Académie royale de Médecine de Belgique


Texte Everardus J. Ariens, correspondant étranger

(Séance du 31 janvier 1981)


by Everardus J. ARIËNS (Nijmegen), Correspondant étranger.

A bioactive agent - pharmaco or drug - can only induce a pharmacodynamic effect in a biological object as the resultant of an interaction between its molecules and certain molecules in the Biological object. An understanding of drug action therefore requires a molecular approach. The chemical properties of the drug are determinant for its action and activity. Thus a relationship between chemical structure and action must exist. The sequence of processes at the basis of drug action can be divided into three main phases : the pharmaceutical phase comprising the release processes of the active drug from the dosage form, and thus determining the concentration available for absorption (the pharmaceutical availability); the pharmacokinetic phase comprising the processes that play a part in the absorption, distribution, metabolic conversion and excretion of the drug, and thus determining the concentration of the active agent at the site of action in the target tissue (the biological availability); the pharmacodynamic phase comprising the molecular interaction between the active agent and its sites of action, which initiates the biological effect. The concept of particular molecules, indicated as receptors, and serving as sites of action form an indispensable concept in the understanding g of drug action. Their characteristics such as chemical properties are still largely unknown. Analogously to the differentiation between the active site on an enzyme and the enzyme molecule as a whole, it makes sense to distinguish between the receptor site and the receptor molecule - the receptor. Receptors my be isolated but not the receptor sites.

Structure-action relationship is based on the interaction between the drug molecule and the receptor site. Binding studies may give information on the properties of this site and on the number and location of receptor molecules in particular tissues.

Drug-receptor interaction is much more dynamic than the classical lock-and-key concept suggests. It is actually based on intermolecular forces, mutually molding drug and receptor. Therewith conformational changes are induced which imply a receptor activation which triggers the sequence of biochemical and biophysical events leading to the effect. Although dynamic, the receptors can yet be regarded as preformed structural entities since, for instance, the activities of optical isomers of bioactive agents often largely differ. 

Pharmaco-receptor Interaction and Receptor Activation :

Bioactive agents may be extremely potent. Then only a few molecules of the active agent have to interact with their receptors to induce a massive response in which tremendous numbers of molecules are involved. This requires amplifier mechanisms. Schematically drug action can be represented by : the input signal (drug concentration), binding to the specific receptor site (the discriminator), resulting in receptor-activation and thus generation of a stimulus which then is conveyed (receptor-effector coupling) to the effector system where it is amplified and transduced to the effect measured, the output signal. The interpretation of the characteristics of dose-effect curves and time-effect curves on basis of the receptor concept boil down to some form of application of the mass-action law. A variety of partially overlapping theoretical models have been worked out. The following sequence of reversible reactions presents a condensate covering essential aspects of these models in the most general way. The symbols represent : D drug; R receptive receptor; R activated receptor; K1, K2, K3, K4 and K5 are defined in such a way that Ki = K-i/Ki. They govern the relative amounts of the different receptor states. Receptor states DR (Ariëns et al. 1964).

In the "hit and run-activation" model it is assumed that the activated state of the receptor exclusively exists in the non-complexed form, R. The affinity and intrinsic activity, parameters indicating the concentration at which 50 % of the maximal effect of a particular drug is obtained and the maximal response obtained after saturation of the system with the drug respectively are operational parameters. In the equations given they boil down to terms composed of rate constants. In a group of related agents the full agonists (high intrinsic activity) give the highest maximal response, partial agonists (intermediate intrinsic activity) give although the receptor system is saturated, only a fraction of that response. Only a fraction of the receptors occupied is activated then. Agents with an intrinsic activity zero but still an affinity for the receptors, behave as competitive antagonists of the agonists. In the "occupation-activation" model, which is most feasible, K3 << K4. For agonist K-1 << K2 and for competitive antagonists k-1 >> k2. Dependent on the response induced by a certain dose of a full agonist addition of increasing concentrations of a partial agonist will result in a synergism or antagonism. At high concentrations the partial agnostic takes over all receptors such as the curves converge to a response equal to the maximal response obtainable with the partial agonist (Ariëns et al, 1977).

Amplifier Systems and Spare Receptors : 

The simplest amplifier unit for assembling strong amplifier systems is the allosteric activation of a proenzyme by binding of the activator, possibly a drug molecule, to its receptor sites on the enzyme molecule. One activated enzyme molecule can convert hundreds of substrate molecules to product molecules. Such an amplifier unit can be coupled to a second one if the product molecules in their turn serve as activators of a second type of enzyme molecules. A sequence of such enzyme activation steps results in a tremendous amplification. Such an amplifier system is involved, for instance, in the action of various hormones and drugs that act by activation or adenylate-cyclase. This enzyme converts ATP to cyclic AMP, which in its turn is an enzyme activator. Amplifier systems have interesting implications. The quantities of intermediate product in an amplifier unit (e.g. cyclic AMP) that can be maximally generated in case of maximal activation of the enzyme (adenylate cyclase) by the drug may be larger than that required to saturate the receptor sites for cyclic AMP on the next amplifier unit. This implies then that the drug needs to activate only a fraction of its receptors to obtain a maximal response from the effector system. There is a spare capacity for the receptors (Ariëns, 1979). The fraction of the receptors that can be irreversibly blocked without a reduction of the maximal effect obtainable is a measure for this spare capacity. For full agonists still differ in their intrinsic activity as far as the induction of the stimulus is concerned. The intrinsic activity on this level is also indicated of the compound (Stephenson, 1956).

