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.     

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.     

3D QSAR models for alpha2a-adrenoceptor agonists

Three-dimensional structure-activity relationship studies of alpha2a-adrenoceptor agonists were carried out by Distance Comparison (DISCOthech) and Comparative Molecular Field Analysis (CoMFA) methods to define the pharmacophore and a quantitative model, respectively, of this class of compounds. The statistical validation of the CoMFA model indicates its high predictive performance for binding affinities of new alpha2a-adrenoceptor agonists.     

2,3,7-Trisubstituted pyrazolo[1,5-d][1,2,4]triazines: functionally selective GABAA alpha3-subtype agonists

Novel synthetic routes have been devised for the preparation of previously inaccessible 2,3,7-trisubstituted pyrazolo[1,5-d][1,2,4]triazines 2. These compounds are high affinity ligands for the GABA(A) benzodiazepine binding site and some analogues show functional selectivity for agonism at alpha3-containing receptors over alpha1-containing receptors with the lead compound being 32.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

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.     

Imidazo[1,2-b][1,2,4]triazines as alpha2/alpha3 subtype selective GABA A agonists for the treatment of anxiety

Imidazo[1,2-a]pyrimidines and imidazo[1,2-b][1,2,4]triazines are ligands for the benzodiazepine binding site of GABA(A) receptors that are functionally selective for the alpha2/alpha3 subtypes over the alpha1 subtype. SAR studies to optimise this functional selectivity, pharmacokinetic and behavioural data are described.     

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.     

N-(4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl,butenyl and butynyl)arylcarboxamides as novel dopamine D(3) receptor antagonists

The dopamine D(3) receptor subtype has been targeted as a potential neurochemical modulator of the behavioral actions of psychomotor stimulants, such as cocaine. Previous synthetic studies provided structural requirements for high affinity binding to D(3) receptors which included a 2,3-dichloro-phenylpiperazine linked to an arylamido function via a butyl chain. To reduce lipophilicity of these agents and further investigate optimal conformation, a second series of 15 novel ligands was designed that included heteroaromatic substitution and unsaturated alkyl linkers. These compounds were synthesized and evaluated for binding at rat D(3) and D(2) receptors stably expressed in Sf9 cells. D(3) binding affinities ranged from K(i)=0.6-1080 nM, with a broad range of D(3)/D(2) selectivities (2-97). The discovery of potent, selective and bioavailable D(3) receptor ligands will provide essential molecular probes to elucidate the role D(3) receptors play in the psychomotor stimulant and reinforcing effects of cocaine.     

Synthesis, alpha 1-adrenoceptor antagonist activity, and SAR study of novel arylpiperazine derivatives of phenytoin

In the search for new antiarrhythmic agents, some active 2-methoxyphenylpiperazine derivatives of phenytoin were obtained as a chemical modification of compound AZ-99 (3-ethyl-1-[2-hydroxy-3-(4-phenylpiperazin-1-yl)-propyl]-2,4-dioxo-5,5-diphenylimidazolidine). These compounds possessed structural properties similar to those of alpha(1)-adrenoceptor antagonists. In the present study, the affinities of the 2-methoxyphenylpiperazine derivatives (1a-3a) for alpha(1)- and alpha(2)-adrenoceptors were evaluated using radioligand ([(3)H]prazosin, [(3)H]clonidine) binding assays. In the next step, a new series of phenylpiperazine derivatives of phenytoin (4a-16a) containing 2-methoxyphenyl-, 2-ethoxyphenyl-, 2-pyridyl- or 2-furoylpiperazine moiety, as well as, various ester or alkyl substituents at 3-position of hydantoin ring were synthesized. The newly synthesized compounds were tested for their affinity to alpha(1)- and alpha(2)-adrenoceptors. They have shown affinities for alpha(1)-adrenoceptors at nanomolar to submicromolar range. Some compounds were moderately selective ligands of alpha(1)-adrenoceptors. Selected compounds (3a-5a, 7a, 13a, 14a) were also evaluated for their alpha(1)-adrenoceptor antagonistic properties in functional bioassays. A SAR study indicated that the most active compounds contain 2-alkoxyphenylpiperazine moieties and methyl or 2-methylpropionate substituent at 3-N position in hydantoin. The exchange of 2-alkoxyphenyl moiety into 2-furoyl or 2-pyridyl group significantly decreased affinities for alpha(1)-adrenoceptors. Molecular modelling results obtained using conformational analysis CONFLEX and PM5 method for geometry optimization, allowed for comparison of the spatial properties of tested compounds with pharmacophore model created by Barbaro et al. for the ideal alpha(1)-adrenoceptor antagonist.     

