Evidence to support a spectrum of active states for the glucagon receptor.

The ternary complex model suggests that G-protein-coupled receptors resonate between inactive (R) and active (R*) forms. Physiologically, R sites ordinarily predominate with a few R* sites giving rise to basal activity. Agonists recognize, stabilize and increase the R* population, thus altering intracellular activity. There is evidence to suggest the possibility of a spectrum of conformations between R and R*. Our aim is to study the consequences of putative GR (glucagon receptor)-activating mutations using glucagon and partial agonist des-His(1)-[Glu(9)]glucagon amide (glucagon-NH(2)). Alanine substitution in TM (transmembrane) helix 2 of Arg(173) or of His(177) detrimentally affected glucagon and glucagon-NH(2) response maxima. TM2 receptor mutant, Phe(181)-Ala, displayed reduced maximum cAMP accumulation in response to glucagon-NH(2). Thr(353)-Cys (TM6) and Glu(406)-Ala (TM7) receptors demonstrated constitutive activity and enhanced EC(50) values for glucagon-NH(2); Arg(346)-Ala (TM6) and Asn(404)-Ala (TM7) receptors were activated by sub-fmol glucagon concentrations, yet were not constitutively active and demonstrated wild-type receptor-like EC(50) values for glucagon-NH(2). Unlike Arg(346)-Ala receptors, Thr(353)-Cys, Asn(404)-Ala and Glu(406)-Ala receptors demonstrated improved EC(50) values for glucagon, whereas their maximal responses to and their affinity for glucagon were comparable with the wild-type receptor. In contrast, despite slightly reduced glucagon-NH(2) affinity, Arg(346)-Ala, Thr(353)-Cys, Asn(404)-Ala and Glu(406)-Ala receptors displayed glucagon-NH(2) response maxima that exceeded those seen for wild-type receptors. Interestingly, we observed biphasic glucagon-mediated signalling responses. Our results are consistent with the concept of different agonists promoting the formation of distinct active states from partially active R*(low) to fully active R*(high) forms.

Stepwise modulation of neurokinin-3 and neurokinin-2 receptor affinity and selectivity in quinoline tachykinin receptor antagonists.

A stepwise chemical modification from human neurokinin-3 receptor (hNK-3R)-selective antagonists to potent and combined hNK-3R and hNK-2R antagonists using the same 2-phenylquinoline template is described. Docking studies with 3-D models of the hNK-3 and hNK-2 receptors were used to drive the chemical design and speed up the identification of potent and combined antagonsits at both receptors. (S)-(+)-N-(1-Cyclohexylethyl)-3-[(4-morpholin-4-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 25, SB-400238: hNK-3R binding affinity, K(i) = 0.8 nM; hNK-2R binding affinity, K(i) = 0.8 nM) emerged as the best example in this approach. Further studies led to the identification of (S)-(+)-N-(1,2,2-trimethylpropyl)-3-[(4-piperidin-1-yl)piperidin-1-yl]methyl-2-phenylquinoline-4-carboxamide (compound 28, SB-414240: hNK-3R binding affinity, K(i) = 193 nM; hNK-2R binding affinity, K(i) = 1.0 nM) as the first hNK-2R-selective antagonist belonging to the 2-phenylquinoline chemical class. Since some members of this chemical series showed a significant binding affinity for the human mu-opioid receptor (hMOR), docking studies were also conducted on a 3-D model of the hMOR, resulting in the identification of a viable chemical strategy to avoid any significant micro-opioid component. Compounds 25 and 28 are therefore suitable pharmacological tools in the tachykinin area to elucidate further the pathophysiological role of NK-3 and NK-2 receptors and the therapeutic potential of selective NK-2 (28) or combined NK-3 and NK-2 (25) receptor antagonists.

Regioselective hydroxylation of debrisoquine by cytochrome P4502D6: implications for active site modelling.

