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2 C H A P T E R Drug Receptors & Pharmacodynamics Mark von Zastrow, MD, PhD* C ASE STUDY...

2 C H A P T E R Drug Receptors & Pharmacodynamics Mark von Zastrow, MD, PhD* C ASE STUDY A 51-year-old man presents to the emergency department medical history is remarkable only for mild hypertension that due to acute difficulty breathing. The patient is afebrile and is being treated with propranolol. The physician instructs the normotensive but anxious, tachycardic, and markedly tachy- patient to discontinue use of propranolol, and changes the pneic. Auscultation of the chest reveals diffuse wheezes. The patient’s antihypertensive medication to verapamil. Why is physician provisionally makes the diagnosis of bronchial the physician correct to discontinue propranolol? Why is asthma and administers epinephrine by intramuscular injec- verapamil a better choice for managing hypertension in this tion, improving the patient’s breathing over several minutes. patient? What alternative treatment change might the physi- A normal chest X-ray is subsequently obtained, and the cian consider? Therapeutic and toxic effects of drugs result from their interac- 1. Receptors largely determine the quantitative relations tions with molecules in the patient. Most drugs act by associating between dose or concentration of drug and pharmacologic with specific macromolecules in ways that alter the macromol- effects. The receptor’s affinity for binding a drug determines the ecules’ biochemical or biophysical activities. This idea, more than concentration of drug required to form a significant number of a century old, is embodied in the term receptor: the component drug-receptor complexes, and the total number of receptors of a cell or organism that interacts with a drug and initiates the may limit the maximal effect a drug may produce. chain of events leading to the drug’s observed effects. 2. Receptors are responsible for selectivity of drug action. Receptors have become the central focus of investigation of The molecular size, shape, and electrical charge of a drug drug effects and their mechanisms of action (pharmacodynamics). determine whether—and with what affinity—it will bind to The receptor concept, extended to endocrinology, immunology, a particular receptor among the vast array of chemically dif- and molecular biology, has proved essential for explaining many ferent binding sites available in a cell, tissue, or patient. aspects of biologic regulation. Many drug receptors have been iso- Accordingly, changes in the chemical structure of a drug can lated and characterized in detail, thus opening the way to precise dramatically increase or decrease a new drug’s affinities for understanding of the molecular basis of drug action. different classes of receptors, with resulting alterations in The receptor concept has important practical consequences for therapeutic and toxic effects. the development of drugs and for arriving at therapeutic decisions 3. Receptors mediate the actions of pharmacologic agonists in clinical practice. These consequences form the basis for under- and antagonists. Some drugs and many natural ligands, such standing the actions and clinical uses of drugs described in almost as hormones and neurotransmitters, regulate the function of every chapter of this book. They may be briefly summarized as receptor macromolecules as agonists; this means that they acti- follows: vate the receptor to signal as a direct result of binding to it. Some agonists activate a single kind of receptor to produce all * The author thanks Henry R. Bourne, MD, for major contributions to their biologic functions, whereas others selectively promote this chapter. one receptor function more than another. 20  CHAPTER 2 Drug Receptors & Pharmacodynamics    21 Other drugs act as pharmacologic antagonists; that is, they RELATION BETWEEN DRUG bind to receptors but do not activate generation of a signal; consequently, they interfere with the ability of an agonist to CONCENTRATION & RESPONSE activate the receptor. Some of the most useful drugs in clinical The relation between dose of a drug and the clinically observed medicine are pharmacologic antagonists. Still other drugs bind response may be complex. In carefully controlled in vitro sys- to a different site on the receptor than that bound by endog- tems, however, the relation between concentration of a drug enous ligands; such drugs can produce useful and quite differ- and its effect is often simple and can be described with math- ent clinical effects by acting as so-called allosteric modulators ematical precision. It is important to understand this idealized of the receptor. relation in some detail because it underlies the more complex relations between dose and effect that occur when drugs are given to patients. MACROMOLECULAR NATURE OF DRUG RECEPTORS Concentration-Effect Curves & Receptor Most receptors for clinically relevant drugs, and almost all of the Binding of Agonists receptors that we discuss in this chapter, are proteins. Tradition- ally, drug binding was used to identify or purify receptor proteins Even in intact animals or patients, responses to low doses of a drug from tissue extracts; consequently, receptors were discovered after usually increase in direct proportion to dose. As doses increase, the drugs that bind to them. Advances in molecular biology and however, the response increment diminishes; finally, doses may be genome sequencing made it possible to identify receptors by pre- reached at which no further increase in response can be achieved. dicted structural homology to other (previously known) receptors. This relation between drug concentration and effect is tradition- This effort revealed that many known drugs bind to a larger diver- ally described by a hyperbolic curve (Figure 2–1A) according to sity of receptors than previously anticipated and motivated efforts the following equation: to develop increasingly selective drugs. It also identified a number of orphan receptors, so-called because their natural ligands are presently unknown; these may prove to be useful targets for future drug development. where E is the effect observed at concentration C, Emax is the The best-characterized drug receptors are regulatory proteins, maximal response that can be produced by the drug, and EC50 which mediate the actions of endogenous chemical signals such as is the concentration of drug that produces 50% of maximal neurotransmitters, autacoids, and hormones. This class of recep- effect. tors mediates the effects of many of the most useful therapeutic This hyperbolic relation resembles the mass action law that agents. The molecular structures and biochemical mechanisms of describes the association between two molecules of a given affin- these regulatory receptors are described in a later section entitled ity. This resemblance suggests that drug agonists act by binding Signaling Mechanisms & Drug Action. to (“occupying”) a distinct class of biologic molecules with a Other classes of proteins have been clearly identified as characteristic affinity for the drug. Radioactive receptor ligands drug receptors. Enzymes may be inhibited (or, less commonly, have been used to confirm this occupancy assumption in many activated) by binding a drug. Examples include dihydrofolate drug-receptor systems. In these systems, drug bound to recep- reductase, the receptor for the antineoplastic drug methotrexate; tors (B) relates to the concentration of free (unbound) drug (C) 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase, as depicted in Figure 2–1B and as described by an analogous the receptor for statins; and various protein and lipid kinases. equation: Transport proteins can be useful drug targets. Examples include Na+/K+-ATPase, the membrane receptor for cardioactive digitalis glycosides; norepinephrine and serotonin transporter proteins that are membrane receptors for antidepressant drugs; and dopa- mine transporters that are membrane receptors for cocaine and a in which Bmax indicates the total concentration of receptor sites number of other psychostimulants. Structural proteins are also (ie, sites bound to the drug at infinitely high concentrations important drug targets, such as tubulin, the receptor for the anti- of free drug) and Kd (the equilibrium dissociation constant) inflammatory agent colchicine. represents the concentration of free drug at which half-maximal This chapter deals with three aspects of drug receptor func- binding is observed. This constant characterizes the receptor’s tion, presented in increasing order of complexity: (1) receptors affinity