Synthesis and structure-activity relationship studies in peripheral benzodiazepine receptor ligands related to alpidem

The exploration of the structure-affinity relationships concerning a new class of peripheral benzodiazepine receptor (PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure-affinity relationships among different classes (and subclasses) of PBR ligands. The target amide derivatives were prepared following a previously published procedure based on the condensation of pyrrolo[3,4-b]quinoline derivatives 11a,b with glyoxylic acid mono-hydrate and the subsequent amidation of the acids obtained via mixed anhydride. On the other hand, the preparation of compound 9g lacking the pharmacophoric (delta1) carbonyl group involved: (a) the double sequential attack of the dimethylmethyleneammonium salt obtained from bis(dimethylamino)methane and acetyl chloride to pyrrolo[3,4-b]quinoline derivative 11b, (b) the quaternization of the obtained allylamine derivative 13 with methyl iodide, and (c) the palladium-catalyzed allylation of N-methyl-p-anisidine by quaternary allylammonium cation 14. The structure-affinity relationship trends observed in this subclass of tricyclic alpidem-related PBR ligands find correlations in other classes (or subclasses) of PBR ligands. This result supports the initial pharmacophoric hypothesis and suggests a common mode of interaction at the PBR binding site.     

Quantitative structure-activity relationship studies on 1-aryl-tetrahydroisoquinoline analogs as active anti-HIV agents

Predictive quantitative structure-activity relationship analysis was developed for a diverse series of recently synthesized 1-aryl-tetrahydroisoquinoline analogs with anti-HIV activities in this study. The conventional 2D-QSAR models were developed by genetic function approximation (GFA) and stepwise multiple linear regression (MLR) with acceptable explanation of 94.9% and 95.5% and good predicted power of 91.7% and 91.7%, respectively. The results of the 2D-QSAR models were further compared with 3D-QSAR model generated by molecular field analysis (MFA), investigating the substitutional requirements for the favorable receptor-drug interaction and quantitatively indicating the important regions of molecules for their activities. The results obtained by combining these methodologies give insights into the key features for designing more potent analogs against HIV.     

Identification and structure-activity studies of novel ultrashort-acting benzodiazepine receptor agonists

The synthesis and evaluation of novel ultrashort-acting benzodiazepine (USA BZD) agonists is described. A BZD scaffold was modified by incorporation of amino acids and derivatives. The propionate side chain of glutamic acid tethers an enzymatically labile functionality where the metabolite carboxylic acid displays markedly reduced BZD receptor affinity. The USA BZDs were characterized by full agonism profiles. Copyright2000 Elsevier Science Ltd.     

Quantitative structure-activity relationship studies on 5-phenyl-3-ureido-1,5-benzodiazepine as cholecystokinin-A receptor antagonists

Quantitative structure-activity relationship (QSAR) studies on a series of 5-phenyl-3-ureido-1,5-benzodiazepine-2,4-diones has been carried out using a pool of distance-based topological indices. Step-wise regression analysis indicated that penta-parametric regression expression containing Sz, B, Ip1, Ip2 and Ip3 is the most potent and selective for CCK-A affinity. The predictive potential of the model is discussed on the basis of cross-validation parameters as well as by estimating root mean square (RMSR) of the residuals.     

Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro-pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (K(D1)=33+/-7 nM, B(MAX1)=1.6+/-0.3 pmol/mg protein, K(D2)=720+/-250 nM and B(MAX2)=7.8+/-4.0 pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1,2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500 microM. In the presence of 100 microM CTZ (K(I(CTZ))=60+/-6 microM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: K(D)=24+/-4 nM, B(MAX)=12.0+/-0.1 pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([3H](S)F-Willardiine: K(D)=140+/-19 nM, B(MAX)=2.9+/-0.5 pmol/mg protein; [3H]NBQX: K(D)=111+/-34 nM, B(MAX)=12+/-3 pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.     

Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria.

Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E.coli. The bacterial [3H]diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli [3H]diazepam binding are those that are active in displacing [3H]benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.     

Quantitative structure-activity relationship studies on some anticancerous, antiviral and cytostatic agents

QSAR studies using molecular connectivity and van der Waal volume have been performed on a new series of hydroxyguanidine derivatives and a series of isoindolediones. Regression analysis has shown that anticancer and antiviral activity of hydroxyguanidines as well as cytostatic activity of isoindolediones correlate well with both the structural parameters.     

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.     

Aging: Effect on neuronal and non-neuronal benzodiazepine binding sites

The frontal cortex, hippocampus, and cerebellum of the Fischer 344 rat were examined for an age-dependent change in neuronal and non-neuronal binding. Clonazepam and Ro5-6669 displaceable [3H]diazepam binding were used as indicators of [3H]diazepam binding on neuronal and non-neuronal membranes, respectively. In both the frontal cortex and the hippocampus, clonazepam displaceable [3H]diazepam binding in the senescent rat was significantly less than the young and mature rat. In the frontal cortex, Ro5-6669 did not significantly displace [3H]diazepam binding in any age group. The Ro5-6669 displaceable [3H]diazepam binding in the hippocampus was not altered with age. In the cerebellum clonazepam and Ro5-6669 displaceable binding in the old rat was significantly less and more, respectively, compared to the young rat.     

Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites.