1. Debrisoquine, a prototypic probe substrate for human cytochrome P4502D6 (CYP2D6), is hydroxylated at the alicyclic C4-position by this enzyme. Phenolic metabolites of debrisoquine (5-, 6-, 7- and 8-hydroxydebrisoquine) have also been reported as in vivo metabolites, but the role of CYP2D6 in their formation is unclear. 2. As part of studies to develop a predictive model of the active site of CYP2D6 using pharmacophore and homology modelling techniques, it became important to determine the precise regioselective hydroxylation of debrisoquine by CYP2D6. 3. Data from studies with human liver microsomes and yeast microsomes containing cDNA-derived CYP2D6 demonstrated unequivocally that debrisoquine was hydroxylated by CYP2D6 at each aromatic site in the molecule, as well as at the alicyclic 4-position. The four phenolic metabolites amounted to > 60% of the total identified products and the pattern of regioselective hydroxylation (4-HD > 7-HD > 6-HD > 8-HD > 5-HD) was similar in both in vitro systems. 4. A pharmacophore model for CYP2D6 indicated that while the hydroxylation of debrisoquine at alternative positions could arise from the substrate adopting multiple binding orientations, the energy constraints for the aromatic hydroxylations were unfavourable. An alternative proposal involving essentially a single binding orientation and a mechanism of hydroxylation based on benzylic radical spin delocalization could satisfactorily rationalize all the hydroxylations of debrisoquine. 5. This latter proposal demonstrates the need to consider the mechanism of oxidation as well as the spatial orientation of the substrate in the development of a predictive model of the active site of CYP2D6.

Biarylcarbamoylindolines are novel and selective 5-HT(2C) receptor inverse agonists: identification of 5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]- 5-pyridyl]carbamoyl]-6-trifluoromethylindoline (SB-243213) as a potential antidepressant/anxiolytic agent.

The evolution, synthesis, and biological activity of a novel series of 5-HT(2C) receptor inverse agonists are reported. Biarylcarbamoylindolines have been identified with excellent 5-HT(2C) affinity and selectivity over 5-HT(2A) receptors. In addition, (pyridyloxypyridyl)carbamoylindolines have been discovered with additional selectivity over the closely related 5-HT(2B) receptor. Compounds from this series are inverse agonists at the human cloned 5-HT(2C) receptor, completely abolishing basal activity in a functional assay. The new series have reduced P450 inhibitory liability compared to a previously described series of 1-(3-pyridylcarbamoyl)indolines (Bromidge et al. J. Med. Chem. 1998, 41, 1598) from which they evolved. Compounds from this series showed excellent oral activity in a rat mCPP hypolocomotion model and in animal models of anxiety. On the basis of their favorable biological profile, 32 (SB-228357) and 40 (SB-243213) have been selected for further evaluation to determine their therapeutic potential for the treatment of CNS disorders such as depression and anxiety.

A molecular mechanism for toxin block in N-type calcium channels.

A series of highly toxic snail venoms, the omega-conotoxins, have been shown to bind selectively, and often irreversibly to the N-type voltage-gated calcium channel alpha-1 subunit. The most potent of these is known as omega-conotoxin GVIA from the species Conus geographus, a marine snail that has been responsible for a number of human fatalities. Using theoretical techniques we present a plausible binding model of the conotoxin to a loop region of the channel. Our model of the toxin binding region also contains a possible EF-hand motif and we suggest that this Ca2+ binding domain lies on the ion permeation pathway, a possible Ca2+ recruitment site.

Novel and selective 5-HT2C/2B receptor antagonists as potential anxiolytic agents: synthesis, quantitative structure-activity relationships, and molecular modeling of substituted 1-(3-pyridylcarbamoyl)indolines.

The synthesis, biological activity, and molecular modeling of a novel series of substituted 1-(3-pyridylcarbamoyl)indolines are reported. These compounds are isosteres of the previously published indole urea 1 (SB-206553) and illustrate the use of aromatic disubstitution as a replacement for fused five-membered rings in the context of 5-HT2C/2B receptor antagonists. By targeting a region of space previously identified as sterically allowed at the 5-HT2C receptor but disallowed at the 5-HT2A receptor, we have identified a number of compounds which are the most potent and selective 5-HT2C/2B receptor antagonists yet reported. 46 (SB-221284) was selected on the basis of its overall biological profile for further evaluation as a novel, potential nonsedating anxiolytic agent. A CoMFA analysis of these compounds produced a model with good predictive value and in addition good qualitative agreement with both our 5-HT2C receptor model and our proposed binding mode for this class of ligands within that model.

The selective 5-HT1B receptor inverse agonist 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydro- spiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289) potently blocks terminal 5-HT autoreceptor function both in vitro and in vivo.

5-HT1 receptors are members of the G-protein-coupled receptor superfamily and are negatively linked to adenylyl cyclase activity. The human 5-HT1B and 5-HT1D receptors (previously known as 5-HT1Dbeta and 5-HT1Dalpha, respectively), although encoded by two distinct genes, are structurally very similar. Pharmacologically, these two receptors have been differentiated using nonselective chemical tools such as ketanserin and ritanserin, but the absence of truly selective agents has meant that the precise function of the 5-HT1B and 5-HT1D receptors has not been defined. In this paper we describe how, using computational chemistry models as a guide, the nonselective 5-HT1B/5-HT1D receptor antagonist 4 was structurally modified to produce the selective 5-HT1B receptor inverse agonist 5, 1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2, 4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6, 7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine] (SB-224289). This compound is a potent antagonist of terminal 5-HT autoreceptor function both in vitro and in vivo.