for binding the drug in a reciprocal fashion: If the Kd as determinants of the quantitative relation between the concen- is low, binding affinity is high, and vice versa. The EC50 and tration of a drug and the pharmacologic response, (2) receptors Kd may be identical but need not be, as discussed below. Dose- as regulatory proteins and components of chemical signaling response data are often presented as a plot of the drug effect mechanisms that provide targets for important drugs, and (3) (ordinate) against the logarithm of the dose or concentration receptors as key determinants of the therapeutic and toxic effects (abscissa), transforming the hyperbolic curve of Figure 2–1 into of drugs in patients. a sigmoid curve with a linear midportion (eg, Figure 2–2). This  22    SECTION I Basic Principles A 1.0 B 1.0 Receptor-bound drug (B) Emax Bmax Drug effect (E) 0.5 0.5 EC50 Kd Drug concentration (C) Drug concentration (C) FIGURE 2–1 Relations between drug concentration and drug effect (A) or receptor-bound drug (B). The drug concentrations at which effect or receptor occupancy is half-maximal are denoted by EC50 and Kd, respectively. transformation is convenient because it expands the scale of Receptor-Effector Coupling & Spare the concentration axis at low concentrations (where the effect Receptors is changing rapidly) and compresses it at high concentrations When an agonist occupies a receptor, conformational changes (where the effect is changing slowly), but otherwise has no bio- occur in the receptor protein that represent the fundamental basis logic or pharmacologic significance. of receptor activation and the first of often many steps required to produce a pharmacologic response. The overall transduction process that links drug occupancy of receptors and pharmacologic response is called coupling. The relative efficiency of occupancy- response coupling is determined, in part, at the receptor itself; full agonists tend to shift the conformational equilibrium of receptors more strongly than partial agonists (described in the text that fol- A B C lows). Coupling is also determined by “downstream” biochemical events that transduce receptor occupancy into cellular response. Agonist effect D For some receptors, such as ligand-gated ion channels, the rela- 0.5 tionship between drug occupancy and response can be simple because the ion current produced by a drug is often directly pro- portional to the number of receptors (ion channels) bound. For other receptors, such as those linked to enzymatic signal transduc- E tion cascades, the occupancy-response relationship is often more complex because the biologic response reaches a maximum before full receptor occupancy is achieved. Many factors can contribute to nonlinear occupancy-response EC50 (A) EC50 (B) EC50 (C) EC50 (D,E) Kd coupling, and often these factors are only partially understood. A useful concept for thinking about this is that of receptor reserve Agonist concentration (C) (log scale) or spare receptors. Receptors are said to be “spare” for a given FIGURE 2–2 Logarithmic transformation of the dose axis and pharmacologic response if it is possible to elicit a maximal bio- experimental demonstration of spare receptors, using different logic response at a concentration of agonist that does not result in concentrations of an irreversible antagonist. Curve A shows ago- occupancy of all of the available receptors. Experimentally, spare nist response in the absence of antagonist. After treatment with a receptors may be demonstrated by using irreversible antagonists low concentration of antagonist (curve B), the curve is shifted to to prevent binding of agonist to a proportion of available recep- the right. Maximal responsiveness is preserved, however, because tors and showing that high concentrations of agonist can still the remaining available receptors are still in excess of the number produce an undiminished maximal response (Figure 2–2). For required. In curve C, produced after treatment with a larger concen- example, the same maximal inotropic response of heart muscle to tration of antagonist, the available receptors are no longer “spare”; catecholamines can be elicited even when 90% of β adrenoceptors instead, they are just sufficient to mediate an undiminished maximal response. Still higher concentrations of antagonist (curves D and E) to which they bind are occupied by a quasi-irreversible antagonist. reduce the number of available receptors to the point that maximal Accordingly, myocardial cells are said to contain a large proportion response is diminished. The apparent EC50 of the agonist in curves D of spare β adrenoceptors. and E may approximate the Kd that characterizes the binding affinity What accounts for the phenomenon of spare receptors? In of the agonist for the receptor. some cases, receptors may be simply spare in number relative to  CHAPTER 2 Drug Receptors & Pharmacodynamics    23 the total number of downstream signaling mediators present in agonist present at a concentration equal to the Kd will occupy 50% the cell, so that a maximal response occurs without occupancy of of the receptors, and half of the effectors will be activated, produc- all receptors. In other cases, “spareness” of receptors appears to be ing a half-maximal response (ie, two receptors stimulate two effec- temporal. For example, β-adrenoceptor activation by an agonist tors). Now imagine that the number of receptors increases tenfold promotes binding of guanosine triphosphate (GTP) to a trimeric to 40 receptors but that the total number of effectors remains con- G protein, producing an activated signaling intermediate whose stant. Most of the receptors are now spare in number. As a result, lifetime may greatly outlast the agonist-receptor interaction (see a much lower concentration of agonist suffices to occupy 2 of the also the following section on G Proteins & Second Messengers). 40 receptors (5% of the receptors), and this same low concentra- Here, maximal response is elicited by activation of relatively few tion of agonist is able to elicit a half-maximal response (two of four receptors because the response initiated by an individual ligand- effectors activated). Thus, it is possible to change the sensitivity of receptor-binding event persists longer than the binding event tissues with spare receptors by changing receptor number. itself. Irrespective of the biochemical basis of receptor reserve, the sensitivity of a cell or tissue to a particular concentration of Competitive & Irreversible Antagonists agonist depends not only on the affinity of the receptor for bind- Receptor antagonists bind to receptors but do not activate them; ing the agonist (characterized by the Kd) but also on the degree of the primary action of antagonists is to reduce the effects of agonists spareness—the total number of receptors present compared with (other drugs or endogenous regulatory molecules) that normally the number actually needed to elicit a maximal biologic response. activate receptors. While antagonists are traditionally thought to The concept of spare receptors is very useful clinically because have no functional effect in the absence of an agonist, some antago- it allows one to think precisely about the effects of drug dosage nists exhibit “inverse agonist” activity (see Chapter 1) because they without having to consider (or even fully understand) biochemical also reduce receptor activity below basal levels observed in the details of the signaling response. The Kd of the agonist-receptor absence of any agonist at all. Antagonist drugs are further divided interaction determines what fraction (B/Bmax) of total receptors into two classes depending on whether or not they act competitively will be occupied at a given free concentration (C) of agonist or noncompetitively relative to an agonist present at the same time. regardless of the receptor concentration: In the presence of a fixed concentration of agonist, increasing concentrations of a competitive antagonist progressively inhibit the agonist response; high antagonist concentrations prevent the response almost completely. Conversely, sufficiently high concen- Imagine a responding cell with four receptors and four effectors. trations of agonist can surmount the effect of a given concentration Here the number of effectors does not limit the maximal response, of the antagonist; that is, the Emax for the agonist remains the same and the receptors are not spare in number. Consequently, an for any fixed concentration of antagonist (Figure 2–3A). Because A B Agonist Agonist alone alone Agonist effect (E) Agonist effect (E) Agonist + competitive antagonist Agonist + noncompetitive antagonist C C' = C (1 + [ l ] / K) EC50 Agonist concentration Agonist concentration FIGURE 2–3 Changes in agonist concentration-effect curves produced by a competitive antagonist (A) or by an irreversible antagonist (B). In the presence of a competitive antagonist, higher concentrations of agonist are required to produce a given effect; thus the agonist concentration (C′) required for a given effect in the presence of concentration [I] of an antagonist is shifted to the right, as shown. High agonist concentrations can overcome inhibition by a competitive antagonist. This is not the case with an irreversible (or noncompetitive) antagonist, which reduces the maximal effect the agonist can achieve, although it may not change its EC50. 