Targets of drug action.

This article considers early work from the author's laboratory on muscarinic receptor specificity, subtypes, and conformational variability, with the use of nuclear magnetic resonance in pharmacology and the conformational variants of dihydrofolate reductase and general questions of receptors. It also considers some current approaches to drug development and receptor function, particularly as influenced by increasing knowledge of three-dimensional structure of receptors.

The action of acetylcholine and other drugs on the efflux of potassium and rubidium from smooth muscle of the guinea-pig intestine. 1968.

1. A method is described for measuring continuously the efflux of potassium or rubidium from smooth muscle of the guinea-pig. 2.

Some considerations of receptor specificity.

Since the amino acid sequences for several muscarinic and adrenergic receptors are known, an attempt was made to correlate side chain hydrophobicity surrounding the aspartate anionic groups with binding structure-activity relationships. No positive correlation was found, suggesting that secondary binding effects of head group substituents are non-local. Displacement of aspartate ionization is unlikely to be due to such neighbour effects.

The effects of agonists on the components of the cardiac muscarinic receptor.

1 The binding of eleven agonists to muscarinic receptors in the rat heart has been measured in competition with [3H]-N-methylscopolamine. 2 Full analysis of binding required the resolution of three components (SH, H and L). 3 The proportion of the H component was independent of agonist structure.

The effect of ionic strength on cardiac muscarinic receptors.

The binding of N-methylscopolamine (NMS) and carbachol to muscarinic receptors in the rat heart has been measured as a function of ionic strength (mu). The binding of NMS was reduced by 3.69 fold for a 10 fold increase in ionic strength.

The effect of sulphydryl block on the binding of H1-antagonists to the muscarinic receptor.

The treatment of brain muscarinic receptors by p-chlormercuribenzoate is known to change structure-activity relationships for antagonists. The changes in binding affinity of H1-antagonists for this receptor have been measured; the changes are similar to those for intrinsic muscarinic antagonists.

Trimethoprim binding to bacterial and mammalian dihydrofolate reductase: a comparison by proton and carbon-13 nuclear magnetic resonance.

The binding of trimethoprim to dihydrofolate reductase from L1210 mouse lymphoma cells has been studied by measuring the changes in chemical shift of nuclei of the ligand that accompanying binding. The 6- and 2',6'-proton chemical shifts of bound trimethoprim have been determined by transfer of saturation experiments, and the 2-carbon chemical shift has been determined by using [2-13C]trimethoprim. The changes in proton chemical shift are substantially smaller than those accompanying binding to bacterial dihydrofolate reductase [Cayley, P.

Ab initio molecular orbital calculations of electron distribution in tetramethylammonium ion.

Ab initio molecular orbital calculations of electron distribution in tetramethylammonium ion and its uncharged isoelectronic analogue, neopentane, have been carried out. Comparison of the two compounds permits a detailed description of the delocalization of the positive charge of tetramethylammonium ion. The van der Waals surface of this ion is found to be characterized by "patches" of positive charge associated with the methyl groups, interspersed with essentially neutral regions.

The effect of p-chloromercuribenzoate on structure-binding relationships of muscarinic receptors in the rat cerebral cortex.

Muscarinic receptors in the rat cerebral cortex, reacted with p-chloromercuribenzoate (PCMB) under different conditions (Phase I and II), have modified binding sites. These exhibit remarkable changes in the structural dependence of the binding of drugs. In Phase I, the structure-binding profile of agonists for both the high and low affinity agonist sites are altered.

The effects of p-chloromercuribenzoate on muscarinic receptors in the cerebral cortex.

The action of p-chloromercuribenzoate (PCMB) on the ligand binding properties of the muscarinic receptors in the rat cerebral cortex has been examined. At low concentrations, PCMB produces a selective change in the binding of agonists without any effect on the binding of antagonists. At higher concentrations, the structure-binding profile for binding antagonists is changed.

Modification of the binding properties of muscarinic receptors by gallamine.

