This material is available at http://www.mc.uky.edu/pharmacology/mtp_research.asp

Background information can be found in chapters 1 and 2 of the Golan text and in chapter 1 of the Yagiela text.

Christopher Thomas is a retired musician. On several visits over a period of a year you note his blood pressure is 142/97, 138/98, 147/99, and 135/95. He has a history of smoking and has chronic bronchitis. He also has elevated lipid and cholesterol levels. You wish to begin therapy with an antihypertensive medication. There are several choices of drugs which can be prescribed in this situation. Two of the potential choices are prazosin, which is a competitive alpha-receptor blocker, and propranolol, which is a competitive beta-receptor antagonist. You choose to prescribe prazosin 1 mg b.i.d. The next day Mr. Thomas calls and complains that he took his medication and shortly thereafter became dizzy and passed out.

Consider the following questions which you will be able to answer after completion of the lecture sequences listed above:

Questions:

What are the various drug classes which can be used to treat hypertension?

What are the pharmacologic and nonpharmacologic approaches to the therapy of hypertension?

What is the agonist blocked by prazosin?

Where are the receptors prazosin blocks located and why does this cause hypotension?

What factors in this case made prazosin a better choice than propranolol?

Why did Mr. Thomas faint and should this be a cause for concern?

Receptor: Any cellular macromolecule that a drug binds to initiate its effects.

Factors Governing Drug Action

Two factors that determine the effect of a drug on physiologic processes are affinity and intrinsic activity.

Affinity is a measure of the tightness that a drug binds to the receptor.

Intrinsic activity is a measure of the ability of a drug once bound to the receptor to generate an effect activating stimulus and producing a change in cellular activity.

Affinity and intrinsic activity are independent properties of drugs. Agonists have both affinity, that is, the ability to bind to the receptor, as well as intrinsic activity, the ability to produce a measurable effect. Antagonists, on the other hand, only have affinity for the receptor. This property allows antagonists to bind to the receptor. However, because antagonists do not have intrinsic activity at the receptor no effect is produced. Because they are bound to the receptor, they can prevent binding of agonists. This is a diagram of a G-protein coupled receptor. Notice how the amino acids that make up the receptor protein can contribute functional groups to allow a drug to bind to this receptor.

The binding of a drug to a receptor is determined by the following forces:

1. Hydrogen bonds
   
2. Ionic bonds
   
3. Van der Waals forces
   
4. Covalent bonds
   

Understanding Affinity

To bind to a receptor the functional group on a drug must interact with complementary surfaces on the receptor. The binding of a drug, illustrated here as D, to the receptor, illustrated as R, can be described by this expression.

 

By appropriate substitution of the equations above we can write:

This equation describes the binding of drugs to receptors and states that the amount of drug bound to the receptor is dependent on the drug concentration and Kd.

Question: WHAT percentage of the total receptor population will be occupied when the concentration of the drug is equal to the dissociation constant? Help  Solution

This points out that when a drug is given at a concentration equal to its dissociation constant, 50% of the receptors will be occupied. The greater the affinity, the less drug will be required to occupy 50% of the receptors.


Understanding the Consequences of Receptor Occupancy

where Emax is the maximal obtainable effect when all receptors are occupied. We can now write:

This equation states that the effect observed, E/Emax, is determined by the concentration of the drug and its affinity (Kd) for the receptor. In other words, the effect is related to the degree of receptor occupancy. This helps us to understand the extreme potency of some drugs. A drug with very high affinity will achieve a large degree of receptor saturation at very low concentrations.

Thus far the effect (E) of a drug has only been related to receptor occupancy. However, drugs once bound to a receptor differ in their ability to initiate a change in receptor conformation and physiologic activity. This is a more difficult parameter to conceptualize. Drug binding to receptors can be measured quite easily and is governed by relatively straightforward biochemical principles. The ability to activate the receptor and induce an effect encompasses much more than the simple chemical process of drug-receptor binding. Let us use the symbol, e to define intrinsic activity. Intrinsic activity describes the ability of a drug induce changes in receptor structure leading to alterations in cellular activity. We can now write:

Therefore, the ability of a drug to produce a physiologic effect is dependent on receptor occupancy (which is in turn governed by [D] and Kd) and the propensity of the drug to activate the receptor (e). While similar, you should understand that equations #1 and #2 calculate different parameters. Equation #1 determines the degree of receptor occupancy. Equation #2 (with the presence of e) calculates the effect of a drug on a functional response.