24    SECTION I Basic Principles the antagonism is competitive, the presence of antagonist increases of remaining unoccupied receptors may be too low for the agonist the agonist concentration required for a given degree of response, (even at high concentrations) to elicit a response comparable to and so the agonist concentration-effect curve is shifted to the right. the previous maximal response (Figure 2–3B). If spare receptors The concentration (C′) of an agonist required to produce a are present, however, a lower dose of an irreversible antagonist given effect in the presence of a fixed concentration ([I]) of com- may leave enough receptors unoccupied to allow achievement of petitive antagonist is greater than the agonist concentration (C) maximum response to agonist, although a higher agonist concen- required to produce the same effect in the absence of the antago- tration will be required (Figure 2–2B and C; see Receptor-Effector nist. The ratio of these two agonist concentrations (called the dose Coupling & Spare Receptors). ratio) is related to the dissociation constant (Ki) of the antagonist Therapeutically, such irreversible antagonists present distinct by the Schild equation: advantages and disadvantages. Once the irreversible antagonist has C′ [l] occupied the receptor, it need not be present in unbound form to =1+ inhibit agonist responses. Consequently, the duration of action of C Ki such an irreversible antagonist is relatively independent of its own rate of elimination and more dependent on the rate of turnover of Pharmacologists often use this relation to determine the Ki of receptor molecules. a competitive antagonist. Even without knowledge of the relation Phenoxybenzamine, an irreversible α-adrenoceptor antagonist, between agonist occupancy of the receptor and response, the Ki is used to control the hypertension caused by catecholamines can be determined simply and accurately. As shown in Figure 2–3, released from pheochromocytoma, a tumor of the adrenal medulla. concentration-response curves are obtained in the presence and in If administration of phenoxybenzamine lowers blood pressure, the absence of a fixed concentration of competitive antagonist; com- blockade will be maintained even when the tumor episodically parison of the agonist concentrations required to produce identical releases very large amounts of catecholamine. In this case, the ability degrees of pharmacologic effect in the two situations reveals the to prevent responses to varying and high concentrations of agonist is antagonist’s Ki. If C′ is twice C, for example, then [I] = Ki. a therapeutic advantage. If overdose occurs, however, a real problem For the clinician, this mathematical relation has two important may arise. If the α-adrenoceptor blockade cannot be overcome, therapeutic implications: excess effects of the drug must be antagonized “physiologically,” ie, 1. The degree of inhibition produced by a competitive antagonist by using a pressor agent that does not act via α adrenoceptors. depends on the concentration of antagonist. The competitive Antagonists can function noncompetitively in a different way; β-adrenoceptor antagonist propranolol provides a useful exam- that is, by binding to a site on the receptor protein separate from ple. Patients receiving a fixed dose of this drug exhibit a wide the agonist binding site; in this way, the drug can modify recep- range of plasma concentrations, owing to differences among tor activity without blocking agonist binding (see Chapter 1, individuals in the clearance of propranolol. As a result, inhibitory Figure 1–2C and D). Although these drugs act noncompetitively, effects on physiologic responses to norepinephrine and epineph- their actions are often reversible. Such drugs are called negative rine (endogenous adrenergic receptor agonists) may vary widely, allosteric modulators because they act through binding to a dif- and the dose of propranolol must be adjusted accordingly. ferent (ie, “allosteric”) site on the receptor relative to the classical 2. Clinical response to a competitive antagonist also depends on (ie, “orthosteric”) site bound by the agonist and reduce activity of the concentration of agonist that is competing for binding to the receptor. Not all allosteric modulators act as antagonists; some receptors. Again, propranolol provides a useful example: When potentiate rather than reduce receptor activity. For example, ben- this drug is administered at moderate doses sufficient to block zodiazepines are considered positive allosteric modulators because the effect of basal levels of the neurotransmitter norepineph- they bind to an allosteric site on the ion channels activated by rine, resting heart rate is decreased. However, the increase in the neurotransmitter γ-aminobutyric acid (GABA) and potenti- the release of norepinephrine and epinephrine that occurs with ate the net activating effect of GABA on channel conductance. exercise, postural changes, or emotional stress may suffice to Benzodiazepines have little activating effect on their own, and this overcome this competitive antagonism. Accordingly, the same property is one reason that benzodiazepines are relatively safe in dose of propranolol may have little effect under these condi- overdose; even at high doses, their ability to increase ion conduc- tions, thereby altering therapeutic response. Conversely, the tance is limited by the release of endogenous neurotransmitter. same dose of propranolol that is useful for treatment of hyper- Allosteric modulation can also occur at targets lacking a known tension in one patient may be excessive and toxic to another, orthosteric binding site. For example, ivacaftor binds to the cystic based on differences between the patients in the amount of fibrosis transmembrane regulator (CFTR) ion channel that is endogenous norepinephrine and epinephrine that they produce. mutated in cystic fibrosis. Certain mutations that render the chan- The actions of a noncompetitive antagonist are different nel hypoactive can be partially rescued by ivacaftor, representing because, once a receptor is bound by such a drug, agonists cannot positive allosteric modulation of a channel for which there is no surmount the inhibitory effect irrespective of their concentration. presently known endogenous ligand. In many cases, noncompetitive antagonists bind to the receptor in an irreversible or nearly irreversible fashion, sometimes by Partial Agonists forming a covalent bond with the receptor. After occupancy of Based on the maximal pharmacologic response that occurs when some proportion of receptors by such an antagonist, the number all receptors are occupied, agonists can be divided into two  CHAPTER 2 Drug Receptors & Pharmacodynamics    25 classes: partial agonists produce a lower response, at full recep- drugs, and it may precipitate a drug withdrawal syndrome in tor occupancy, than do full agonists. Partial agonists produce opioid-dependent patients. concentration-effect curves that resemble those observed with full agonists in the presence of an antagonist that irreversibly blocks some of the receptor sites (compare Figures 2–2 [curve Other Mechanisms of Drug Antagonism D] and 2–4B). It is important to emphasize that the failure Not all mechanisms of antagonism involve interactions of drugs of partial agonists to produce a maximal response is not due or endogenous ligands at a single type of receptor, and some to decreased affinity for binding to receptors. Indeed, a partial types of antagonism do not involve a receptor at all. For example, agonist’s inability to cause a maximal pharmacologic response, protamine, a protein that is positively charged at physiologic pH, even when present at high concentrations that effectively satu- can be used clinically to counteract the effects of heparin, an anti- rate binding to all receptors, is indicated by the fact that partial