Mechanisms and kinetics of drug receptor interaction
There are several typical mechanisms of action that apply to the different types of receptor proteins. For enzymes, these are
• Competitive inhibition: The drug occupies the active site and prevents binding of the physiological substrate. Example: The inhibition of angiotensin convertase by enalapril.
• Irreversible (covalent) inhibition: The drug again binds to the active site of the enzyme and then covalently reacts with it, so that the active site becomes irreversibly blocked. Example: Inhibition of cyclooxygenase by acetylsalicylic acid.
• Allosteric inhibition: The drug binds outside the active
site but prevents the enzyme from adopting its active conformation. Example: Inhibition of Na+/K+-ATP'ase by digitoxin or digoxin.
The allosteric behaviour seen with many enzymes is also typically observed with ion channels and metabolic recep-tors. In the absence of physiological agonists, these proteins typically prefer their inactive conformation; channels will be closed, and metabolic receptors will not stimulate their downstream cascades. The physiological agonists act al-losteric activators, promoting conversion to the active state. Drugs acting on these targets typically belong to one of the following classes:
• Reversible agonists (activators), i.e. the drug mimics the physiological agonist. Example: Isoproterenol, an agonist at β-adrenergic receptors.
• Reversible inhibitors: The drug, typically in a competitive way, prevents binding of the physiological agonist. Example: Propranolol, an antagonist at β-adrenergic re-ceptors.
• Reversible partial agonists: The drug has activity intermediate between that of an inhibitor and an agonist. Ex-ample: Dobutamine, a partial agonist at β-adrenergic re-ceptors. Partial agonists may be used for their agonistic properties or their antagonistic properties.
• Irreversible (covalent) inhibitors. This case is less common than reversible inhibition or activation. Example: Phenoxybenzamine, an antagonist at α-adrenergic re-ceptors.
With few exeptions, all drugs we are going to consider in the rest of this course will fall into one of the above cat-egories.
In the simplest possible case, one effector molecule, which may be either the physiological agonist or a drug, will bind to one target molecule, and all target molecules will bind the effector with the same affinity. It is noteworthy that there are numerous deviations from this simple situation1. Nevertheless, we will confine ourselves to this simple mod-el, which will still take us to some important conclusions.
With the above assumptions, the binding will be subject to the law of mass action, and a single parameter – the disso-ciation constant, typically called K – will describe the inter-action. K will be an empirical value, depending on both the ligand and the receptor molecule in question. K is inversely related to the affinity; the higher it is, the lower the binding affinity. The law of mass action can be rearranged to give us the receptor occupancy, i.e. the fraction of all receptors saturated with the ligand (Figure 3.1a). You will recognize the formal similarity to Michaelis-Menten enzyme kinetics. Accordingly, if we plot the receptor occupancy as a func-tion of the ligand concentration, we get the same hyperbolic type of curve (Figure 3.1b, top).
Shown are three curves, differing in their respective values for K. The bottom panel shows that plotting the same num-bers on a logarithmic scale for the ligand yields nice sig-moidal plots, which are now distinguished solely by their parallel offsets along the x-axis. From these plots, K can be determined as the ligand concentration of half-maximal receptor occupancy.
If a drug activates its receptor, it simply assumes the role of the ligand in the above model, albeit its affinity will most likely differ from that of the physiological ligand. What we can see, then, is that very little benefit can be expected from increasing the drug concentration beyond, say, five times its K value, since the receptor will already be saturated. The only thing that will happen upon further increase is that secondary, less affine and specific sites will be bound, potentially evoking unwanted side effects.
If the drug is an inhibitor, we are dealing with a ternary sys-tem of receptor, physiological agonist, and our inhibitory drug. We will examine two cases: Reversible competitive inhibitors (Fig. 3.2, top) and irreversible ones (Fig. 3.2, bottom).
If a drug does not undergo a covalent reaction with its re-ceptor, binding will almost always be reversible2. There fore, the total number of functional receptor molecules will not change, but we now have two linked, competing equi-libria squeezed into the same pool. This gives rise to a mod-ified relationship of receptor occupancy to ligand concen-tration, as stated and illustrated in Figure 3.3. Again, the situation is entirely analogous to reversible inhibition in Michaelis-Menten kinetics3, and you may want to consult your biochemistry textbook for the derivation – or just do it yourself, as an exercise.
