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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

To link to this article: http://dx.doi.org/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 α1-α6, β1-β4, γ1-γ4, δ, ρ1-ρ3, π, ε and θ (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 0.01Å.

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 distance matrix comparisons included in previously validated methods forpharmacophore development embodied in computer programs like DISCO (26, 27)and DISTCOMP (28). Specifically, the computation of a pharmacophore involvescomputing the similarities in distance matrix elements between dissimilar ligands.In the MOLMOD implementation, exclusive conformational families are formed bydistance comparison between the pharmacophoric moieties of each of the conform-ers in a given ligand conformational library. Standard deviations of these averagedistances are then computed and stored and the uncertainty of the candidate phar-macophore calculated as sum of the variances of the interpharmacophore distances.

Database searches for validation of the 3D pharmacophore identified in the presentwork were performed using the Tripos Inc. SYBYL/ UNITY package(SYBYL/UNITY, Tripos Associates, Inc., St. Louis, MO, 1999). The 3D databasessearched were the Cambridge Structural Database, the Chapmann and HallChemical Database, and the NCI and Maybridge Databases.

Results and Discussion

In the present work, seventeen different BDZR ligands were used for the develop-ment of a 3D pharmacophore for recognition of BDZR/GABAA receptors initiat-ing hyperphagia. The chemical structures of all these compounds are given inFigure 1. As can be seen from this figure, the selected benzodiazepine ligandsbelong to diverse chemical families. These families include 1,4 benzodiazepines(flunitrazepam), pyrazoloquinolines (CGS 9896 and CGS 8216), β-carbolines(abercanil), imidazoquinolines (U78875 and RU31719), imidazobenzodiazepines(Ro 16-6028, Ro 15-1788, Ro 41-7812, Ro 42-8773, and FG8205), annelated 1,4benzodiazepines (Ro 23-0364), quinolines (Ro 23-1590), imidazopyrimidines(RU32698), pyrroloquinazolones (AHR11797), imidazopyridines (Zolpidem), andaryl-imidazopyridines (AHR14797). In addition, among the BDZR ligands select-ed for the present work, are eleven agonists, three antagonists, one inverse agonist,and two compounds with no effect at the hyperphagia behavioral endpoint.

Also shown in Figure 1, labeled by the letters A-D, are the four chemical moietiesselected for each compound as potential components of the 3D pharmacophore.These components, common to the BDZR ligands with some effect at the hyper-phagia endpoint, were used as input to the 3D pharmacophore generating program.Specifically, A and B indicate two proton acceptor atoms, the letter C marks thecentroid of a hydrophobic group and the letter D marks the centroid of an aromat-ic ring. This aromatic ring in each compound is the major component of the lowestenergy unoccupied molecular orbitals (LUMO), and hence corresponds to the mostfavorable electron accepting region. This property has been ascertained for thesecompounds from quantum chemical calculations recently carried out in our labora-tory, details of which are reported elsewhere (6).

Use of the candidate chemical moieties and conformational libraries for each com-pound as input to the 3D pharmacophore generating program MOLMOD (6),

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Figure 1: Chemical structures of the seventeenBDZR ligands included in the present work. Shownin color are the four candidate chemical moieties (A,B, C, and D) selected as possible components of the3D pharmacophore for recognition of BDZR/GABAA

receptors initiating hyperphagia. Specifically, A andB indicate two proton acceptor atoms, the letter Cmarks the centroid of a hydrophobic group and theletter D marks the centroid of an aromatic ring.

Figure 2: Schematic representation of the 3D phar-macophore developed for ligand recognition ofBDZR/GABAA receptors initiating hyperphagia interms of its components A, B, C , and D and the dis-tances between them.

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resulted in the identification of a common spatial arrangement of the four candidatechemical moieties in all compounds with a specific effect (agonist, antagonist orinverse agonist) at the hyperphagia endpoint. The common spatial arrangement ofthese components thus constitutes a 3D pharmacophore for ligand recognition of

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Figure 3: Spatial overlap of the 4 common recognition moieties A, B, C, and Din the fifteen BDZR ligands with specific effect at the hyperphagia endpoint usedfor the pharmacophore development. In this superposition, the lowest energyconformer of each ligand satisfying the pharmacophore requirements was used.

Figure 4: Superposition between the common four recognition moieties of thefull agonist flunitrazepam and the two no-effect compounds, Zolpidem and AHR14797, at the hyperphagia endpoint.

Figure 5: Chemical structures of the BDZR ligandsused as positive controls for pharmacophore validation.

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BDZR/GABAA receptors initiating hyperphagia. Specifically, the computation ofthis pharmacophore is provided by computing the similarities in distance matrixelements between the diverse ligands used for the development.

Figure 2 shows a schematic representation of the 3D pharmacophore for ligandrecognition of BDZR/GABAA receptors initiating hyperphagia characterized in thepresent work in terms of its defining recognition moieties and the pairwise distancesbetween them. Specifically, these moieties are labeled by the letters A, B, C, and D,according to the definition provided in Figure 1. In addition, the pairwise distancesbetween these moieties and the standard deviation of the mean values of these dis-tances are also reported. The relative magnitude of these standard deviations is ameasure of the uncertainties in the geometric descriptors of the 3D pharmacophore.

