David J. Triggle

High affinity binding of a calcium channel antagonist to smooth and cardiac muscle

Abstract Specific binding of the Ca 2+ -channel antagonist nitrendipine, a close structural analog of nifedipine, has been measured in microsomal membrane fractions from guinea pig ileal longitudinal smooth muscle. The dissociation constant was 0.18 nanomole per liter and maximum binding was 1.14 picomoles per milligram of protein. Binding with very similar characteristics was found in a rat ventricle preparation. This high affinity binding was sensitive to displacement by a series of 1,4-dihydropyridine analogs of nifedipine with an activity sequence correlating well with that determined for inhibition of mechanical responses in the intact smooth muscle.

The pharmacology of ion channels: with particular reference to voltage-gated Ca2+ channels.

Ion channels are molecular machines that serve as principal integrating and regulatory devices for the control of cellular excitability. They are also major targets for drug action. The basic aspects of ion channel structure and pharmacological control are reviewed and illustrated with specific reference to a major class of therapeutic agents and molecular tools--the clinically available Ca2+ channel antagonists.

Benzodiazepines and calcium channel function

Abstract Although the neuronally mediated pharmacological and therapeutic effects of benzodiazepines exerted through central sites are well defined, there is evidence for other sites of action, including peripheral sites, whose functions are less clear. A role for benzodiazepines as regulators of Ca 2+ channel activity has been suggested for both centrally and peripherally acting benzodiazepine ligands. David Rampe and David Triggle review this evidence. Although the properties of the several receptor sites indicate clearly their separate character, the benzodiazepines obviously modulate some Ca 2+ channel processes. Such observations may provide a clue to new structures active at one or more of the several classes of Ca 2+ channels or may indicate a new category of benzodiazepine interaction.

Binding of [3H]nimodipine to cardiac and smooth muscle membranes

Specific binding of [3H]nimodipine to membranes from rat ventricle and guinea pig ileal longitudinal smooth muscle was studied. Dissociation constants were 0.24 and 0.12 nM, and the maximal number of binding sites were 0.4 and 0.75 pmol/mg protein for cardiac and smooth muscle, respectively. The values obtained for both types of muscle were similar to those obtained for [3H]nitrendipine binding, as were the potencies of a series of dihydropyridines for competing with [3H]nimodipine. These results support the hypothesis that the binding site characterized is that mediating the pharmacological effects of these compounds.

Stereoselectivity of drug action

Chirality is a fundamental property of biological systems and reflects the underlying asymmetry of matter. Drug- receptor interactions have long been known to be stereo- selective, and it is increasingly recognized that both pharmacodynamic and pharmacokinetic events contribute to the overall clinically observed stereoselectivity. The importance of this phenomenon has now been explicitly recognized by drug regulatory agencies who are issuing guidelines for drug development. This review outlines some of the scientific issues surrounding drug stereo- selectivity, with particular emphasis being paid to drug inter- actions at ion channels where, because of state-dependent interactions, stereoselectivity is a moving target.

New ligands for L-type Ca2+ channels

Although the clinically important categories of drug represented by verapamil, nifedipine and diltiazem are defined as acting at three major and discrete sites on the L class of voltage-dependent Ca2+ channel, it is likely that some new classes of drug modulate channel activity by acting at additional sites. David Rampe and David Triggle describe the actions of some of these new drugs, which may both offer improved therapeutic and side-effect profiles over existing agents and provide information to define further the structure and function of this channel class. These drugs may mimic the actions of endogenous ligand(s) and such ligands could provide new directions for Ca2+ channel drug structures.

Effect of thyroid status on \-adrenoceptors and calcium channels in rat cardiac and vascular tissue

Fig. 1. Mean concentration-response curves for the positive inotropic responses of rat papillary muscles to (A) isoproterenol, (B) calcium, (C) Bay K 8644. Tissues were taken from euthyroid (untreated) (O), hypothyroid ( 0 ) and hyperthyroid (A) animals. Responses to isoproterenol and calcium are plotted as an increase in tension expressed as a percentage of the maximal increase. Responses to Bay K 8644 were plotted as a percent of the maximal response to isoproterenol (Iso). Vertical bars represent SE (n = 7 9)

Regulation by chronic drug administration of neuronal and cardiac calcium channel, beta-adrenoceptor and muscarinic receptor levels

Chronic administration of atropine (40-100 mg/kg, 23 days) produced a 29-33% increase in muscarinic receptors, measured by [3H]quinuclidinyl benzilate binding, in rat brain. Diisopropyl phosphorofluoridate (0.9 mg/kg, 14 days) produced a 35% decrease in muscarinic receptors. Propranolol administration (800 micrograms/kg/hr, 10 days) increased beta-adrenoceptors, measured by [3H]dihydroalprenolol binding, by 69 and 50% in brain and heart respectively. Isoproterenol administration (800 micrograms/kg/hr, 10 days) produced a 50% reduction in cardiac beta-adrenoceptors but did not alter brain receptors. These drug treatments were without effect on binding of the Ca2+ channel ligands, [3H]nimodipine and [3H]nitrendipine, to brain or heart respectively. However, chronic administration of nifedipine for 20 days (36 and 360 micrograms/kg/hr) did produce down-regulation of both cardiac and neuronal Ca2+ channels and a similar down-regulation of beta-adrenoceptors. Co-regulation of Ca2+ channels and neurotransmitter receptors may occur but may not be an automatic consequence of either receptor or channel regulation.

The chemist as astronaut: Searching for biologically useful space in the chemical universe

Chemical space whether defined by small molecules or large proteins is larger than can be usefully explored. One of the challenges of drug discovery is thus the definition of the overlap between chemical space, biologically useful space and pharmacological space and how this may be employed in the discovery of new small molecule drugs. Despite the decrease in drug discovery productivity in recent years there is no shortage of targets for small molecule intervention, including stroke, pain, neurodegenerative diseases, inflammation and bacterial and viral infections. Only an extremely small fraction of available chemical space has thus far been explored and it is likely that prior synthetic constraints and bias to existing frameworks and scaffolds have contributed to this. Several approaches are being employed to explore more fruitful paths to discovery. These include recognition that existing therapeutic entities already occupy validated pharmacological space and thus are good leads, the use of molecular fragments that permits a broader exploration of chemical space, and the role of templates that permit fragments to combine to generate active species. Finally, a new focus on natural product-like scaffolds from both synthetic methodologies and the genetic reengineering of biosynthetic pathways is likely to prove valuable. However the exploration of chemical space will alone not solve the current deficit in drug discovery productivity. It is necessary to recognize that cellular environments are not the dilute homogeneous solutions of many screening systems and that a more integrated systems approach will serve to maximize any success of chemical space exploration.

Binding of a 1,4-dihydropyridine calcium channel activator, (-) S bay K 8644, to cardiac preparations

The 1,4-dihydropyridine Ca2+ channel activator, (-) [3H]Bay K 8644, binds to cardiac membranes and polarized [5 mM K+] and depolarized [50 mM K+] cardiac cells. Binding to microsomal membranes at 25 degrees C indicates a single set of binding sites, KD = 2.9 x 10(-9) M and a site density, 337 fmoles/mg protein, not different from that measured by antagonist 1,4-dihydropyridines. Binding to neonatal rat myocytes at 37 degrees C was independent of membrane potential with a KD value of 5 x 10(-8)M and a site density, 63 fmoles/mg protein, not significantly different from that measured by PN 200 110. These results indicate that 1,4-dihydropyridine activators and antagonists label the same number of binding sites in cardiac tissue, but that activator binding to intact myocytes is voltage-independent.