coagulant that is negatively charged. In this case, one drug acts as agonists competitively inhibit the responses produced by full a chemical antagonist of the other simply by ionic binding that agonists (Figure 2–4). This mixed “agonist-antagonist” prop- makes the other drug unavailable for interactions with proteins erty of partial agonists can have both beneficial and deleteri- involved in blood clotting. ous effects in the clinic. For example, buprenorphine, a partial Another type of antagonism is physiologic antagonism agonist of μ-opioid receptors, is a generally safer analgesic drug between endogenous regulatory pathways mediated by different than morphine because it produces less respiratory depression receptors. For example, several catabolic actions of the glucocor- in overdose. However, buprenorphine is effectively antianalgesic ticoid hormones lead to increased blood sugar, an effect that is when administered in combination with more efficacious opioid physiologically opposed by insulin. Although glucocorticoids and A B 100 1.0 Percentage of maximal binding 80 0.8 Full agonist Response 60 Full agonist Partial agonist 0.6 40 0.4 20 0.2 Partial agonist 0 0.0 –10 –8 –6 –10 –8 –6 log (Partial agonist) log (Full agonist or partial agonist) C 1.0 Total response 0.8 Full agonist Response 0.6 component 0.4 Partial agonist 0.2 component 0.0 –10 –8 –6 log (Partial agonist) FIGURE 2–4 A: The percentage of receptor occupancy resulting from full agonist (present at a single concentration) binding to receptors in the presence of increasing concentrations of a partial agonist. Because the full agonist (blue line) and the partial agonist (green line) compete to bind to the same receptor sites, when occupancy by the partial agonist increases, binding of the full agonist decreases. B: When each of the two drugs is used alone and response is measured, occupancy of all the receptors by the partial agonist produces a lower maximal response than does similar occupancy by the full agonist. C: Simultaneous treatment with a single concentration of full agonist and increasing concentra- tions of the partial agonist produces the response patterns shown in the bottom panel. The fractional response caused by a single high concen- tration of the full agonist decreases as increasing concentrations of the partial agonist compete to bind to the receptor with increasing success; at the same time, the portion of the response caused by the partial agonist increases, while the total response—ie, the sum of responses to the two drugs (red line)—gradually decreases, eventually reaching the value produced by partial agonist alone (compare with B). 26    SECTION I Basic Principles insulin act on quite distinct receptor-effector systems, the clinician How do cellular mechanisms for amplifying external chemical must sometimes administer insulin to oppose the hyperglycemic signals explain the phenomenon of spare receptors? effects of a glucocorticoid hormone, whether the latter is elevated Why do chemically similar drugs often exhibit extraordinary by endogenous synthesis (eg, a tumor of the adrenal cortex) or as selectivity in their actions? a result of glucocorticoid therapy. Do these mechanisms provide targets for developing new drugs? In general, use of a drug as a physiologic antagonist produces effects that are less specific and less easy to control than are the effects Most transmembrane signaling is accomplished by a small of a receptor-specific antagonist. Thus, for example, to treat brady- number of different molecular mechanisms. Each type of mecha- cardia caused by increased release of acetylcholine from vagus nerve nism has been adapted, through the evolution of distinctive protein endings, the physician could use isoproterenol, a β-adrenoceptor families, to transduce many different signals. These protein families agonist that increases heart rate by mimicking sympathetic stimula- include receptors on the cell surface and within the cell, as well as tion of the heart. However, use of this physiologic antagonist would enzymes and other components that generate, amplify, coordinate, be less rational—and potentially more dangerous—than use of a and terminate postreceptor signaling by chemical second messen- receptor-specific antagonist such as atropine (a competitive antago- gers in the cytoplasm. This section first discusses the mechanisms nist of acetylcholine receptors that slow heart rate as the direct targets for carrying chemical information across the plasma membrane of acetylcholine released from vagus nerve endings). and then outlines key features of cytoplasmic second messengers. Five basic mechanisms of transmembrane signaling are well SIGNALING MECHANISMS & DRUG understood (Figure 2–5). Each represents a different family of receptor protein and uses a different strategy to circumvent the ACTION barrier posed by the lipid bilayer of the plasma membrane. These strategies use (1) a lipid-soluble ligand that crosses the membrane Until now we have considered receptor interactions and drug effects and acts on an intracellular receptor; (2) a transmembrane recep- in terms of equations and concentration-effect curves. We must tor protein whose intracellular enzymatic activity is allosterically also understand the molecular mechanisms by which a drug acts. regulated by a ligand that binds to a site on the protein’s extra- We should also consider different structural families of receptor cellular domain; (3) a transmembrane receptor that binds and protein, and this allows us to ask basic questions with important stimulates an intracellular protein tyrosine kinase; (4) a ligand- clinical implications: gated transmembrane ion channel that can be induced to open or Why do some drugs produce effects that persist for minutes, close by the binding of a ligand; or (5) a transmembrane receptor hours, or even days after the drug is no longer present? protein that stimulates a GTP-binding signal transducer protein Why do responses to other drugs diminish rapidly with prolonged (G protein), which in turn modulates production of an intracel- or repeated administration? lular second messenger. 1 2 3 4 5 Drug Outside cell R Membrane R R R E G Inside cell A B Y Y~P C D R FIGURE 2–5 Known transmembrane signaling mechanisms: 1: A lipid-soluble chemical signal crosses the plasma membrane and acts on an intracellular receptor (which may be an enzyme or a regulator of gene transcription); 2: the signal binds to the extracellular domain of a transmembrane protein, thereby activating an enzymatic activity of its cytoplasmic domain; 3: the signal binds to the extracellular domain of a transmembrane receptor bound to a separate protein tyrosine kinase, which it activates; 4: the signal binds to and directly regulates the open- ing of an ion channel; 5: the signal binds to a cell-surface receptor linked to an effector enzyme by a G protein. (A, C, substrates; B, D, products; R, receptor; G, G protein; E, effector [enzyme or ion channel]; Y, tyrosine; P, phosphate.)  CHAPTER 2 Drug Receptors & Pharmacodynamics    27 Although the five established mechanisms do not account for all the chemical signals conveyed across cell membranes, they do transduce many of the most important signals exploited in pharmacotherapy. Ligand-binding domain Intracellular Receptors for Lipid-Soluble Agents hsp90 Several biologic ligands are sufficiently lipid-soluble to cross the Steroid plasma membrane and act on intracellular receptors. One class of such ligands includes steroids (corticosteroids, mineralocorticoids, sex steroids, vitamin D) and thyroid hormone, whose receptors stimulate the transcription of genes by binding to specific DNA hsp90 sequences (often called response elements) near the gene whose expression is to be regulated. These “gene-active” receptors belong to a protein family that evolved from a common precursor. Dissection of the receptors by recombinant DNA techniques has provided insights into their molecular mechanism. For example, binding of glucocorticoid Transcription- activating hormone to its normal receptor protein relieves an inhibitory domain constraint on the transcription-stimulating activity of the protein. Figure 2–6 schematically depicts the molecular mechanism of DNA-binding glucocorticoid action: In the absence of hormone, the receptor is domain bound to hsp90, a protein that prevents normal folding of several structural domains of the receptor. Binding of hormone to the ligand-binding domain triggers release of hsp90. This allows the Altered transcription of specific genes DNA-binding and transcription-activating domains of the recep- tor to fold into their