Sonographic measurement of diaphragmatic displacement during tidal breathing manoeuvres - a reliability study.

The aim of this investigation was to establish a reliable method of measuring diaphragm movement during relaxed tidal breathing. This study follows a previous study in which test-retest reliability was established for maximal breaths but not for tidal breaths (Blaney and Sawyer 1997). Twelve normal subjects were tested in a semi-reclined long sitting position. Sonographic measurements were taken for tidal breaths during four different patterns of breathing. All breaths were controlled for inspired volume. Testing was repeated one week later under the same conditions. The results showed that the method for measuring diaphragmatic movement at tidal breathing during a given pattern of breathing was reliable. This method will allow further investigation of the diaphragm during different breathing manoeuvres at tidal volume.

Synthesis, biological activity, and molecular modeling of selective 5-HT(2C/2B) receptor antagonists.

The synthesis and biological activity are reported for a series of analogues of the previously published indole urea 2 (SB-206553), designed to probe the 5-HT(2C) receptor binding site. Small molecule modeling studies have been used to define a region in space which is allowed at the 5-HT(2C) receptor but disallowed at the 5-HT(2A) receptor. In a complementary approach, docking of 2 into our model of the 5-HT(2C) receptor has allowed us to propose a novel primary binding interaction for this series of diaryl ureas, involving a potential double hydrogen-bonding interaction between the urea carbonyl oxygen of the ligand and two serine residues in the receptor. The difference of two valine residues in the 5-HT(2C) receptor for leucine residues in the 5-HT(2A) receptor is believed to account for the observed 5-HT(2C)/5-HT(2A) selectivity with 2.

A group program for obese, infertile women: weight loss and improved psychological health.

We report on a group program for obese infertile women. Sixty-four women completed the 24-week program, which included exercise, information about healthy eating and group discussion sessions. Their mean initial weight was 101.9 +/- 18.14 kg. The mean weight loss on completion of the program was 5.2 +/- 5.11 kg (p < 0.0001). There was significant improvement on ratings of self-esteem and depression. Changes to life-style and health which are known to improve fertility may be a useful precursor to invasive, high technology infertility treatment procedures.

Improved pregnancy rates for obese, infertile women following a group treatment program. An open pilot study.

Thirty-seven women with a mean initial weight of 98.5 +/- 18.7 kg completed a group treatment program for obese, infertile women. The program lasted for 24 weeks and included regular exercise and group discussion of topics such as coping with the psychological impact of infertility, developing healthy eating patterns, and the effects of obesity on reproductive physiology. There was significant weight loss (mean weight loss 6.2 +/- 4.5 kg, p < 0.001) and improvement on measures of self-esteem, anxiety, depression, and general health. Twenty-nine women became pregnant during the follow-up period (21-36 months). Two women were avoiding pregnancy, so only six who had completed the group program and wished to become pregnant had not conceived by the end of the follow-up period. A further five women did not complete the program as they became pregnant while attending the group. Our results suggest that active measures to improve mood and self-esteem, along with better nutrition and weight reduction through diet and exercise, can produce considerable improvement in the outcome of treatment for infertility in obese women.

Models of ion pores in N-type voltage-gated calcium channels.

Two computer models of the outer vestibule of the pore of the N-type voltage-gated Ca2+ channel are predicted. The models are constructed from beta-hairpin peptide segments in the S5-S6 loops of each of the four domains that produce the channel. These hairpins together are modeled to form a short eight-stranded beta barrel. The models contain a ring of glutamates at the base of the barrel, which have been shown by mutagenesis experiments to function as a selectivity filter. These filters are suggested by the models to be of the correct dimensions to allow the permeation of a hydrated calcium ion, where the filter glutamates may substitute for molecules of water from the hydration shell of the ion. The models also suggest that a ring of threonines and an aspartate might be present between the mouth of the pore and the filter, and hence the models may prove useful in suggesting future mutagenesis experiments.

Weight loss results in significant improvement in pregnancy and ovulation rates in anovulatory obese women.

