The rapidly expanding range of neuronal benzodiazepine receptor ligands.

Comparative Quantitative Structure−Activity Relationship Studies (QSAR) on Non-Benzodiazepine Compounds Binding to Benzodiazepine Receptor (BzR)

The gastric H,K ATPase is an alpha beta heterodimeric member of the eukaryotic alkali-cation P-type ion-motive ATPase family. The alpha subunit is composed of 1033 amino acids and the beta subunit of 291 amino acids with 6 or 7 potential N-linked glycosylation sites. Much effort has been expended to define the membrane domain of P-type ATPases. A membrane domain of the large subunit consisting of 10 membrane-spanning sequences is suggested by a combination of methods such as (1) tryptic digestion, separation, and sequencing of membrane peptides, (2) labeling with extracytoplasmic reagents, and (3) in vitro translation of hydrophobic segments. The beta subunit has a single transmembrane segment with strong hydrophobic interactions with the alpha subunit. Blue native gel electrophoresis shows that the enzyme is an (alpha-beta)2 dimer. Cross-linking with Cu-phenanthroline provides evidence that association is between the alpha subunits, and the potential SH groups that are Cu sensitive are at cysteine 565 and cysteine 615, in the region of the large cytoplasmic loop between the fourth and fifth transmembrane segments. No cross-linking is observed in the membrane domain. ATP prevents cross-linking because of a conformational change at the surface of the protein induced by ATP or by direct binding of the nucleotide at the site of cross-linking. The WGA binding properties of the beta subunit allow investigation of the region of interaction with the alpha subunit. Thus, digestion of the enzyme by trypsin followed by SDS solubilization and selective elution from a WGA column resulted in coelution of the membrane fragment containing TM7 and TM8. This result demonstrates major hydrophobic interaction between the seventh and eighth transmembrane segments and the beta subunit. An antibody generated against rat parietal cells also recognized shared epitopes in the same region of both the alpha and beta subunits. Biochemical investigation of the arrangement of the transmembrane segments has been hindered by the lack of effective cross-linking reagents probably because of the compact arrangement of this domain, preventing even Cu access. A series of antiulcer drugs has been developed that have a unique chemistry related to their inhibition of the gastric H,K ATPase. They are 2-(substituted pyridyl methylsulfinyl) benzimidazoles, weak bases with a pKa of 4.0. After accumulation in the acidic space generated by the H,K ATPase either in vivo or in vitro, they undergo acid-catalyzed conversion to a tetracyclic sulfenamide which reacts with luminally accessible SH residues to form stable disulfide derivatives. In the particular case of pantoprazole, 2-(3,4-dimethoxy-2-pyridyl-methylsulfinyl)-5-difluoromethoxy benzimidazole, reaction is confined largely to cysteine 813, placed between the fifth and sixth transmembrane segments. The 5 azido analog of pantoprazole provided acid transport-dependent inhibition of the isolated transporting ATPase by this photoactivatable covalent SH reagent. The inhibited enzyme was then photolyzed, cleaved with trypsin, and the membrane fragments compared before and after photolysis. Disappearance of the segment corresponding to TM3,4 and a relative loss of the segment corresponding to TM7,8 suggests close proximity of these two membrane pairs to the loop joining the fifth and sixth transmembrane segments, in particular TM3,4. Use of this type of covalent, photoactivatable site-specific reagent to determine loop proximity can be extended to other acid transporters.

Structural aspects of the gastric H,K ATPase.

Benzodiazepine (BZ) binding site ligands are important central nervous system (CNS) drugs. Their numbers and our knowledge of how they interact with the BZ-binding site of GABAA receptors are both rapidly expanding. The GABAA receptor is a member of the ligand-gated ion channel superfamily. In general, it consists of an assembly of transmembrane pentamers of different subunit compositions (1). To date, at least 15 types of subunits (α1–6, β1–3, γ1–3, δ, e, and π) have been identified using molecular cloning techniques that combine in various combinations to give rise to the different GABAA-receptor subtypes (1–3). The central BZ-binding site is a part of the GABAA-receptor-associated chloride ion channel. Most functional subtypes of the GABAA receptor contain α, β, and γ subunits, with the different receptor subtypes displaying marked variations in their binding capacity to different BZ-binding site ligands (4,5). When GABA, the major inhibitory transmitter in the CNS, binds to a GABAA receptor, the chloride ion flux through the channel is increased. This leads to membrane hyperpolarization that results in a reduction in the excitability potential of the neuron (6). As a consequence, GABAA receptors are the molecular targets of a variety of pharmacologically and clinically important drugs, such as the anxiolytic, anticonvulsant, sedative-hypnotic benzodiazepines, some anxiogenic, convulsant β-carbolines, and the convulsants bicuculline or picrotoxinin. Furthermore, multiple recognition sites that exist within the three-dimensional structure of the various GABAA-receptor subtypes possess the capacity to interact with a host of different ligands (6). These sites include: the GABA site; the benzodiazepine site; the barbiturate site; the general anesthetic site; the picrotoxinin site; and the storage site for GABA. BZ-binding site ligands act through mechanisms

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Ligands of the GABAA Receptor Benzodiazepine Binding Site

There has recently been a large increase in the number of new benzodiazepine receptor ligands, some with benzodiazepine structures, but many with other chemical structures. The pharmacological activities of these ligands have been interpreted on the basis of a continuum of efficacy ranging from full agonist through different degrees of partial agonism to antagonist and through partial to full inverse agonists. Inconsistencies with this hypothesis are considered in terms of alternative hypotheses, particularly the existence of functionally separable receptor subtypes. The potential of partial agonists as non-sedative anxiolytic agents with reduced potential of dependence and of weak partial inverse agonists as pro-cognitive agents is discussed. A pharmacophore for benzodiazepine receptors is proposed and supporting evidence presented.