functionally active conformations, so that the activated receptor can initiate transcription of target genes. FIGURE 2–6 Mechanism of glucocorticoid action. The gluco- The mechanism used by hormones that act by regulating gene corticoid receptor polypeptide is schematically depicted as a protein with three distinct domains. A heat-shock protein, hsp90, binds to expression has two therapeutically important consequences: the receptor in the absence of hormone and prevents folding into 1. All of these hormones produce their effects after a characteristic the active conformation of the receptor. Binding of a hormone ligand lag period of 30 minutes to several hours—the time required (steroid) causes dissociation of the hsp90 stabilizer and permits for the synthesis of new proteins. This means that the gene- conversion to the active configuration. active hormones cannot be expected to alter a pathologic state within minutes (eg, glucocorticoids will not immediately relieve the symptoms of bronchial asthma). hormone-binding domain and a cytoplasmic enzyme domain, 2. The effects of these agents can persist for hours or days after which may be a protein tyrosine kinase, a serine kinase, or a gua- the agonist concentration has been reduced to zero. The persis- nylyl cyclase (Figure 2–7). In all these receptors, the two domains tence of effect is primarily due to the relatively slow turnover are connected by a hydrophobic segment of the polypeptide that of most enzymes and proteins, which can remain active in cells resides in the lipid bilayer of the plasma membrane. for hours or days after they have been synthesized. Conse- The receptor tyrosine kinase signaling function begins with quently, it means that the beneficial (or toxic) effects of a gene- binding of ligand, typically a polypeptide hormone or growth fac- active hormone usually decrease slowly when administration of tor, to the receptor’s extracellular domain. The resulting change in the hormone is stopped. receptor conformation causes two receptor molecules to bind to one another (dimerize). This activates the tyrosine kinase enzyme Ligand-Regulated Transmembrane activity present in the cytoplasmic domain of the dimer, leading to Enzymes Including Receptor phosphorylation of the receptor as well as additional downstream signaling proteins. Activated receptors catalyze phosphorylation Tyrosine Kinases of tyrosine residues on different target signaling proteins, thereby This class of receptor molecules mediates the first steps in signaling allowing a single type of activated receptor to modulate a number by insulin, epidermal growth factor (EGF), platelet-derived growth of biochemical processes. (Some receptor tyrosine kinases form factor (PDGF), atrial natriuretic peptide (ANP), transforming oligomeric complexes larger than dimers upon activation by growth factor-β (TGF-β), and many other trophic hormones. ligand, but the pharmacologic significance of such higher-order These receptors are polypeptides consisting of an extracellular complexes is presently unclear.) 28    SECTION I Basic Principles EGF molecules +EGF –EGF Outside Inside P P Y Y Y Y S S~P ATP ADP FIGURE 2–7 Mechanism of activation of the epidermal growth factor (EGF) receptor, a representative receptor tyrosine kinase. The receptor polypeptide has extracellular and cytoplasmic domains, depicted above and below the plasma membrane. Upon binding of EGF (circle), the receptor converts from its inactive monomeric state (left) to an active dimeric state (right), in which two receptor polypeptides bind nonco- valently. The cytoplasmic domains become phosphorylated (P) on specific tyrosine residues (Y), and their enzymatic activities are activated, catalyzing phosphorylation of substrate proteins (S). Insulin, for example, uses a single class of tyrosine kinase recep- notably receptors for nerve growth factor, serves a very different tors to trigger increased uptake of glucose and amino acids and function. Internalized nerve growth factor receptors are not rap- to regulate metabolism of glycogen and triglycerides in the cell. idly degraded but are translocated in endocytic vesicles from the Activation of the receptor in specific target cells drives a complex distal axon, where receptors are activated by nerve growth factor program of cellular events ranging from altered membrane transport released from the innervated tissue, to the cell body. In the cell of ions and metabolites to changes in the expression of many genes. body, the growth factor signal is transduced to transcription fac- Inhibitors of particular receptor tyrosine kinases are finding tors regulating the expression of genes controlling cell survival. increased use in neoplastic disorders in which excessive growth This process, effectively opposite to down-regulation, transports a factor signaling is often involved. Some of these inhibitors are critical survival signal from its site of agonist release to the site of monoclonal antibodies (eg, trastuzumab, cetuximab), which bind a critical downstream signaling effect and can do so over a remark- to the extracellular domain of a particular receptor and interfere ably long distance—up to a meter in some neurons. with binding of growth factor. Other inhibitors are membrane- A number of regulators of growth and differentiation, includ- permeant small molecule chemicals (eg, gefitinib, erlotinib), ing TGF-β, act on another class of transmembrane receptor which inhibit the receptor’s kinase activity in the cytoplasm. enzymes that phosphorylate serine and threonine residues. Atrial The intensity and duration of action of EGF, PDGF, and other natriuretic peptide (ANP), an important regulator of blood agents that act via receptor tyrosine kinases are often limited by volume and vascular tone, acts on a transmembrane receptor a process called receptor down-regulation. Ligand binding often whose intracellular domain, a guanylyl cyclase, generates cGMP induces accelerated endocytosis of receptors from the cell surface, (see below). Receptors in both groups, like the receptor tyrosine followed by the degradation of those receptors (and their bound kinases, are active in their dimeric forms. ligands). When this process occurs at a rate faster than de novo synthesis of receptors, the total number of cell-surface receptors is reduced (down-regulated), and the cell’s responsiveness to ligand Cytokine Receptors is correspondingly diminished. A well-understood example is the Cytokine receptors respond to a heterogeneous group of peptide EGF receptor tyrosine kinase, which internalizes from the plasma ligands, which include growth hormone, erythropoietin, several membrane at a greatly accelerated rate after activation by EGF kinds of interferon, and other regulators of growth and differ- and then is delivered to lysosomes and proteolyzed. This down- entiation. These receptors use a mechanism (Figure 2–8) closely regulation process is essential physiologically to limit the strength resembling that of receptor tyrosine kinases, except that in this and duration of the growth factor signal; genetic mutations that case, the protein tyrosine kinase activity is not intrinsic to the interfere with the down-regulation process cause excessive and receptor molecule. Instead, a separate protein tyrosine kinase, prolonged responses that underlie or contribute to many forms from the Janus-kinase (JAK) family, binds noncovalently to the of cancer. Endocytosis of other receptor tyrosine kinases, most receptor. As in the case of the EGF receptor, cytokine receptors  CHAPTER 2 Drug Receptors & Pharmacodynamics    29 Cytokine molecules + Cytokine P~Y Y~P R R R R JAK JAK JAK JAK Y~P P~Y STAT STAT Y~P STAT STAT P~Y FIGURE 2–8 Cytokine receptors, like receptor tyrosine kinases, have extracellular and intracellular domains and form dimers. However, after activation by an appropriate ligand, separate mobile protein tyrosine kinase molecules (JAK) are activated, resulting in phosphorylation of signal transducers and activation of transcription (STAT) molecules. STAT dimers then travel to the nucleus, where they regulate transcription. dimerize after they bind the activating ligand, allowing the bound These polypeptides, each of which crosses the lipid bilayer four JAKs to become activated and to phosphorylate tyrosine residues times, form a cylindrical structure that is approximately 10 nm in on the receptor. Phosphorylated tyrosine residues on the receptor’s diameter but is impermeable to ions. When acetylcholine binds cytoplasmic surface then set in motion a complex signaling dance to sites on the α subunits, a conformational change occurs that by binding another