alpha4beta3delta GABA(A) receptors characterized by fluorescence resonance energy transfer-derived measurements of membrane potential

Selective modulators of gamma-aminobutyric acid, type A (GABA(A)) receptors containing alpha(4) subunits may provide new treatments for epilepsy and premenstrual syndrome. Using mouse L(-tk) cells, we stably expressed the native GABA(A) receptor subunit combinations alpha(3)beta(3)gamma(2,) alpha(4)beta(3)gamma(2), and, for the first time, alpha(4)beta(3)delta and characterized their properties using a novel fluorescence resonance energy transfer assay of GABA-evoked depolarizations. GABA evoked concentration-dependent decreases in fluorescence resonance energy transfer that were blocked by GABA(A) receptor antagonists and, for alpha(3)beta(3)gamma(2) and alpha(4)beta(3)gamma(2) receptors, modulated by benzodiazepines with the expected subtype specificity. When combined with alpha(4) and beta(3), delta subunits, compared with gamma(2), conferred greater sensitivity to the agonists GABA, 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), and muscimol and greater maximal efficacy to THIP. alpha(4)beta(3)delta responses were markedly modulated by steroids and anesthetics. Alphaxalone, pentobarbital, and pregnanolone were all 3-7-fold more efficacious at alpha(4)beta(3)delta compared with alpha(4)beta(3)gamma(2.) The fluorescence technique used in this study has proven valuable for extensive characterization of a novel GABA(A) receptor. For GABA(A) receptors containing alpha(4) subunits, our experiments reveal that inclusion of delta instead of gamma(2) subunits can increase the affinity and in some cases the efficacy of agonists and can increase the efficacy of allosteric modulators. Pregnanolone was a particularly efficacious modulator of alpha(4)beta(3)delta receptors, consistent with a central role for this subunit combination in premenstrual syndrome.     

Tricyclic pyridones as functionally selective human GABAA alpha 2/3 receptor-ion channel ligands

A series of tricyclic pyridones has been evaluated as benzodiazepine site ligands with functional selectivity for the alpha(3) over the alpha(1) containing subtype of the human GABA(A) receptor ion channel. This investigation led to the identification of a high affinity, functionally selective, orally bioavailable benzodiazepine site ligand that demonstrated activity in rodent anxiolysis models and reduced sedation relative to diazepam.     

Ligand recognition of serine-cysteine amino acid exchanges in transmembrane domain 5 of alpha2-adrenergic receptors by UK 14,304

Ligand binding of UK 14,304 reveals notable species (i.e., human-rodent) and receptor-subtype differences of alpha2-adrenergic receptors (alpha2-ARs). To study the molecular basis of the selectivity of UK 14,304, we compared a series of conservative serine-cysteine exchange mutants at ligand-accessible positions in transmembrane domain 5 of the human and mouse alpha2A-ARs. UK 14,304 bound with approximately 200-fold higher affinity to the human alpha2A-AR wild-type receptor compared with the human alpha2A-ARSer201 mutant, but only an approximately fivefold difference was seen with the corresponding mouse alpha2A-AR variant. These effects of cysteine-serine exchanges only involved the agonist low-affinity forms of the receptors, as the affinity of [3H]UK 14,304 for the agonist high-affinity receptor populations was not influenced. The apparent affinities of a set of eight structurally diverse alpha2-AR ligands (six agonists and two antagonists) were not influenced significantly by the cysteine-serine exchanges (except for oxymetazoline and yohimbine, with up to nine- and eightfold differences in affinity, respectively). We conclude that position 201 (a) plays a primary role in determining observed subtype/species selectivity of UK 14,304 in competitive antagonist radioligand binding assays and (b) does not determine the subtype selectivity of chlorpromazine.     

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.     

Ligand design for alpha1 adrenoceptor subtype selective antagonists

Alpha1 adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring D analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human alpha1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (alpha1A, alpha1B, alpha1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the alpha1D adrenoceptor subtype is also reported.     

3-Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione derivatives as novel, high-affinity and selective alpha(1)-adrenoceptor ligands

The discovery of a new series of selective and high-affinity alpha(1)-adrenoceptor (alpha(1)-AR) ligands, characterized by a 1H-pyrrolo[2,3-d]-pyrimidine-2,4(3H,7H)-dione system, is described in this paper. Some synthesized compounds, including 20, 22, and 30, displayed affinity in the nanomolar range for alpha(1)-ARs and substantial selectivity with respect to 5-HT(1A) and dopaminergic D(1) and D(2) receptors. Functional assays, performed on selected derivatives, showed antagonistic properties.     