Obesity can affect ovulation and the chances of pregnancy. In this prospective study, a weight loss programme was assessed to determine whether it could help infertile overweight anovulatory women to establish ovulation and assist in achieving pregnancy, ideally without further medical intervention. The subjects acted as their own historical controls. They underwent a weekly programme of behavioural change in relation to exercise and diet over 6 months; those who did not complete the 6 months were treated as the comparison group. Women in the study group lost an average of 6.3 kg, with 12 of the 13 subjects resuming ovulation and 11 becoming pregnant, five of these spontaneously. Fitness, diet and psychometric measurements all improved. Fasting insulin and testosterone concentrations dropped significantly, while sex hormone binding globulin concentrations rose. None of these changes occurred in the comparison group. Thus, weight loss with a resultant improvement in ovulation, pregnancy outcome, self-esteem and endocrine parameters is the first therapeutic option for women who are infertile and overweight.

Molecular surface comparison. 2. Similarity of electrostatic vector fields in drug design.

In the first article of this series a real-time graphics method was described for molecular similarity of scalar properties. This has now been extended for the comparison of molecular vector properties, most notably electrostatic field. A comparison of the various techniques of calculating fields is presented that includes a new method based on natural orbital fitted point charges. In the two examples described, namely, a series of benzodiazepine agonists and a set of serotonin 5-HT3 antagonists, the program has been shown to produce useful pharmacophoric overlaps that can be used in the design of novel therapeutic agents.

5-HT2 receptor subtypes: a family re-united?

The current classification for 5-HT2 receptors accommodates three subtypes. In addition to the originally defined 5-HT2 receptor, sanctuary is now provided for the structurally related 5-HT1c receptor (now 5-HT2c) and at least one atypical 5-HT receptor subtype. The strong functional union of this family is reflected in the paucity of ligands that will discriminate between its subtypes and prompts some re-evaluation of the activities of compounds which may now be regarded as nonselective for the receptor subtypes in this class. In this article, Gordon Baxter and colleagues examine the pharmacology of both officially recognized and atypical 5-HT2 receptor subtypes. A number of novel selective agents are highlighted, some of which may prove useful for 5-HT2 receptor classification and, ultimately, clarify the mechanistic basis for current and future therapeutic strategies which target this receptor family.

MAMBAs: a real-time graphics environment for QSAR.

MAMBAs (Multivariate Analysis Methods in Biomechanistic Activity Studies) is an integrated workstation-based graphics program designed for the investigation of quantitative structure activity relationships (QSAR). It combines many of the commonly used statistical techniques with an extensive database of substituent constants, a variety of molecular and substituent property calculations and detailed graphics-based table and graph editors. Graphical representations of standard substituent generation and optimization techniques are also included. These are all utilized within a state-of-the-art real-time graphics environment.

Molecular surface comparison: application to drug design.

An interactive system for the display and manipulation of molecular surface properties is presented. The property at the molecular surface is mapped onto the sphere by gnomonic projection. This representation allows direct comparison of the surface properties of pairs of molecules. The system allows the user to explore the similarities between a pair of molecules in an interactive manner, and provides extensive visual (color-coded field and field difference maps) and numerical (rms difference value) aids to complement the user's chemical intuition. Examples of the use of the system to study beta-lactam compounds and phosphodiesterase inhibitors are presented.

Conformational studies on (+)-anatoxin-a and derivatives.

Anatoxin-a (AnTX) is a highly potent agonist acting at the nicotinic acetylcholine receptor (nAChR) and represents a valuable tool in the study of this receptor. Molecular mechanics, semi-empirical and ab initio molecular orbital energy minimization procedures were conducted to investigate the conformation of AnTX. For each minimization procedure, the s-trans enone isomer of protonated AnTX was the energetically favoured conformer due to intramolecular electrostatic interactions. Our studies are discussed in the light of previous experimental observations and conformational studies, in addition to their importance in the development of future pharmacophore models for nAChR agonist binding.

Comparison of azabicyclic esters and oxadiazoles as ligands for the muscarinic receptor.

The link between the cognitive deficit associated with Alzheimer type dementia and the loss of cholinergic function in the disease provides a basis for examining muscarinic agonists as potential therapeutic agents. This paper describes the design and synthesis of novel azabicyclic methyl esters as ligands for the muscarinic receptor. Replacement of the methyl ester by a 3-methyl-1,2,4-oxadiazole ring produces potent metabolically more stable muscarinic agonists capable of penetrating the central nervous system. These compounds generally show improved affinity relative to the corresponding methyl esters. 3-Methyl-1,2,4-oxadiazole 7b has an affinity 4 times that of acetylcholine. Receptor affinity is discussed in relation to the size and geometry of the azabicyclic ring and the electronic properties of the heteroaromatic ring.