set of proteins, called STATs (signal transduc- ers and activators of transcription). The bound STATs are them- selves phosphorylated by the JAKs, two STAT molecules dimerize Na+ (attaching to one another’s tyrosine phosphates), and finally the ACh ACh STAT/STAT dimer dissociates from the receptor and travels to the δ nucleus, where it regulates transcription of specific genes. γ α α Ion Channels Outside β Many of the most useful drugs in clinical medicine act on ion channels. For ligand-gated ion channels, drugs often mimic or block the actions of natural agonists. Natural ligands of such receptors include acetylcholine, serotonin, GABA, and glutamate; all are synaptic transmitters. Each of their receptors transmits its signal across the plasma membrane by increasing transmembrane conductance of the relevant ion and thereby altering the electrical potential across the membrane. For example, acetylcholine causes the opening of Inside the ion channel in the nicotinic acetylcholine receptor (nAChR), which allows Na+ to flow down its concentration gradient into Na+ cells, producing a localized excitatory postsynaptic potential—a depolarization. FIGURE 2–9 The nicotinic acetylcholine (ACh) receptor, a ligand- The nAChR is one of the best characterized of all cell-surface gated ion channel. The receptor molecule is depicted as embedded in receptors for hormones or neurotransmitters (Figure 2–9). One a rectangular piece of plasma membrane, with extracellular fluid above form of this receptor is a pentamer made up of four different and cytoplasm below. Composed of five subunits (two α, one β, one γ, polypeptide subunits (eg, two α chains plus one β, one γ, and one and one δ), the receptor opens a central transmembrane ion channel δ chain, all with molecular weights ranging from 43,000–50,000). when ACh binds to sites on the extracellular domain of its α subunits. 30    SECTION I Basic Principles results in the transient opening of a central aqueous channel, concentration of the intracellular second messenger. For cAMP, approximately 0.5 nm in diameter, through which sodium ions the effector enzyme is adenylyl cyclase, a membrane protein that penetrate from the extracellular fluid to cause electrical depolar- converts intracellular adenosine triphosphate (ATP) to cAMP. ization of the cell. The structural basis for activating other ligand- The corresponding G protein, Gs, stimulates adenylyl cyclase after gated ion channels has been determined recently, and similar being activated by hormones and neurotransmitters that act via general principles apply, but there are differences in key details specific Gs-coupled receptors. There are many examples of such that may open new opportunities for drug action. For example, receptors, including α and β adrenoceptors, glucagon receptors, receptors that mediate excitatory neurotransmission at central thyrotropin receptors, and certain subtypes of dopamine and nervous system synapses bind glutamate, a major excitatory neu- serotonin receptors. rotransmitter, through a large appendage domain that protrudes Gs and other G proteins activate their downstream effectors from the receptor and has been called a “flytrap” because it physi- when bound by GTP and also have the ability to hydrolyze GTP cally closes around the glutamate molecule; the glutamate-loaded (Figure 2–10); this hydrolysis reaction inactivates the G protein flytrap domain then moves as a unit to control pore opening. but can occur at a relatively slow rate, effectively amplifying the Drugs can regulate the activity of such glutamate receptors by transduced signal by allowing the activated (GTP-bound) G protein binding to the flytrap domain, to surfaces on the membrane- to have a longer lifetime in the cell than the activated receptor embedded portion around the pore, or within the pore itself. itself. For example, a neurotransmitter such as norepinephrine The time elapsed between the binding of the agonist to a may encounter its membrane receptor for only a few milliseconds. ligand-gated channel and the cellular response can often be mea- When the encounter generates a GTP-bound Gs molecule, how- sured in milliseconds. The rapidity of this signaling mechanism is ever, the duration of activation of adenylyl cyclase depends on the crucially important for moment-to-moment transfer of informa- longevity of GTP binding to Gs rather than on the duration of tion across synapses. Ligand-gated ion channels can be regulated norepinephrine’s binding to the receptor. Indeed, like other by multiple mechanisms, including phosphorylation and endocy- G proteins, GTP-bound Gs may remain active for tens of seconds, tosis. In the central nervous system, these mechanisms contribute enormously amplifying the original signal. This mechanism also to synaptic plasticity involved in learning and memory. helps explain how signaling by G proteins produces the phenom- Voltage-gated ion channels do not bind neurotransmitters enon of spare receptors. The family of G proteins contains several directly but are controlled by membrane potential; such channels functionally diverse subfamilies (Table 2–1), each of which medi- are also important drug targets. Drugs that regulate voltage-gated ates effects of a particular set of receptors to a distinctive group channels typically bind to a site of the receptor different from of effectors. Note that an endogenous ligand (eg, norepinephrine, the charged amino acids that constitute the “voltage sensor” acetylcholine, serotonin, many others not listed in Table 2–1) domain of the protein used for channel opening by membrane may bind and stimulate receptors that couple to different subsets potential. For example, verapamil binds to a region in the pore of voltage-gated calcium channels that are present in the heart and in vascular smooth muscle, inhibiting the ion conductance sepa- Agonist rately from the voltage sensor, producing antiarrhythmic effects, and reducing blood pressure without mimicking or antagonizing any known endogenous transmitter. Other channels, such as the CFTR, although not strongly sensitive to either a known natural R R* Cell membrane ligand or voltage, are still important drug targets. Lumacaftor binds CFTR and promotes its delivery to the plasma membrane GTP E after biosynthesis. Ivacaftor binds to a different site and enhances GDP channel conductance. Both drugs act as allosteric modulators of G–GDP G–GTP the CFTR and were recently approved for treatment of cystic fibrosis, but each has a different effect. E* G Proteins & Second Messengers Pi Many extracellular ligands act by increasing the intracellular con- centrations of second messengers such as cyclic adenosine-3′,5′- monophosphate (cAMP), calcium ion, or the phosphoinositides FIGURE 2–10 The guanine nucleotide-dependent activation- inactivation cycle of G proteins. The agonist activates the receptor (described below). In most cases, they use a transmembrane signaling (R→R*), which promotes release of GDP from the G protein (G), system with three separate components. First, the extracellular ligand allowing entry of GTP into the nucleotide binding site. In its GTP- is selectively detected by a cell-surface receptor. The receptor in turn bound state (G-GTP), the G protein regulates activity of an effector triggers the activation of a GTP-binding protein (G protein) located enzyme or ion channel (E→E*). The signal is terminated by hydrolysis on the cytoplasmic face of the plasma membrane. The activated of GTP, followed by return of the system to the basal unstimu- G protein then changes the activity of an effector element, usu- lated state. Open arrows denote regulatory effects. (Pi, inorganic ally an enzyme or ion channel. This element then changes the phosphate.)  