The interaction of gallamine with muscarinic receptors from different tissues has been investigated. Gallamine binds to a site distinct from the conventional muscarinic ligand binding site and modulates the binding of agonists and antagonists to the conventional binding site. In agreement with reported pharmacological studies, the effects of gallamine on the binding of muscarinic ligands are much greater in heart than in other tissues.

Use of transferred nuclear Overhauser effect measurements to compare binding of coenzyme analogues to dihydrofolate reductase.

Transferred nuclear Overhauser effect measurements have been made on complexes of NADP+ and thioNADP+ with Lactobacillus casei dihydrofolate reductase to provide information about the glycosidic bond conformations in these complexes. Both NADP+ and thioNADP+ are shown to have very similar anti conformations about their adenine glycosidic bonds when bound to the enzyme. However, their nicotinamide glycosidic bond conformations are very different: while NADP+ binds in an exclusively anti conformation, thioNADP+ binds with a distribution of syn/anti conformations very similar to that observed in nicotinamide mononucleotides in free solution (approximately 50:50).

The effect of McN-A-343 on muscarinic receptors in the cerebral cortex and heart.

McN-A-343 behaves as a competitive agonist in binding to muscarinic receptors in the cerebral cortex. In its interaction with myocardial muscarinic receptors it is not competitive but it retains features of agonist binding.

Solubilisation and molecular characterisation of muscarinic acetylcholine receptors.

Stable, soluble preparations of rat brain muscarinic receptors can be prepared by extracting membranes with digitonin, or with combinations of sodium cholate and sodium chloride. The stability of the cholate/NaCl extract is enhanced by the addition of egg phosphatidylcholine, which, at the same time, suppresses the considerable dispersity apparent in the hydrodynamic behaviour of the solubilised receptor. The Stokes radius of the brain muscarinic receptor in cholate/NaCl/lecithin extracts is 6.

The binding properties of the muscarinic receptors of the cynomolgus monkey ciliary body and the response to the induction of agonist subsensitivity.

1 The binding properties of the muscarinic receptors in the ciliary muscle of cynomolgus monkeys have been evaluated. 2 The concentration of receptor binding sites is the highest yet reported. As found in many species and tissues, there are subclasses of agonist binding sites.

Hydrogen-1, carbon-13, and phosphorus-31 nuclear magnetic resonance studies of the dihydrofolate reductase-nicotinamide adenine dinucleotide phosphate-folate complex: characterization of three coexisting conformational states.

The Lactobacillus casei dihydrofolate reductase-folate-NADP+ complex is shown by 1H and 13C NMR to exist in three interconverting conformational states, I, IIa, and IIb. The proportions of the three states, as estimated from the intensities of the three separate 13C resonances observed in the complex containing [3-carboxamido-13C]NADP+, are pH dependent. State I predominates at low pH and states IIa and IIb predominate at high pH; the ratio IIa:IIb is pH independent.

The theory, methods and relevance of muscarinic receptor binding studies.

The development of receptor-specific ligands, radiolabelled to high specific activity has made possible the investigation of the binding of anti-muscarinic compounds to the muscarinic receptor in vitro. Binding of these compounds satisfies the criteria of saturability, and specificity for the muscarinic receptor, and their potencies for binding correlate with their potencies in inhibiting muscarinic responses in vivo. Whilst analysis of the binding data for 'classical' muscarinic antagonists indicate that they bind to a single, uniform population of binding sites within a given tissue, some anti-muscarinic compounds have been found to bind selectively to receptors in different tissues.

Negative cooperativity between folinic acid and coenzyme in their binding to Lactobacillus casei dihydrofolate reductase.

The binding of folinic acid (5-formyl-5,6,7,8-tetrahydrofolate) to Lactobacillus casei dihydrofolate reductase has been measured. The natural 6S, alpha S diastereoisomer has a binding constant of 1.3 (+/- 0.

Conformational changes and drug action.

Conformational changes induced or selected by drugs in receptors may be coupled to second sites on receptor molecules giving negative or positive cooperative binding. The structure-activity relationships may be changed depending on the predominant conformation. Some conformational changes can be identified by nuclear magnetic resonance spectroscopy.