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.

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 proportion of the SH component ranged from 2-36% of the total and was dependent on the agonist. The proportion of the L component varied in a complementary way from 59-22% of the total. 4 The ratios of the affinities of ten of the agonists to the three components of the receptors were constant; the weakest binding agonist, choline, had lower ratios of affinity. 5 When saturated with guanylylimidodiphosphate (GppNHp) the SH receptor population was no longer detectable and the H receptor population was reduced by 63%: about 85% of the receptors was in the L population. 6 The affinity constants of agonists for the H and L forms of the receptors were not changed by GppNHp. 7 The results are interpreted in terms of the effects of accessory proteins on the proportions of binding and non-binding conformations of the receptor.

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 binding of carbachol was affected in two ways. Firstly, the proportions of the subsites were changed. Above mu = 0.5 M, the superhigh (SH) subsite was converted into the low (L) subsite and above mu = 0.8 M, the high (H) subsite was also converted into the L subsite. Therefore, at high ionic strength, no agonist-determined subsites can be detected. In addition, increase in ionic strength reduced the binding of carbachol to all subsites and to a much greater extent than for NMS.

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. J., Albrand, J. P., Feeney, J., Robert, G. C. K., Piper, E. A., & Burgen, A. S. V. (1979) Biochemistry 18, 3886]. It is shown that this difference arises largely from the fact that trimethoprim adopts different conformations when bound to mammalian and to bacterial dihydrofolate reductase. The proton chemical shifts are interpreted in terms of ring-current contributions from the two aromatic rings of trimethoprim itself and the nearby aromatic amino acid residues of the enzyme. The latter have been located by using the refined crystallographic coordinates of the Lactobacillus casei and Escherichia coli reductases in their complexes with methotrexate [Bolin, J. T., Filman, D. J., Matthews, D. A. & Kraut, J. (1982) J. Biol. Chem. 257, 13650], under the assumption that, as indicated by the 13C chemical shifts, the diaminopyrimidine ring of trimethoprim binds in the same way as does the corresponding part of methotrexate. With use of these assumptions, the conformation of trimethoprim bound to the dihydrofolate reductases from L. casei, E. coli, and L1210 cells has been calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

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 consequences of this nonspherical charge distribution for interaction with anions have been explored by calculations of the interaction energy of fluoride ion with tetramethylammonium ion and neopentane in several mutual orientations. The lowest-energy orientation is found to be one in which the anion approaches a "face" of the tetrahedral cation (opposite to a C--N bond direction). The origins of this preference and the electron redistribution produced by the interaction with fluoride are discussed. The tetramethylammonium ion is clearly not a featureless positively charged sphere but will have appreciable geometrical specificity in its interaction with a presumed anionic group on the acetylcholine receptor.

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. The affinity of agonists is greatly reduced and the heterogeneity of binding eliminated. The effects of both high and low concentrations of PCMB can be reversed by dithiothreitol. Inactivation of receptors proceeds in parallel and is kinetically complex. It can only be partially reversed by dithiothreitol. Evidence is presented connecting the low affinity agonist binding site with the high affinity pirenzepine binding site. The changes produced by PCMB have been interpreted in terms of the modification of receptor conformation.

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. In Phase II, the structure-binding profile of antagonists is also observed. In Phase II, the ability of potent agonists to discriminate between sub-classes of agonist binding sites is eliminated. There is also a loss of heterogeneity in the binding of the selective antagonist pirenzepine. Of the 16 agonists examined, only pilocarpine has a heterogeneous binding profile in Phase II, the dispersity of binding being increased. The changes in binding properties of the receptors are discussed in terms of general theories of drug-receptor interactions.

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. These findings suggest the possibility of developing novel and selective muscarinic drugs.

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). Thus for thioNADP+, binding to the enzyme does not significantly perturb the potential function for rotation about the nicotinamide glycosidic bond. Earlier NMR studies [Hyde, E. I., Birdsall, B., Roberts, G. C. K., Feeney, J., & Burgen, A. S. V. (1980) Biochemistry 19, 3738] had indicated that large downfield 1H shifts of the nicotinamide ring protons (0.61-1.36 ppm) are detected on binding NADP+ while only very small shifts (less than 0.1 ppm) are observed in complexes with thioNADP+. The chemical shift and conformational findings are best explained if the thionicotinamide ring extends into solution making essentially no contacts with the enzyme.

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 6.7 nm, with very similar values in other detergents, including digitonin and sodium dodecyl sulphate. Its sedimentation coefficient is 3.78s, and its molecular weight approximately 110,000 after correction for detergent binding. The isoelectric point of the digitonin - solubilised receptor is approximately 4.5.

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. Agonist binding is not affected by the non-hydrolysable guanosine triphosphate (GTP) analogue, GppNHp, suggesting that these receptors are not linked to adenylate cyclase. 3 Ciliary muscles made subsensitive by treatment with muscarinic agonists have a decreased receptor concentration but no other changes in the binding properties of the receptors could be detected.

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 pH dependence of the interconversion of states I and IIa + IIb can be explained by a model in which a group on the enzyme has a pK of less than 5 in state IIa + IIb and greater than 7 in state I. 1H, 13C, and 31P NMR has been used to characterize the structural differences between the three states of the complex. As judged by the 1H and 13C chemical shifts of the bound coenzyme, states I and IIa are similar to one another but quite different from state IIb. This difference appears to be a localized one, since only the nicotinamide 2 and 4 protons, nicotinamide 3-carboxamide 13C, and pteridine 7 proton show differences in chemical shift between these states. These differences are, however, large--up to 1.4 ppm for 1H and 2 ppm for 13C. The remaining coenzyme protons, as well as the three 31P nuclei, are unaffected. Studies of the C2 proton resonances of the seven histidine residues show that the ionizable group responsible for the interconversion of states I and IIa + IIb is not a histidine (although two histidines show slight differences in environment between states IIa and IIb); the possible identity of this ionizable group and the nature of the conformational differences between the states are discussed.