Question: AT WHAT concentration of agonist will the effect (E/Emax) be equal to 50 % of the maximal response? .Help  Solution:

This concentration is also referred to as the Effective Dose-50 or ED₅₀.

Full and Partial Agonists

While the precise mechanism is not known, agonists have the ability to impart a stimulus to the receptor such that cellular signaling is activated. Agonists differ in their propensity to deliver an activating stimulus to receptors. As a result, agonists can be further divided into full and partial agonists:

Full Agonists: Compounds that are able to elicit a maximal response following receptor occupation and activation.

Partial Agonists: Compounds that can activate receptors but are unable to elicit the maximal response of the receptor system.

Drugs which are full agonists are arbitrarily assigned an intrinsic activity value of 1. Partial agonists, which cannot produce the same maximal effect as full agonists will have intrinsic activity values less than 1. The effect of partial and full agonists on equation # 2 is apparent. Because partial agonists have e values less than 1, the value of E/Emax will be some fraction of the value obtained with a full agonist.

As illustrated below, the position and shape of the log-dose response curve is dependent on the affinity of the ligand for the receptor and its intrinsic activity. Affinity determines the position of the dose-response curve on the X-axis, while intrinsic activity affects the magnitude of the response.

	Norepinephrine and phenylephrine are full agonists with intrinsic activity values of 1. However, Norepinephrine has a higher affinity for the receptor. As is illustrated, affinity affects the position of the dose-response curve on the x-axis.
	Clonidine and Methoxamine are partial agonists. Clonidine has a higher affinity but a lower intrinsic activity than does Methoxamine. Intrinsic activity affects the magnitude of the response.

Thus far we have made the assumption that the relationship between receptor occupancy [DR]/[RT] and response E/Emax is linear. This linear relationship can be expressed by equation # 2 and is shown in the graph below. In this type of response system, all receptors must be occupied to produce a maximal response.

In most physiological systems in which drugs will be administered, the relationship between receptor occupancy and response is not linear but some unknown function f of receptor occupancy. In the graph, this unknown function is presented as being hyperbolic. As the graph depicts in this type of system, all receptors do not have to be occupied to produce a full response. Because of this hyperbolic relationship between occupancy and response, maximal responses are elicited at less than maximal receptor occupancy. A certain number of receptors are "spare." Spare receptors are receptors which exist in excess of those required to produce a full effect. There is nothing different about spare receptors. They are not hidden or in any way different from other receptors.

Assume an agonist with a K_D = 50 nM and an e=1.

A= High Receptor Reserve B=Medium Receptor reserve C=No Receptor Reserve

Antagonists exhibit affinity for the receptor but do not have intrinsic activity at the receptor. An antagonist that binds to the receptor in a reversible mass-action manner is referred to as a competitive antagonist. Because the antagonist does not have intrinsic activity, once it binds to the receptor, it blocks binding of agonists to the receptor. A key point about competitive antagonists is that like agonists, they bind in a reversible manner. This has important implications regarding the effect competitive antagonists have on the configuration of the dose-response curve of agonists. Because competitive antagonists bind in a reversible manner, agonists, if given in high concentrations, can displace the antagonist from the receptor and the agonist can then produce its effect. The antagonist action can, in effect, be surmounted. Because the antagonist can be completely displaced, the agonist is still able to produce the same maximal effect observed prior to antagonist treatment. However, because higher agonist concentrations were necessary to displace the antagonist, the agonist dose-response curve is shifted to the right in the presence of a competitive antagonist. This can be illustrated with two equilibrium equations:

The antagonist [B] and agonist [D] are competing for the same limited number of receptors [R]. The drug that binds to the receptor in the highest concentration will be determined by two factors.