A review of recently-developed ligands for neuronal benzodiazepine receptors and their pharmacological activities.

Quantitative structure-activity relationship (QSAR) analyses of three different series of benzodiazepine ligands, pyridodiindoles, and beta-carbolines, having inverse agonist/antagonist and agonist actions have been performed with a view to understanding their likely mode of action at the benzodiazepine-receptor (BzR). From this study, the in vitro radioligand displacement activities of substituted 7,12-dihydropyrido[3,2-b: 5,4-b']diindoles (Figure 1) were found to be significantly correlated with resonance effect of R-substituents, van der Waals volumes at positions-1 and -3, hydrophobic constant at position-2 and field effect at position-4 of X-substituents. For 3-substituted beta-carbolines, a similar analysis has established that R-substituents at position-3 of beta-carbolines (Figure 2) are involved in strong hydrophobic interaction with some hydrophobic pocket on the receptor. For the analogues of 6-(benzyloxy)-4-(methoxymethyl)-beta-carbolines-3-carboxylic acid ethyl ester (Figure 3), however, the combined hydrophobicities of 6- and 4-substituents were shown to be important in determining ligand binding behaviour. The significant correlations so obtained are in agreement with the proposed models (Figures 4 and 5) of pharmacophores of inverse agonist/antagonist and of agonist sites at the BzR.

Quantitative structure-activity relationship study of some benzodiazepine-receptor ligands having inverse agonist/antagonist and agonist actions.

The (H+/K+)-ATPase enzyme inhibitory activity of omeprazole analogues (Figure 1) and 1-aryl-4-methyl-2,3-dihydropyrrolo[3,2-c]quinolines (Figure 2) was found to be significantly correlated with electronic (sigma) or pKa parameter that governs the basicity of the molecules. The former compounds are representative of irreversible blockers, and the latter of reversible blockers. Inclusion of hydrophobic (pi) and/or steric (Es) parameters sometimes led to improvement in the correlations, suggesting that these parameters may play a role in the formation of a cyclic intermediate. The derived significant correlation equations strongly support a mechanism of action, first proposed by Lindberg et al., involving such a cyclic intermediate.

Quantitative structure-activity relationship studies of inhibitors of gastric (H+/K+)-ATPase.

Previously reported quantitative structure-activity study on 1-aryl-3-methylpyrazolo[4,5-c]quinolin-4-ones is extended to include recently reported 1-aryl[1]benzopyrano-pyrazol-4-ones. It has been shown that the ability of these compounds to displace [3H]-flunitrazepam from bovine brain membrane is significantly correlated with steric and hydrophobic constants of aryl substituents. For 1,3-diaryl-pyrazolo[4,5-c]quinolin-4-ones, however, only hydrophobic interaction of meta-substituents is found to be relevant.

Quantitative structure-activity relationship study on some ligands acting as inhibitors of benzodiazepine-receptor binding.

Binding affinities, pKi's, of 1,3-dipropyl-8-phenylxanthines and 8-substituted xanthines as selective antagonists at A(1)- and A(2)- adenosine receptors, were quantitatively analysed in terms of hydrophobic parameter, pi, and van der Waals volume, Vw. For ligands of the first series, the hydrophobicity of para-substituents and the bulk of meta-substituents are shown to be the deciding factors. Similarly, for the other series, the binary substitutions at X-, Y- and R-positions, highlighted by respective dummy variables, and the bulk rendered by groups at R(1)-position, are found to be significantly correlated with A(1)- and A(2)-receptor affinities. Additionally, the Free-Wilson study of this series resulting into individual substituent contribution, provides similar inferences to these, but in a more exact manner. This study also hints at the possibility of a different accommodation site at the receptor for the R(1)-substituents of the congeners on account of conformational dissimilarity of A(1)- and A(2)-receptors.

A quantitative analysis of binding affinities of ligands active at adenosine receptors.

A quantitative structure-activity relationship (QSAR) analysis of two related series of the derivatives of 7-substituted imidazo [4,5-b]-quinolin-2-one active as inhibitors of human blood platelet cAMP phosphodiesterase (PDE) is presented with a view to reflecting upon the parametric requirements of the side chain as well as of the N-1 and N-3 substitutions on the heterocycle. For the first series (Figure 1) consisting of 114 congeners and having a more flexible functionalized side chain at the 7-position and only binary variations (Me or H) at N-1 and N-3, it has been shown that bulk (Vw), of the functionalized side chain tends to potentiate the inhibitory activity of a derivative while positions N-1 and N-3 should better remain unsubstituted, as reflected through the dummy variables. A similar analysis of the compounds of the second series (Figure 2), where a less flexible side chain is linked through a basic nitrogen, has provided a parabolic dependence of inhibition activity on Vw. From this relationship, a limiting size of the side chain, seems to be necessary for triggering a minimal response.

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Papers

Quantitative structure-activity relationship study of some benzodiazepine-receptor ligands having inverse agonist/antagonist and agonist actions.

Quantitative structure-activity relationship studies of inhibitors of gastric (H+/K+)-ATPase.

Quantitative structure-activity relationship study on some ligands acting as inhibitors of benzodiazepine-receptor binding.

A quantitative analysis of binding affinities of ligands active at adenosine receptors.

Inhibitors of blood platelet cAMP phosphodiesterase: a QSAR analysis.