The spare capacity is composed of contributions therein of various amplifier units. The characteristics of the dose-effect curves therefore may differ if, in the sequence of events initiated by the receptor activation in the effector system different end-points are chosen as effect, since then the number of amplifier units involved may differ. So, for instance, ACTH analogues showing a low intrinsic activity in the dose-effect curves for the cyclic AMP generation, appear to be practically full agonists in the curves for the generation of the end-product cortisone, both measured on the same receptor-effector system (adrenal cortex tissue) (Seelig et al., 1973). The order of the intrinsic actives to the various compounds in the series do not depend on the end-point chosen. Clear correlations between receptor occupancy and effect have been demonstrated extensively if adenylate cyclase activity is taken as an end-point. This in contrast to more distal responses where usually the number of amplifier steps and therewith the spare capacity for receptors differ (Catt et al, 1977).

A spare capacity for receptors implies that an irreversible blockade of a certain fraction of the receptors is possible without a reduction in the maximal response obtained. The degree of receptor elimination that is tolerated before the maximal response is reduced elimination that is tolerated before the maximal response is reduced is a measure for the reserve capacity. For partial agonists the maximal response requires full receptor saturation, there is no spare capacity, such that receptor elimination always results in a reduction in the maximal response then (Ariëns et al., 1964).

Chemial Structure and Action : 

Agonists and their competitive antagonists are assumed to act on common receptor sites and thus are expected to be chemically complementary to these sites. They therefore should show a chemical relationship. This indeed holds true for various metabolites and antimetabolites, hormones and anti hormones, vitamins and anti-vitamins, etc…, but this is definitely not the case for compounds such as acetylcholine, competitive antagonists, anticholinergics,  -adrenergic blocking agents, H1-antihistaminics, and antiserotonins (Ariëns et al., 1979).

Structure-activity relationship studies in a series of compounds in which acetylcholine, histamine, and norepinephrine are gradually converted by chemical manipulation into their competitive antagonists reveal that with gradual change in structure the affinity of the agonist derivative for its receptor decreases until a hydrophobic double-ring system is introduced. This results in a strong increase in the affinity and indicates that in the competitive antagonists obtained the moiety representing the structure of the original agonist interacts only loosely with the receptor, while the hydrophobic moiety is tightly bound.

Information on this point can be obtained by introduction of a center of asymmetry in e.b. the choline moiety and/or in the acyl moiety. If a high degree of complementarity between the moiety concerned and the receptor is required, a large ratio for the activities of the stereoisomers may be expected. Moiety of acetylcholine, concerting this drug to acetyl- -methylcholine, results in two isomers which show a large 1/d ratio for the activities is found to be close to 1. In the anticholinergic compound benzilyl-methylcholine, the choline part of the molecule does not behave as a critical moiety anymore (Ellenbrook et al.,1965).

A further possibility in the introduction of two centers of asymmetry in the potent anticholinergic esters : one in the choline and one in the ring bearing moiety. The phenylcyclohexyl glycolic acid ester of  b-methylcholine is an example (Ellenbrook et al., 1965; Ariëns et al., 1967). As a consequence four different isomers are obtained. It will be clear that the two hydrophobic rings impossibly can bind on the receptor site for acetylcholine itself. Accessory binding sites are involved. The results show that for the pairs of isomers differing in the configuration of the choline moiety a low activity ratio is found; for the pairs differing in the configuration of the ring bearing moiety, large activity ratios are observed (Ellenbrook et al., 1965; Ariëns et al., 1967). This again indicate that in these anticholinergic agents the interaction of the ring bearing moiety with an "accessory receptor area" is of greater importance that the interaction of the choline moiety with the cholinergic receptor. As studies indicate, stereoselectivity as observed in dose-response curves correlates well with that observed in binding studies (Beld et al., 1974).