Where [B] is the concentration of antagonist and Kb is the affinity exhibited by the antagonist for the receptor. Inspection of this equation will reveal that the affinity of the agonist, Kd, is modified by the term (1+[B]/Kb). If the concentration of antagonist [B] is large in relation to its affinity Kb, the term (1+[B]/Kb) will be large. Therefore, the major effect of an antagonist is to shift the dose-response curve for an agonist to the right. The dose-response curve obtained in the presence of a competitive antagonist is parallel to the dose-response curve obtained in the absence of antagonist. If the Kb is small and the concentration high, the antagonist will have a more pronounced effect than if the Kb is large and the antagonist concentration is small. This also points out that large concentrations of the agonist can overcome the actions of a competitive antagonist. Assume that the agonist, D, and the antagonist, B, have equal affinity for the receptor. If the concentration of D is much larger than B, the value of E/Emax will not be significantly decreased by the presence of the antagonist. This again illustrates that the actions of the competitive antagonist can be surmounted by the agonist.

Prazosin is a competitive antagonist of the action of agonist PE

1. Reversible binding to the receptor.
2. The blockade can be overcome by increasing the agonist concentration.
3. The maximal response of the agonist is not decreased.
4. The agonist dose-response curve in the presence of a competitive antagonist is displaced to the right parallel to the curve in the absence of agonist.

Irreversible Receptor Antagonists

Another type of antagonist is referred to as an irreversible receptor antagonist. The properties of irreversible antagonists are markedly different from competitive antagonists. Irreversible receptor antagonists are chemically reactive compounds. These ligands first bind to the receptor. Following this binding step, the ligand then reacts with the functional groups of the receptor. The consequence of this chemical reaction is that the ligand becomes covalently bound to the receptor. Because a chemical bond is formed, an irreversible ligand does not freely dissociate from the receptor. It remains attached to the receptor for a long period of time. The synthesis of new receptor protein may be required to generate a receptor free of an irreversible blocker. Because the ligand is covalently bound to the receptor, the binding of agonists, and hence their pharmacologic activity, are blocked. Unlike competitive antagonists, the blocking activity of irreversible receptor antagonists can not be overcome by increasing the agonist concentration. The antagonism therefore cannot be overcome by increasing the agonist concentration. Recall, that the effect of an agonist is proportional to the active drug-receptor complexes formed. Because an irreversible receptor antagonist reduces the total number of active receptors, [RT], the maximal pharmacologic effect Emax is also decreased. The reduction in maximal agonist reponse is the hallmark of irreversible antagonists. The shape of the dose-response curve is also altered because of this decrease in maximal effect. The dose-response is shifted to the right and the maximal response is depressed.

Few drugs interact with one and only one receptor. Such a drug would be said to be specific, that is producing effects by specifically interacting with a single receptor. Most drugs interact with several receptors and thus have the capability to produce distinctly different pharmacologic effects. Some of these effects could be beneficial, some could be toxic. Such a drug would be said to be a selective. The factors that determine which particular effect of a drug will be observed are the affinity and intrinsic activity of a drug .

To illustrate this point consider the following example. A drug is capable of producing actions at 2 distinct receptors. At each of these receptors, the ligand has a different affinity as well as pharmacologic effect.

Receptor System # 1:

K_D = 40.0, intrinsic activity 1.0,effect- lowering of systemic arterial blood pressure.

K_D = 40.0, intrinsic activity 1.0,effect- lethal ventricular arrhythmias.

Thus, this drug could either be a highly beneficial therapeutic agent or a lethal poison. An overwhelming majority of drugs used in clinical practice produce their therapeutic effects due

The therapeutic index is the ratio of the ED50 of a drug to produce a toxic effect to the ED50 to produce a therapeutic effect. For the drug example above, the ED50 for the beneficial effect of blood pressure lowering is 0.4 nM while the ED50 for toxicity is 40 nM. Therefore, the therapeutic index will be;

Partial agonists have lower intrinsic activities than full agonists but values greater than competitive antagonists. At certain concentrations partial agonists actually can be antagonists.

To understand how the relationship between occupancy and response can be non linear let us analyze the components which contribute to the response.

G-Protein Coupled Receptors

G-protein coupled receptors are a large family of receptors that serve as the site of action for many drugs. The name reflects the fact that the activity of these receptors is regulated by interaction with guanine nucleotide regulatory proteins (hence G-proteins). Despite major differences in the physiologic responses they activate and the variety of second messengers involved, the structure of all G-protein coupled receptors is similar. G-protein coupled receptors have a single polypeptide chain which passes through the cell membrane seven times. This arrangement results in the formation of loops on both the extracellular and intracellular sides of the membrane. Seven clusters of hydrophobic amino acids make up the membrane spanning domains of the receptor. The membrane spanning regions also form a binding pocket with which agonists and antagonists interact. The intracellular loops are thought to be necessary for the interaction with G-proteins and second messenger systems.

