competitive antagonist. For example, diphenhydramine exhibits clinically significant levels of H, antihistaminic, anticholinergic, and a-adrenergic blockage, CNS depression, and (at least topically) local anesthetic activity. It would be difficult to accept that diphenhydramine is chemically related to agonists as different as histamine, acetylcholine, norepinephrine, and y-aminobutyric acid. Acceptance of the accessory-binding concept would increase our understanding of structural incongruity of agonists and competitive antagonists as well as existence of such multifaceted competitive antagonists as diphenhydramine. The breadth of action may be due in part to the fact that diphenhydramine is such a flexible molecule. This may give it the capability to interact hydrophobically with, or accommodate itself to, different accessory sites with facility even though the sites obviously differ. Decreasing this high degree of conformational freedom by the introduction of an ortho methyl group on one of the two aromatic ring brings about a 15-fold decrease in antihistaminic activity while simultaneously increasing anticholinergic activity by an almost equivalent factor of 16. The much bulkier tert-butyl group practically eliminates antihistaminic action while maintaining high anticholinergic activity. Complete loss of conformational flexibility is obtained with the cyclic analog nefopam, which exhibits a dramatic increase in skeletal muscle relaxation ability while showing a 90-fold decrease in antihistaminic activity and an 83% decline in anticholinergic character (Fig. 1-13). What we may be seeing here is the fact that increased rigidity increases the possibility of a better (accessory) receptor fit and with it better selectivity of action.
The illustration of diphenhydramine and acetylcholine competition indicates an overlap in that the positively charged nitrogen atoms are assumed to bind the same anionic site on the cholinergic receptor, while the rings interact hydrophobically at a neighboring accessory site. In other situations no such overlap need exist. A competitive antagonist occupying only an accessory site or sites may perturb the agonist receptors sites sufficiently to alter affinities, much as allosteric site substrate binding on enzymes can alter catalytic
AH- 0 07; AC-18; MR * 2*5 AH-0 011; AC - 0-17; MR - 20
Figure 1-13. Decreased conformational freedom of diphenhydramine activity. AH, antihistaminic; AC, anticholinergic; MR, muscle relaxant activity (see text).
activity. In either case, it can be seen that a competitive antagonist can interfere with normal access by the agonist to its receptor sites.
The allosteric receptor is closely related to receptor sites, and it is modeled by analogy to a similar concept in enzymology. Here we visualize the agonist and antagonist binding to separate, nonoverlapping receptor sites that nevertheless affect each other's affinity for their respective ligands in an allosteric manner. Thus a mutual exclusion of binding ability results. Interest is in that situation where the antagonists binding to an allosteric site prevents the agonist from doing so at its receptor. It would, of course, be very difficult to differentiate experimentally between accessory and allosteric competitive interactions. It should be pointed out that whether a particular receptor will be activated or deactivated by these binding interactions is not predictable from these ideas.
From the foregoing discussion, it should not be assumed that competitive antagonists never exhibit structural similarity to agonists involved in affecting physiological functions. A prominent exception to the "rule" is in the field of opiate analgetics, where it will be seen that morphine antagonists show very great structural likeness to morphine and its congeners (Chapter 5), yet at the same time the endogenous ligands for these so-called opiate receptors are peptides, called enkephalins, which are quite dissimilar.
As the receptor model discussed to this point evolved deficiencies, it also required modifications. While maintaining the idea that drug-receptor interactions result in activation of the receptor from a nonactivated (resting) state R to an activated state R*, the concept was expanded to state that an equilibrium exists between these two states prior to any interactions with ligands. It is proposed that agonist molecules exhibit affinity only for the activated state and will not bind to the resting state. Similarly, antagonists will not bind to the activated receptor state; rather, they will only interact with the "relaxed" receptor state. Furthermore, the agonist by its presence will bind to the R* state and will also shift the equilibrium to it, whereas competitive antagonists will displace the equilibrium toward R. Partial agonists are assumed to have affinity for both types of binding sites. Figure 1-14 schematically summarizes the two-state receptor model.
The ratio of affinities for R* and R needs to be determined to evaluate the intrinsic activity of a partial agonist. This, then, would also indicate the fraction of the receptors in the activated form when sufficient partial agonist is present to saturate all sites. It should be pointed out in considering this two-state model that the two receptor states for agonists and competitive antagonists are assumed to be unrelated (i.e., they each interact only with their putative ligands).12
To test the validity of the proposal that in the dual-receptor model the sites for the agonist and corresponding antagonist are actually distinct, experiments to reductively alkylate muscarinic acetylcholine receptors in neural membranes with N-ethylmaleimide, a sulfhydryl-group blocking agent for proteins were carried out. This action distinctly altered the affinity of the receptor preparation toward cholinergic agonists, yet affinity toward antagonists remained unaffected. Other experiments were able to destroy membrane sensitivity of the cholinergic receptor population in the electroplax of the electric eel with
12 For a detailed description and kinetic derivation, see Triggle (1978) and Ariens and Rodrigues De Miranda (1979).
Figure 1-14. Simplified two-state receptor scheme. /?*, activated receptor state; R, nonactivated state; Ag, agonist; At, antagonist; P-Ag, partial agonist.
dithiothreitol, which is a reducing agent. This desensitization could be prevented by prior treatment with cholinergic agonists. The potent antagonist d-tubocurarine, however, could not protect the receptors against this effect.
An additional factor that should be brought into our thinking about receptors is their mobility within the bilayer membrane structure. Many membrane glycoproteins, presumably including those that are components of receptors that bridge this double layer, could therefore respond to effects on the outside of the cell by effecting, at least transiently, alterations inside the cell. Such transmembrane effects by the nicotinic acetylcholine receptor on the endplate region of skeletal muscle cells were demonstrated. Following acetylcholine release at the motor neuron innervation point and its interaction with the receptor protein, a pore forms across the membrane (for several milliseconds) and allowed a cation flux through it. At some point sufficient ions migrate by this mechanism to depolarize the muscle cell until an action potential results. It can be stated that the effect of acetylcholine on a receptor induces a microfluctuation in the current passing across the membrane. Cooperative receptor interactions involving rapid movement of membrane proteins across a two-dimensional matrix can now be visualized. This type of receptor mobility is conceivably involved in various aspects of cell behavior.
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