CHAPTER 2 Drug Receptors & Pharmacodynamics    31 TABLE 2–1 G proteins and their receptors and effectors. G Protein Receptors for Effector/Signaling Pathway Gs β-Adrenergic amines, histamine, serotonin, glucagon, and many ↑ Adenylyl cyclase →↑ cAMP other hormones Gi1, Gi2, Gi3 α2-Adrenergic amines, acetylcholine (muscarinic), opioids, Several, including: serotonin, and many others ↓ Adenylyl cyclase →↓ cAMP + Open cardiac K channels →↓ heart rate Golf Odorants (olfactory epithelium) ↑ Adenylyl cyclase →↑ cAMP Go Neurotransmitters in brain (not yet specifically identified) Not yet clear Gq Acetylcholine (muscarinic), bombesin, serotonin (5-HT2), and ↑ Phospholipase C →↑ IP3, diacylglycerol, cytoplasmic Ca2+ many others Gt1, Gt2 Photons (rhodopsin and color opsins in retinal rod and ↑ cGMP phosphodiesterase →↓ cGMP (phototransduction) cone cells) cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; IP3, inositol-1,4,5-trisphosphate. of G proteins. The apparent promiscuity of such a ligand allows both proteins, allowing agonist binding to the receptor to effec- it to elicit different G protein-dependent responses in different tively “drive” a nucleotide exchange reaction that “switches” the cells. For instance, the body responds to danger by using catechol- G protein from its inactive (GDP-bound) to active (GTP-bound) amines (norepinephrine and epinephrine) both to increase heart form. Figure 2–11 shows the main components schematically. rate and to induce constriction of blood vessels in the skin, by acting on Gs-coupled β adrenoceptors and Gq-coupled α1 adreno- ceptors, respectively. Ligand promiscuity also offers opportunities Agonist in drug development (see Receptor Classes & Drug Development in the following text). Receptors that signal via G proteins are often called “G Outside protein-coupled receptors” (GPCRs). GPCRs make up the largest N receptor family and are also called “seven-transmembrane” (7TM) II or “serpentine” receptors because the receptor polypeptide chain I “snakes” across the plasma membrane seven times (Figure 2–11). III Receptors for adrenergic amines, serotonin, acetylcholine (musca- VII Ag IV VI V rinic but not nicotinic), many peptide hormones, odorants, and even visual receptors (in retinal rod and cone cells) all belong to the GPCR family. All were derived from a common evolutionary Inside HO C precursor. A few GPCRs (eg, GABAB and metabotropic glutamate G OH receptors) require stable assembly into homodimers (complexes of protein OH OH two identical receptor polypeptides) or heterodimers (complexes of different isoforms) for functional activity. However, in contrast to FIGURE 2–11 Transmembrane topology of a typical “serpen- tyrosine kinase and cytokine receptors, dimerization is not univer- tine” GPCR. The receptor’s amino (N) terminal is extracellular (above sally required for GPCR activation, and many GPCRs are thought the plane of the membrane), and its carboxyl (C) terminal intracellu- to function as monomers. lar, with the polypeptide chain “snaking” across the membrane seven GPCRs can bind agonists in a variety of ways, but they all times. The hydrophobic transmembrane segments (light color) are appear to transduce signals across the plasma membrane in a simi- designated by Roman numerals (I–VII). Agonist (Ag) approaches the lar way. Agonist binding (eg, a catecholamine or acetylcholine) receptor from the extracellular fluid and binds to a site surrounded stabilizes a conformational state of the receptor in which the cyto- by the transmembrane regions of the receptor protein. G protein plasmic ends of the transmembrane helices spread apart by about interacts with cytoplasmic regions of the receptor, especially around 1 nm, opening a cavity in the receptor’s cytoplasmic surface that the third cytoplasmic loop connecting transmembrane regions V and VI. Lateral movement of these helices during activation exposes an binds a critical regulatory surface of the G protein. This reduces otherwise buried cytoplasmic surface of the receptor that promotes nucleotide affinity for the G protein, allowing GDP to dissociate guanine nucleotide exchange on the G protein and thereby activates and GTP to replace it (this occurs because GTP is normally pres- the G protein, as discussed in the text. The receptor’s cytoplasmic ent in the cytoplasm at much higher concentration than GDP). terminal tail contains numerous serine and threonine residues whose The GTP-bound form of G protein then dissociates from the hydroxyl (-OH) groups can be phosphorylated. This phosphorylation receptor and can engage downstream mediators. Thus GPCR–G is associated with diminished receptor-G protein coupling and can protein coupling involves coordinated conformational change in promote receptor endocytosis. 32    SECTION I Basic Principles Many high-resolution structures of GPCRs are available from the orthosteric agonists, but differ from conventional agonists in Protein Data Bank (www.rcsb.org). An animated model depicting effects on receptor conformation after binding. Allosteric ligands the conformational change associated with activation is available may also stabilize different conformational states of the receptor, from the Protein Data Bank in Europe (http://www.ebi.ac.uk/ but differ from functionally selective ligands by binding noncom- pdbe/quips?story=B2AR). petitively to a different site. Receptor Regulation Well-Established Second Messengers G protein-mediated responses to drugs and hormonal agonists A. Cyclic Adenosine Monophosphate (cAMP) often attenuate with time (Figure 2–12A). After reaching an Acting as an intracellular second messenger, cAMP mediates such initial high level, the response (eg, cellular cAMP accumulation, hormonal responses as the mobilization of stored energy (the break- Na+ influx, contractility, etc) diminishes over seconds or minutes, down of carbohydrates in liver or triglycerides in fat cells stimulated even in the continued presence of the agonist. In some cases, this by β-adrenomimetic catecholamines), conservation of water by the desensitization phenomenon is rapidly reversible; a second expo- kidney (mediated by vasopressin), Ca2+ homeostasis (regulated by sure to agonist, if provided a few minutes after termination of the parathyroid hormone), and increased rate and contractile force of first exposure, results in a response similar to the initial response. heart muscle (β-adrenomimetic catecholamines). It also regulates Multiple mechanisms contribute to desensitization of GPCRs. the production of adrenal and sex steroids (in response to corti- One well-understood mechanism involves phosphorylation of cotropin or follicle-stimulating hormone), relaxation of smooth the receptor. The agonist-induced change in conformation of muscle, and many other endocrine and neural processes. the β-adrenoceptor causes it not only to activate G protein, but cAMP exerts most of its effects by stimulating cAMP-depen- also to recruit and activate a family of protein kinases called G dent protein kinases (Figure 2–13). These kinases are composed protein-coupled receptor kinases (GRKs). GRKs phosphorylate of a cAMP-binding regulatory (R) dimer and two catalytic (C) serine and threonine residues in the receptor’s cytoplasmic tail chains. When cAMP binds to the R dimer, active C chains are (Figure 2–12B), diminishing the ability of activated β adrenocep- released to diffuse through the cytoplasm and nucleus, where they tors to activate Gs and also increasing the receptor’s affinity for transfer phosphate from ATP to appropriate substrate proteins, binding a third protein, β-arrestin. Binding of β-arrestin to the often enzymes. The specificity of the regulatory effects of cAMP receptor further diminishes the receptor’s ability to interact with resides in the distinct protein substrates of the kinases that are Gs, attenuating the cellular response (ie, stimulation of adenylyl expressed in different cells. For example, the liver is rich in phos- cyclase as discussed below). Upon removal of agonist, phosphory- phorylase kinase and glycogen synthase, enzymes whose reciprocal lation by the GRK is terminated, β-arrestin can dissociate, and regulation by cAMP-dependent phosphorylation governs carbo- cellular phosphatases remove the phosphorylations, reversing the hydrate storage and release. desensitized state and allowing activation to occur again upon When the hormonal stimulus stops, the intracellular actions another encounter with agonist. of cAMP are terminated by an elaborate series of enzymes. For β adrenoceptors, and for many other GPCRs, β-arrestin can cAMP-stimulated phosphorylation of enzyme substrates is rapidly produce other effects. One effect is to accelerate endocytosis of reversed by a diverse group of specific and nonspecific phos- β adrenoceptors from the plasma membrane. This can down-regulate phatases. cAMP itself is degraded to 5′-AMP by several cyclic β adrenoceptors if receptors subsequently travel to lysosomes, nucleotide phosphodiesterases (PDEs; Figure 2–13). Milrinone, a similar to down-regulation of EGF receptors, but it can also help selective inhibitor of type 3 phosphodiesterases that are expressed reverse the desensitized state for those receptors returned to the in cardiac muscle cells, has been used as an adjunctive agent in plasma membrane by exposing receptors to phosphatase enzymes treating acute heart failure. Competitive inhibition of cAMP deg- in endosomes (Figure 2–12B). In some cases, β-arrestin can itself radation is one way that caffeine, theophylline, and other methyl- act