The G-protein Regulatory Cycle

In cellular signaling pathways involving G-proteins, the receptor/agonist complex does not interact directly with the enzyme which generates the second messenger. Rather, an intermediate or transducing protein couples the receptor to the second messenger generating system. This is the role of the G-protein . There is not a single G-protein, but a family of G-proteins which functions to regulate second messenger systems. G-proteins consist of three subunits: alpha, beta and gamma. In the resting state the receptor is not occupied by an agonist and the G-protein exists as trimer of the alpha, beta and gamma subunits with GDP bound to the alpha subunit. In this state, G-proteins are poor activators of intracellular signaling systems. Agonist binding to the receptor promotes the dissociation of the GDP and binding of GTP. GTP binding promotes the dissociation of the alpha subunit from the beta and gamma subunits. It is the GTP bound alpha subunit that activates effector enzyme systems. The alpha subunit is also a GTPase and is thus able to hydrolyze the GTP. The hydrolysis of GTP to GDP deactivates the alpha subunit and terminates the activation effector systems. The alpha subunit/GDP complex is then re-associated with the beta and gamma subunits to complete the regulatory cycle. The G-protein heterotrimer is again available for interaction with a receptor and activation of second messenger generating systems. Therefore, the rate at which the GTP is hydrolyzed regulates the time the G-protein is active. The longer the G-protein is active, the more second messenger can be generated

In a responding system which has a linear relationship between occupancy and physiologic response, there is a direct proportionality between the degree of receptor activation and the generation of second messengers. While this is difficult to conceptualize, it can be thought of as a small amount of receptor occupancy producing a small increase in the level of the second messenger. This small amount of second messenger activates a small increase in physiologic response. In the more realistic nonlinear occupancy versus response system, a small degree of receptor occupancy generates a large increase in second messenger levels which in turn generate an even larger physiologic response. The signal is amplified at every step of the signal transduction process. In this fashion, then, a small degree of receptor occupancy leads to a large physiologic response. Consider the following example. In a given beta-receptor system, 50,000 cAMP molecules are needed to yield a full response. In a linear response relationship, 50,000 receptors would have to be occupied to give a full response. However, in a nonlinear system, only 100 would be required to achieve a full response.

Regulation of Receptor Function

Continuous exposure of an agonist results in a phenomenon referred to as desensitization. The same concentration of agonist becomes less and less effective at producing the same level of effect. When this desensitization occurs very rapidly, it is referred to as tachyphylaxis. Recent evidence has suggested potential mechanisms by which the process of tachyphylaxis and desensitization occur. The receptor becomes phosphorylated in the third cytoplasmic loop and c-terminal tail. The phosphorylated receptor is less efficient at activating G-protein and also exhibits lower affinity for agonists. The receptors can also be removed from and sequestered away from the cell surface. These events indicate that second messengers not only regulate intracellular processes but are also capable of regulating the receptor systems which generate them.

Inverse Agonists

Traditionally, G-protein coupled receptors were thought to be inactive and that agonist occupation was required to allow the receptor to assume an active conformation. Recently, though, it has been suggested that the receptor can be active without the presence of agonist. The term for this is constitutive activity. Constitutively active receptors are thought to be coupled to second messenger pathways in the absence of agonists.

This has led to the postulate that in addition to traditional agonists, drugs can function as inverse agonists. Inverse agonists bind to constitutively active receptors and shift the equilibrium to the formation of the inactive conformer. In this system an inverse agonist would actually reverse receptor activity. The concept of inverse agonism has added a level of complexity to our thinking of drug action. As the diagram below illustrates, the spectrum of drug activity can range from a full agonist to a full inverse agonist.

The relevance of constitutively active receptors and inverse agonists to normal physiology and pathophysiology has not been established. That being stated, the concept of a constitutively active receptor does offer insights which could help to explain pathophysiologic conditions. If the process of disease induced the expression of a constitutively active receptor, the receptor would no longer be under the influence of the sympathetic nervous system. This could occur in hypertension with a constitutively active GPCR being expressed in any number of areas including the brain, kidneys or peripheral blood vessels. In this scenario, drugs with inverse agonist properties could prove to be safe, rational therapeutics.