique conformers, using a 5-degrees rms torsion crite-rion; and iii) energy minimizing the resulting unique conformers. In test assess-

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  • ments in our laboratory we have determined that for fairly rigid compounds withfew rotatable bonds, a thorough exploration of the conformational space is oftenachieved after 6-8 cycles of this three-steps procedure.

    The additional input required for the pharmacophore generating programMOLMOD consists of a set of selected candidate chemical moieties common toeach ligand with an effect at the hyperphagia endpoint. Using these inputs,MOLMOD performs systematic pairwise comparisons between all the low energyconformers characterized for each ligand. The result of such comparisons consistsof identifying a common 3D arrangement of the candidate chemical moieties, i.e. a3D pharmacophore. The concepts on which this in-house program is based arereported in details elsewhere (6). Basically, it uses the same principles of clusteringand dist...