Inspection of the amino acid-base interactions in protein-DNA complexes is essential to the understanding of specific recognition of DNA target sites by regulatory proteins. The accumulation of information on protein-DNA co-crystals challenges the derivation of quantitative parameters for amino acid-base interaction based on these data. Here we use the coordinates of 53 solved protein-DNA complexes to extract all non-homologous pairs of amino acid-base that are in close contact, including hydrogen bonds and hydrophobic interactions. By comparing the frequency distribution of the different pairs to a theoretical distribution and calculating the log odds, a quantitative measure that expresses the likelihood of interaction for each pair of amino acid-base could be extracted. A score that reflects the compatibility between a protein and its DNA target can be calculated by summing up the individual measures of the pairs of amino acid-base involved in the complex, assuming additivity in their contributions to binding. This score enables ranking of different DNA binding sites given a protein binding site and vice versa and can be used in molecular design protocols. We demonstrate its validity by comparing the predictions using this score with experimental binding results of sequence variants of zif268 zinc fingers and their DNA binding sites.     

Quantitative structure-activity relationship study on some nonpeptidal cholecystokinin antagonists.

A quantitative structure-activity relationship (QSAR) analysis has been performed on a series of 1,4-benzodiazepine derivatives, which were found to act as antagonists of cholecystokinin (CCK), a gastrointestinal peptide hormone. The CCK acts with three different receptor subtypes termed as CCK-A, CCK-B, and gastrin receptor, which can be found in peripheral system, brain, and stomach, respectively. With all the three subtypes, the binding of the compounds is found to significantly depend on the lipophilicity of the compounds and their ability to form the hydrogen bonds with the receptor. However, the binding sites in CCK-A receptor seem to be slightly rigid as compared to those in CCK-B or gastrin receptor. The latter two appear to have similar binding features.     

Quantitative structure-activity relationship studies on cyclic cyanoguanidines acting as HIV-1 protease inhibitors

A quantitative structure-activity relationship study has been performed on some cyclic cyanoguanidines that inhibit the enzyme HIV-1 protease (HIV-1-PR) and exhibit antiviral potency, and the results have been compared with those of cyclic urea derivatives. Both the enzyme inhibition activity and antiviral potency in cyclic cyanoguanidines as well as in cyclic urea derivatives are found to be primarily governed by hydrophobic property of substituents attached to nitrogen (P2/P2') and further enhanced by OH or NH2 group, if any, present in the substituents. However, aromatic substituents are found to be unfavourable to both the activities of cyclic cyanoguanidines but not to any activity of cyclic urea derivatives. Cyclic urea derivatives are indicated to be more potent than cyclic cyanoguanidines. A model for the interaction of cyclic cyanoguanidines with the receptor is proposed.     

Uniform regulation of central and peripheral benzodiazepine binding sites by GABA receptor agonists in rats

Central and peripheral benzodiazepine binding sites were studied in vitro after the administration of GABAA and GABAB agonists. Cerebral cortex and kidney homogenates were used in this study. Muscimol (1.5 mg/kg, intraperitoneally) pretreatment significantly increased the affinity of benzodiazepine binding sites not only in the cerebral cortex but also in the kidneys. Similar changes were obtained with (-) and (+) baclofen (5 mg/kg, intraperitoneally), with the only exception that (-) baclofen in addition to changes in the affinity caused a marked decrease in the number of binding sites in both structures studied. The mechanism of action of GABA agonists on peripheral benzodiazepine binding sites is discussed.     

Effect of picrotoxinin on benzodiazepine receptor binding

The effect of picrotoxinin on [³H]flunitrazepam binding to benzodiazepine receptors was investigated. In mouse forebrain membranes, picrotoxinin inhibited basal, GABA- and pentobarbital-stimulated [³H]flunitrazepam binding; this inhibitory activity was temperature- and chloride ion-dependent. Scatchard analysis of the data indicates that picrotoxinin decreases the number of binding sites without alterating binding affinity. In cerebellar membranes, picrotoxinin did not alter [³H]flunitrazepam receptor binding.     

Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites

The statistics of base-pair usage within known recognition sites for a particular DNA-binding protein can be used to estimate the relative protein binding affinities to these sites, as well as to sites containing any other combinations of base-pairs. As has been described elsewhere, the connection between base-pair statistics and binding free energy is made by an equal probability selection assumption; i.e. that all base-pair sequences that provide appropriate binding strength are equally likely to have been chosen as recognition sites in the course of evolution. This is analogous to a statistical-mechanical system where all configurations with the same energy are equally likely to occur. In this communication, we apply the statistical-mechanical selection theory to analyze the base-pair statistics of the known recognition sequences for the cyclic AMP receptor protein (CRP). The theoretical predictions are found to be in reasonable agreement with binding data for those sequences for which experimental binding information is available, thus lending support to the basic assumptions of the selection theory. On the basis of this agreement, we can predict the affinity for CRP binding to any base-pair sequence, albeit with a large statistical uncertainty. When the known recognition sites for CRP are ranked according to predicted binding affinities, we find that the ranking is consistent with the hypothesis that the level of function of these sites parallels their fractional saturation with CRP-cAMP under in-vivo conditions. When applied to the entire genome, the theory predicts the existence of a large number of randomly occurring "pseudosites" with strong binding affinity for CRP. It appears that most CRP molecules are engaged in non-productive binding at non-specific or pseudospecific sites under in-vivo conditions. In this sense, the specificity of the CRP binding site is very low. Relative specificity requirements for polymerases, repressors and activators are compared in light of the results of this and the first paper in this series.     

Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.