Binding Constants for Agonists and Antagonists : 

A receptor reserve implies that the apparent affinity constants of the agonists derived from dose-response curves (acetylcholine and metacholine plus ou moins 10-7 mol) are flattered as compared to the binding constants obtained in binding studies (plus ou moins 3 x 10-5 mol) (Beld et al., 1974). For the competitive antagonists, as expected, the constants do not differ in this respect. Binding constants for a specific antagonist, e.g. atropine, measured in different tissues do not differ which indicates identity of these receptor systems (Beld et al., 1975). A further remarkable aspect is that binding constants measured for agonists (methacholine 10-5 mol) are usually much lower than those of the competitive antagonists (atropine 2.6 x 10-9 mol, benzetimide 4.2 x10-10 mol). Such a phenomenon is also observed for other groups of drugs, such as histamine and H1-antihistamines. This may be due to the fact that in agonist binding - which implies induction of conformational changes in the receptor protein and thus activation thereof - a fraction of the total binding energy is consumed as activation energy, indicated as "productive" binding energy. The binding constants represent the total binding energy, the "non-productive" (binding-stabilizing) energy. In case of competitive antagonists no receptor activation takes place such that the binding constant then represents the total binding energy (Franklin, 1980). This may also solve the riddle on the high and low affinity binding sites observed in binding studies for agonists but not observed for competitive antagonists (Kent et al.,1980; Beld 1980). If a fraction of the receptor molecules in the membrane preparation is not coupled to the effector molecules,no receptor activating energy may be involved even in agonist binding such that for that fraction of the receptors the total binding energy is manifested in the binding constant (the hight affinity binding). Agonist binding to effector-coupled receptors requires activation energy with as a consequence a low affinity binding. Receptor desensitization, if based on receptor elimination, may result in a relative increase in the fraction of effector-coupled receptors and therewith an increase in the procent of receptors involved in high affinity binding without changing the affinity constants (Kent et al.,1980).

Receptor Differentiation : 

In the foregoing different receptor-states for one type of receptors were discussed. Another possibility is different types of receptors for one type of agonist like a and b-adrenergic receptors and H1- and H2-histamine receptors. For each of these receptor types specific blockers are available, a-and b-blockers and H1- and H2-blockers. This receptor differentiation is correlated with the type of receptor-effector coupling. This is based on activation of adenylate-cyclase activation and generation of the second messenger cyclic AMP, in case of the b-adrenergic and H2-histaminergic receptors and a mobilization of calcium as second messenger in case of the b-adrenergic and H1-histaminergic receptors. A further type of receptor differentiation is that between b1- and b2- adrenergic receptors representing the receptors for the neurotransmitter norepinephrine, predominantly involved in circulatory homeostasis and the receptors for the hormone epinephrine involved in the "fight, fright and flight" reaction (Ariëns et al, 1976). Also for other messengers such as dopamine (Calne, 1980) and the endorphines (opiate receptors) such differentiation are reported.

Receptor Regulation and Receptor Pathology : 

There is a good deal of analogy between enzymology and receptorology. In both cases the macromolecules involved fundamentally underlie the dynamics of life. In the case of enzymes, the interaction between "substrate" and active site, implies a mutual molding on the basis of intermolecular forces such that the substrate molecule is activated and chemically converted. In the case of receptors, the interaction of the messenger with the receptor site results in an activation of the messenger with the receptors, the interaction of the messenger with the receptor site results in an activation of the receptor molecule, an activation conveyed to the effector system. In both cases agents are Known that bind to the corresponding sites but do not result in an activation; they are antimetabolites and receptor blockers respectively. 

Taken into account the highly complex inter- and intracellular communication, receptorology may well develop to a field as large and complex as enzymology. Examples of messengers are transmitters, mediators, hormones, enzyme activity regulators, repressors, derepressors, etc… Like for enzyme systems, also for receptor systems regulation in capacity takes place under the influence of various factors. Lasting exposure of a receptor system to its specific messenger or agonist usually results in a down-regulation of the capacity - a decrease in the number of receptors - these are homologous regulations. Up- and down-regulation in a certain receptor system can also take place by messengers acting on other receptor systems, heterologous regulation. The b-adrenergic receptor capacity, for instance, is expanded (up-regulation) under the influence of thyroxine acting on its own specific receptors, whereas the receptor capacity for oxytocin in the myometrium is decreased (down-regulation) under the influence of progestagens (Catt et al., 1977; Synder 1979 and Baxter et al., 1979). Like in enzymology where "inborn errors of metabolism" are known based on genetically determined deficiencies in the enzyme structure, also genetically determined "inborn errors of receptor function" are know, for instance for the androgen receptors (Griffin et al., 1980). Inborn errors of polypeptide messenger function are also reported, e.g. the biosynthesis of an abnormal nonfunctional insulin (Tager et al., 1979). Other examples of diseases based on pathological impaired interveullar communication (Rubinstein 1980) are the autoimmune diseases in which antibodies are formed against receptor proteins. The antibody-receptor interaction can result in a receptor blockade, e.g. in myasthenia gravis, or in receptor activation such as in the case of Graves disease in which thyroid-stimulating immunoglobulins are involved. Another example of involvement of receptors in pathology are the differences in receptor density of tumors e.g. estrogen receptors on mama tumor tissue, which appears be correlated with the therapeutic responsiveness (Henderson et al., 1980). 

The field of receptorology is rapidly expanding and, like enzymology some decades ago, gaining physiologial, pathological and clinical redevance. It opens intriguing perspectives for both fundamental and applied science.