The preparation of HEK293 alpha1A or HEK293 alpha1B cell membrane stationary phase and the chromatographic affinity study of ligands of alpha1 adrenoceptor

As a novel bioaffinity chromatographic technique, cell membrane chromatography (CMC) originated in 1996. The cell membrane stationary phase (CMSP) consists of porous silica coated with active cell membranes. By immersing silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes. The present study repeatedly investigated the interaction between ligands and receptors by employing the system of CMC and especially evaluated the accuracy and feasibility of the CMC model in the study of subtype receptors. The cDNA encoding alpha1A or alpha1B adrenergic receptors (ARs) was transfected into human embryonic kidney 293 (HEK293) cell lines; cell lines stably overexpressing subtype receptors were obtained. HEK293 alpha1A or HEK293 alpha1B ARs CMSP were prepared by immobilizing relevant cell membranes on silica. In the described chromatography-based CMSP, the retention times of nine alpha1 adrenoceptor ligands and calculated capacity factors, as chromatographic parameters, were recorded carefully. These results showed a good correlation with the affinity of the same compounds for the corresponding cloned alpha1 adrenoceptor subtype. The rank order of capacity factors was consistent with the affinity rank order obtained from radioligand binding assays. The immobilized subtype-selective CMSP was stable and reproducible. The study demonstrates that the HEK293 alpha1A and HEK293 alpha1B CMSP can be utilized for initial screening of drug candidates.     

Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine derivatives as 5-HT1A serotonin receptor ligands

A series of novel 1,2,3-benzotriazin-4-one derivatives was prepared and evaluated as ligands for 5-HT receptors. Radioligand binding assays proved that the majority of the novel compounds behaved as good to excellent ligands at the 5-HT1A receptor, some of which were selective with respect 5-HT2A and 5-HT2C receptors. Six analogues (1a, 2a, 2b, 2c, 2e and 2i) were selected and further evaluated for their binding affinities on D1, D2 dopaminergic and alpha1-, alpha2-adrenergic receptors. A o-OCH3 derivative (2e) bound at 5-HT1A sites with subnanomolar affinity (IC50 = 0.059 nM) and shows high selectivity over all considered receptors and may offer a new lead for the development of therapeutically efficacious agents.     

Synthesis and structure–affinity relationships of novel small molecule natural product derivatives capable of discriminating between serotonin 5-HT1A, 5-HT2A, 5-HT2C receptor subtypes

Efforts to develop ligands that distinguish between clinically relevant 5-HT2A and 5-HT2C serotonin receptor subtypes have been challenging, because their sequences have high homology. Previous studies reported that a novel aplysinopsin belonging to a chemical class of natural products isolated from a marine sponge was selective for the 5-HT2C over the 5-HT2A receptor subtype. Our goal was to explore the 5-HT2A/2C receptor structure–affinity relationships of derivatives based on the aplysinopsin natural product pharmacophore. Twenty aplysinopsin derivatives were synthesized, purified and tested for their affinities for cloned human serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptor subtypes. Four compounds in this series had >30-fold selectivity for 5-HT2A or 5-HT2C receptors. The compound (E)-5-((5,6-dichloro-1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one (UNT-TWU-22, 16) had approximately 2100-fold selectivity for the serotonin 5-HT2C receptor subtype: an affinity for 5-HT2C equal to 46 nM and no detectable affinity for the 5-HT1A or 5-HT2A receptor subtypes. The two most important factors controlling 5-HT2A or 5-HT2C receptor subtype selectivity were the combined R1,R3-alkylation of the imidazolidinone ring and the type and number of halogens on the indole ring of the aplysinopsin pharmacophore.     

Yohimbine dimers exhibiting binding selectivities for human alpha2a- versus alpha2b-adrenergic receptors

A series of yohimbine dimers was prepared and evaluated at the human alpha2a- and alpha2b-adrenergic receptors (ARs) expressed in Chinese hamster ovary (CHO) cells. All dimers display higher binding selectivities for alpha2a versus alpha2b subtype than yohimbine, and four compounds (3d, 3e, 3g and 3i) represent the most potent and alpha2a versus alpha2b-AR selective ligands identified so far.