as a positive signal transducer, analogous to G proteins but xanthines produce their effects (see Chapter 20). through a different mechanism, by serving as a molecular scaffold to bind other signaling proteins (rather than through binding B. Phosphoinositides and Calcium GTP). In this way, β-arrestin can confer on GPCRs a great deal Another well-studied second messenger system involves hormonal of flexibility in signaling and regulation. This flexibility is still stimulation of phosphoinositide hydrolysis (Figure 2–14). Some poorly understood but is presently thought to underlie the ability of the hormones, neurotransmitters, and growth factors that of some drugs to produce a different spectrum of downstream trigger this pathway bind to receptors linked to G proteins, effects from other drugs, despite binding to the same GPCR. Cur- whereas others bind to receptor tyrosine kinases. In all cases, the rent drug development efforts are exploring the potential of this crucial step is stimulation of a membrane enzyme, phospholi- phenomenon, called functional selectivity or agonist bias, as a pase C (PLC), which splits a minor phospholipid component means to achieve specificity in drug action beyond that presently of the plasma membrane, phosphatidylinositol-4,5-bisphosphate possible using conventional agonists and antagonists. Functionally (PIP2), into two second messengers, diacylglycerol (DAG) and selective agonists are thought to occupy the orthosteric ligand- inositol-1,4,5-trisphosphate (IP3 or InsP3). Diacylglycerol is binding site, making their binding competitive with conventional confined to the membrane, where it activates a phospholipid- and  CHAPTER 2 Drug Receptors & Pharmacodynamics    33 A Agonist Response (cAMP) 1 2 3 4 5 1 2 Time B Agonist in extracellular space 1 2 -OH -OH GRK -OH -OH ATP -OH -OH Coated pit P P P GS 5 β−Arr 3 4 6 Lysosome P'ase -OH P -OH Endosomes P -OH P FIGURE 2–12 Rapid desensitization, resensitization, and down-regulation of β adrenoceptors. A: Response to a β-adrenoceptor agonist (ordinate) versus time (abscissa). (Numbers refer to the phases of receptor function in B.) Exposure of cells to agonist (indicated by the light- colored bar) produces a cyclic AMP (cAMP) response. A reduced cAMP response is observed in the continued presence of agonist; this “desensi- tization” typically occurs within a few minutes. If agonist is removed after a short time (typically several to tens of minutes, indicated by broken line on abscissa), cells recover full responsiveness to a subsequent addition of agonist (second light-colored bar). This “resensitization” fails to occur, or occurs incompletely, if cells are exposed to agonist repeatedly or over a more prolonged time period. B: Agonist binding to receptors initiates signaling by promoting receptor interaction with G proteins (Gs) located in the cytoplasm (step 1 in the diagram). Agonist-activated receptors are phosphorylated by a G protein-coupled receptor kinase (GRK), preventing receptor interaction with Gs and promoting binding of a different protein, β-arrestin (β-Arr), to the receptor (step 2). The receptor-arrestin complex binds to coated pits, promoting receptor internal- ization (step 3). Dissociation of agonist from internalized receptors reduces β-Arr binding affinity, allowing dephosphorylation of receptors by a phosphatase (P’ase, step 4) and return of receptors to the plasma membrane (step 5); together, these events result in the efficient resensitiza- tion of cellular responsiveness. Repeated or prolonged exposure of cells to agonist favors the delivery of internalized receptors to lysosomes (step 6), promoting receptor down-regulation rather than resensitization. calcium-sensitive protein kinase called protein kinase C. IP3 is channels promotes the binding of Ca2+ to the calcium-binding water-soluble and diffuses through the cytoplasm to trigger release protein calmodulin, which regulates activities of other enzymes, of Ca2+ by binding to ligand-gated calcium channels in the limit- including calcium-dependent protein kinases. ing membranes of internal storage vesicles. Elevated cytoplasmic With its multiple second messengers and protein kinases, the Ca2+ concentration resulting from IP3-promoted opening of these phosphoinositide signaling pathway is much more complex than 34    SECTION I Basic Principles Agonist the cAMP pathway. For example, different cell types may contain one or more specialized calcium- and calmodulin-dependent kinases with limited substrate specificity (eg, myosin light-chain kinase) in addition to a general calcium- and calmodulin- Gs AC Membrane dependent kinase that can phosphorylate a wide variety of protein substrates. Furthermore, at least nine structurally distinct types of protein kinase C have been identified. Rec As in the cAMP system, multiple mechanisms damp or ter- ATP cAMP 5'-AMP minate signaling by this pathway. IP3 is inactivated by dephos- phorylation; diacylglycerol is either phosphorylated to yield PDE phosphatidic acid, which is then converted back into phospholip- R2 cAMP4 ids, or it is deacylated to yield arachidonic acid; Ca2+ is actively R2C2 removed from the cytoplasm by Ca2+ pumps. 2C * These and other nonreceptor elements of the calcium- ATP ADP phosphoinositide signaling pathway are of considerable importance S~P in pharmacotherapy. For example, lithium ion, used in treatment of S bipolar (manic-depressive) disorder, affects the cellular metabolism Pi of phosphoinositides (see Chapter 29). P'ase C. Cyclic Guanosine Monophosphate (cGMP) Response Unlike cAMP, the ubiquitous and versatile carrier of diverse messages, cGMP has established signaling roles in only a few cell types. In intestinal mucosa and vascular smooth muscle, the FIGURE 2–13 The cAMP second messenger pathway. Key cGMP-based signal transduction mechanism closely parallels the proteins include hormone receptors (Rec), a stimulatory G protein (Gs), cAMP-mediated signaling mechanism. Ligands detected by cell- catalytic adenylyl cyclase (AC), phosphodiesterases (PDE) that hydro- lyze cAMP, cAMP-dependent kinases, with regulatory (R) and catalytic surface receptors stimulate membrane-bound guanylyl cyclase (C) subunits, protein substrates (S) of the kinases, and phosphatases to produce cGMP, and cGMP acts by stimulating a cGMP- (P’ase), which remove phosphates from substrate proteins. Open dependent protein kinase. The actions of cGMP in these cells are arrows denote regulatory effects. terminated by enzymatic degradation of the cyclic nucleotide and by dephosphorylation of kinase substrates. Increased cGMP concentration causes relaxation of vascular Agonist smooth muscle by a kinase-mediated mechanism that results in dephosphorylation of myosin light chains (see Figure 12–2). In these smooth muscle cells, cGMP synthesis can be elevated by two trans- PIP2 membrane signaling mechanisms utilizing two different guanylyl R G PLC Membrane DAG cyclases. Atrial natriuretic peptide, a blood-borne peptide hormone, stimulates a transmembrane receptor by binding to its extracellular IP3 domain, thereby activating the guanylyl cyclase activity that resides PK-C * in the receptor’s intracellular domain. The other mechanism medi- ATP ADP ates responses to nitric oxide (NO; see Chapter 19), which is gener- Ca2+ ated in vascular endothelial cells in response to natural vasodilator CaM S S~P agents such as acetylcholine and histamine. After entering the target Pi cell, nitric oxide binds to and activates a cytoplasmic guanylyl cyclase (see Figure 19–2). A number of useful vasodilating drugs, such as E CaM-E * nitroglycerin and sodium nitroprusside used in treating cardiac isch- emia and acute hypertension, act by generating or mimicking nitric Response oxide. Other drugs produce vasodilation by inhibiting specific phos- phodiesterases, thereby interfering with the metabolic breakdown of cGMP. One such drug is sildenafil, used in treating erectile dysfunc- FIGURE 2–14 The Ca2+-phosphoinositide signaling pathway. Key proteins include hormone receptors (R), a G protein (G), a tion and pulmonary hypertension (see Chapter 12). phosphoinositide-specific phospholipase C (PLC), protein kinase C substrates of the kinase (S), calmodulin (CaM), and calmodulin- Interplay among Signaling Mechanisms binding enzymes (E), including kinases, phosphodiesterases, etc. (PIP2, phosphatidylinositol-4,5-bisphosphate; DAG, diacylglycerol; IP3, The calcium-phosphoinositide and cAMP signaling pathways inositol trisphosphate. Asterisk denotes activated state. Open arrows oppose one another in some cells and are complementary in others. denote regulatory effects.) For example, vasopr