An important aspect of competitive inhibition is that, with sufficiently high concentrations of physiological ligand, the receptor can still be maximally activated. Competitive inhibition thus reduces the receptor's sensitivity to the ago-nist but does not diminish the maximum effect that can be attained at very high agonist concentrations. This means that, in case of an accidental overdose of the inhibitor, the endogenous agonist or a drug that mimics it could be used to overcome the inhibition.
If a drug undergoes a covalent reaction with its receptor, the receptor molecules affected will be irreversibly blocked and thus altogether removed from the total receptor pool available for the interaction with the agonist. Thus, the agonist-receptor equilibrium now plays out in that reduced total pool. The number of occupied receptors will therefore be proportionally reduced (Figure 3.4).
For an experimental illustration of the foregoing, let us look at the inhibition of α-adrenergic receptors. These re-ceptors are stimulated by epinephrine and norepinephrine; stimulation will increase the tension of blood vessel walls and therefore enhance blood pressure. α-Adrenergic re-ceptors are very numerous in the spleen. The spleen has a sponge-like structure and stores about half a litre of blood, which upon adrenergic stimulation will get squeezed out into the circulation4. This extrusion of blood is effected by the contraction of smooth muscle cells that are embedded in the spleen tissue. Accordingly, if we take a fresh slice of spleen and bathe it in solutions of mediators or drugs, we can measure its mechanical tension to quantify the extent of α-adrenergic stimulation. Figure 3.5a shows the force of contraction developed by such spleen strips in response to varying concentrations of norepinephrine, in the presence of tolazoline or phenoxybenzamine, respectively. By com parison to the theoretical plots above (Fig. 3.3, 3.4), you will be able to decide which of the two inhibitors is the re-versible one, and which is the covalent one.
Let us consider the molecular principles behind the two modes of inhibition. Fig. 3.5b shows the structures of the agonist (norepinephrine) and of the two inhibitors. With some imagination, one can spot the similarity between agonist and inhibitors, so that it is understandable that they all bind to the same site on the α-adrenoceptor. Tolazoline has no obvious reactive groups, and it will therefore bind non-covalently and reversibly.
Phenoxybenzamine, on the other hand, has a chloroethyl group (indicated in red) attached to the nitrogen that is quite reactive. It will undergo the reactions depicted in Figure 3.5c. The initial step results in the formation of an ethylen-imine group, which is quite reactive because of the ring tension. In a second step, after binding to the receptor, the ring is opened by some nucleophile, most probably the SH group of a cysteine5 that is part of the receptor molecule. In this way, the drug becomes covalently attached to the recep-tor and permanently inactivates it.
Several things are notable about the action of phenoxyben-zamine:
• The initial circularization (formation of the aziridine ring) is rather slow, causing the pharmacological action to lag behind the plasma levels. On the other hand, re-ceptor blockade will persist long after any excess drug has been eliminated. With most drugs that act by non-covalent association with their receptors, plasma levels correlate much more closely with the intensity of drug action.
• While the benzylamino moiety of phenoxybenzamine (blue in Figure 3.5b) targets it to the α-adrenoceptor, the chemical reactivity of the ethyleneimino group is rather non-selective and will cause molecules not bound to the receptor to react in random locations, potentially causing harm including genetic damage. Accordingly, phenoxybenzamine is not the drug of first choice in most clinical indications of α-adrenoceptor blockade.
Phenoxybenzamine is the drug of choice in one particu-lar disease called phaeochromocytoma. This is a tumour of the adrenal glands that produces and intermittently releas-es very large amounts of epinephrine and norepinephrine, causing dangerous spikes in blood pressure. The superior effect of phenoxybenzamine in phaeochromocytoma is a direct consequence of its covalent mode of binding: The inactivated receptor cannot be reactivated by whatever amounts of hormone released (cf. Figure 3.5a). In contrast, reversible inhibition could be overridden in this particular situation.
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