Figure 3 shows superposition of the 15 BDZR ligands serving as agonists, antago-nists or inverse agonists at the hyperphagia endpoint using for each the lowest ener-gy conformation that satisfies the pharmacophore. This figure illustrates the goodspatial overlap of the four common recognition moieties, despite the structuraldiversity of the ligands. As shown in this figure, in all these compounds with a spe-cific effect at the hyperphagia endpoint, both of the acceptor atoms A or B are onthe same side of the molecule with respect to the two hydrophobic moieties. Thusthis arrangement can be assumed to be complementary to the position of the pro-ton donating residues of the BDZR/GABAA receptors initiating hyperphagia.

Three different criteria of assessment of the validity of the 3D pharmacophore forrecognition of BDZR/GABAA receptors involved in the hyperphagic response wereused. First, the two compounds with no effect at the hyperphagia endpoint, Zolpidemand AHR14797, whose chemical structures are shown in Figure 1, were used as neg-ative controls for the characterized 3D pharmacophore. As can be seen in Figure 1,both these no-effect compounds contain all the four chemical moieties common to theBDZR ligands with effect at the hyperphagia endpoint. In addition, the pairwise dis-tances defining the developed 3D pharmacophore are satisfied. However, as shownin Figure 4, the two polar groups A and B in the no-effect compounds have a quali-tatively different spatial arrangement than in all the compounds with some effect atthis endpoint. Shown in this figure is the superposition of the four moieties A, B, C,and D of one of the active compounds, the agonist flunitrazepam, with the no-effectcompounds, Zolpidem, and AHR14797. This superposition clearly shows thatZolpidem and AHR14797 have a different spatial orientation of the two polar accep-tor atoms, A (colored red), and B (colored blue). Specifically in both Zolpidem andAHR14797, the two corresponding moieties A and B are on opposite sides of themolecule. Therefore they are not in a favorable spatial orientation to interact with thesame complementary proton donors in the binding site the BDZR/GABAA receptorsinitiating hyperphagia as the active compounds.

The 3D pharmacophore shown in Figure 2 was further assessed by use of some BDZRligands reported to be agonists, inverse agonists, or antagonists at the hyperphagiaendpoint, which were intentionally excluded from the set of compounds used for thepharmacophore development. These specific compounds used, whose chemical struc-tures are shown in Figure 5, include the agonists chlordiazepoxide or librium (11,14),midazolam (17,18), Ro 17-1812 (21,22), ZK 91296 (22), and ZK 93423 (22), theinverse agonist Ro 15-4513 (24), and the antagonist CGS-9895 (29). All these com-pounds were found to satisfy the pharmacophore requirements. Specifically, as shownin Figure 5, they contain the four chemical moieties A, B, C, and D common to allactive compounds in the same relative spatial arrangement as the 15 compounds usedto develop the pharmacophore and the pairwise distances between them are within thetolerance values of the 3D pharmacophore showed in Figure 2.

The third validation of the 3D pharmacophore developed in the present study wasmade by using it to search the Cambridge Structural 3D Database for compounds

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Figure 6: Chemical structures and reference codes ofthe BDZR ligands found in the 3D CambridgeStructural Database using the criteria of the 3D phar-macophore developed in the present work.

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that satisfied the requirements determined for binding to receptor subtypes respon-sible for hyperphagia. As a result, more than fifty BDZR ligands were found. Thechemical structures of these compounds, as well as their database reference codes,are reported in Figure 6. Among them, BDZR ligands such as diazepam, clon-azepam, and lorazepam, known to have a specific effect at the hyperphagia end-point (12,16,30), but that were not included in the initial set, were identified.

These results taken together provide convincing validation of the 3D pharma-cophore developed in the present work, which can be used to distinguish com-pounds with specific or no-effect at the hyperphagia endpoint. This differentiationmay constitute a first step to the design of new molecules, which can selectivelyrecognize the BDZR/GABAA receptors initiating hyperphagia.

Conclusions

A 3D pharmacophore for recognition of BDZR/GABAA receptors initiating hyper-phagia was developed and assessed in the present work. Development was basedon the use of seventeen different BDZR ligands, eleven of which are agonists, threeare antagonists, one is an inverse agonist, and two have no-effect at the hyperpha-gia endpoint. The conformational libraries of all these compounds as well as thesets of candidate chemical moieties common to all agonists, inverse agonists, andantagonists at the hyperphagia endpoint were used as input information for a novelin-house computer program, MOLMOD. This program allows the identification ofa common spatial arrangement of the selected candidate chemical moieties, lead-ing to the characterization of a 3D pharmacophore.

The 3D pharmacophore developed was then subjected to three different types ofassessment. First, the two BDZR ligands, which were included as negative controlsin the set of seventeen compounds used for the pharmacophore development, didnot fit the pharmacophore. Second, some GABAA/BDZR ligands known to be ago-nists, antagonists or inverse agonists at the hyperphagia endpoint and excludedfrom the pharmacophore development were used as positive controls and demon-strated to fit the pharmacophore. Finally, using the 3D pharmacophore developedin the present work to search 3D databases, over 50 classical benzodiazepines werefound. Among them, are benzodiazepine ligands known in the literature to havehyperphagic effects.

Acknowledgements

Support for this research by NIDA grant DA06304 is gratefully acknowledged.

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Date Received: November 23, 1999

Communicated by the Editor Ramaswamy H. Sarma

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