CHAPTER 1: Basic Principles of Neuropharmacology

• An understanding of drug action in the brain must integrate knowledge of the molecular and cellular actions of a drug with their effects on brain circuitry.

• The clinical actions of a drug in the brain often are due to neural plasticity—the long-term adaptations of neurons to the sustained short-term actions of a drug.

• The binding of a drug to its specific target(s) normally is saturable and stereoselective.

• The specific binding of a drug to its target is quantified according to its affinity for the target, expressed as a dissociation constant (K_d), and the total amount of binding (B_max).

• Potency of a drug describes the strength of binding between the drug and its target; efficacy describes the maximal biologic effects that the drug exerts by binding to its target.

• Drugs can be classified as agonists, partial agonists, inverse agonists, partial inverse agonists, or antagonists.

• Modern neuropharmacology takes advantage of the tools of molecular biology, genetics, and cell biology as well as combinatorial chemistry, which is used to generate novel molecules that may function as new drugs.

• Functional genomics and proteomics will help identify novel drug targets.

• Pharmacogenetics will guide the choice of drug treatments based on an individual’s genetic constitution.

Neuropharmacology is the scientific study of the effects of drugs on the nervous system. Its primary focus is the actions of medications for psychiatric and neurologic disorders as well as those of drugs of abuse. Neuropharmacology also uses drugs as tools to form a better understanding of normal nervous system functioning. The goal of neuropharmacology is to apply information about drugs and their mechanisms of action to develop safer, more effective treatments and eventually curative and preventive measures for a host of nervous system abnormalities. The importance of neuropharmacology to medical practice, and to society at large, is difficult to overstate. Drugs that act on the nervous system, including antidepressant, antianxiety, anticonvulsant, and antipsychotic agents, are among the most widely prescribed medications. Moreover, commonly prescribed medications that act on other organ systems often are associated with side effects that involve the nervous system and in turn may limit their clinical utility. In addition, a substantial number of individuals use common substances, such as caffeine, alcohol, and nicotine, that are included in the domain of neuropharmacology because of their effects on the central nervous system (CNS). In a much smaller fraction of the population, these and other drugs are used compulsively, in a manner that constitutes an addiction. Drug abuse and addiction exact an astoundingly high financial and human toll on society through direct adverse effects, such as lung cancer and hepatic cirrhosis, and indirect adverse effects—for example, accidents and AIDS—on health and productivity. Still other common afflictions of the nervous system, such as Alzheimer disease as just one example, are awaiting effective medications, further emphasizing the importance of neuropharmacology.

Neuropsychopharmacology is an all-encompassing term that typically is applied to all types of drug effects that influence nervous system functioning. The term psychopharmacology is often used to describe the effect of drugs on psychologic parameters such as emotions and cognition. Drugs that influence behavior are known as psychotropic agents. In this book we use the term neuropharmacology to describe the study of all drugs that affect the nervous system, whether they affect sensory perception, motor function, seizure activity, mood, higher cognitive function, or other forms of nervous system functioning.

The actions of drugs that affect the nervous system are considerably more complicated than those of drugs that act on other organ systems. To understand how drugs act on the nervous system, it is critical to integrate information about the molecular and cellular actions of a drug with knowledge of how these actions affect brain circuitry—a circuitry that is constantly changing in structure and function in response to both pharmacologic and nonpharmacologic input from the environment. The complexity that underlies such actions can be illustrated by consideration of fluoxetine, a widely prescribed antidepressant, and furosemide, a widely prescribed diuretic. The chemical actions of these drugs are fairly simple. Both drugs initially bind to their specific protein target: fluoxetine binds to and inhibits serotonin transporters, which normally inactivate the actions of the neurotransmitter serotonin (Chapter 6), and furosemide binds to and inhibits Cl⁻ channels located in the ascending loop of Henle in nephrons of the kidney. However, the relation between the chemical and clinical actions of these drugs—particularly those of fluoxetine—requires a more elaborate explanation.

The association between furosemide’s chemical and clinical activity is relatively straightforward. By inhibiting Cl⁻ transport in Henle’s loop, furosemide causes more Cl⁻ to remain in the lumen of the nephron tubule, which in turn requires more H₂O to remain in the tubule. Furosemide exerts this same effect on all nephrons in the kidney, and the increase in H₂O in individual nephron tubules combines to cause diuresis at the level of the kidney. Diuresis is achieved as soon as effective concentrations of the drug reach the kidney’s extracellular fluid, and is maintained with repeated use of the drug—for example, in the treatment of chronic congestive heart failure.

The relationship between the chemical and clinical actions of fluoxetine is more intricate and also more speculative. Most drugs that act on the nervous system interact with only the minute subset of the brain’s neurons that express the initial protein target of the drug. Fluoxetine directly affects only those neurons that use serotonin as a neurotransmitter—a few 100,000 out of approximately 100 billion neurons in the brain. By inhibiting serotonin reuptake by these neurons, fluoxetine enhances serotonergic transmission throughout the brain, but it is not known with certainty where in the brain enhanced serotonin function causes an antidepressant effect. Similarly, little is known about which of serotonin’s 14 known receptors must be activated to achieve an antidepressant response. Moreover, the mood-elevating effects of fluoxetine are not evident after initial exposure to the drug but require its continued use for several weeks. This delayed effect suggests that it is not the inhibition of serotonin transporters per se, but some adaptation to sustained increases in serotonin function that mediates the clinical actions of fluoxetine. However, where these adaptations occur in the brain and the nature of the adaptations at the molecular level have yet to be identified definitively.

The clinical actions of fluoxetine, like those of many neuropharmacologic agents, reflect drug-induced neural plasticity, which is the process by which neurons adapt over time in response to chronic disturbance. Consequently, to understand fully the effects of a neuropharmacologic drug, we must determine not only the initial effects of the drug but also the intraneuronal signals that control a neuron’s adaptations over time, the interneuronal signals through which neurons communicate with one another, and the ways in which large groups of neurons operate in circuits to produce complex brain functions.

Parts I and II of this book explore the intraneuronal and interneuronal signals that enable communication among neurons, which are fairly well understood. Part III addresses the relationships between circuits of neurons and complex brain functions, about which much remains to be discovered.

Neuropharmacology has contributed to many important advances in the neurosciences during the past several decades. Drugs have been used as tools to dissect the functions of the brain and of individual nerve cells under normal and pathophysiologic conditions. Historically, neuropharmacology has involved the delineation of diverse molecules that function as neurotransmitters in the nervous system, including monoamines, amino acids, purines, and peptides. The identification of many of these neurotransmitters and the elucidation of their synthesis, degradation, and receptors occurred in conjunction with studies of synthetic and plant substances that were known to exert profound effects on behavior. The neuropharmacology of ergot alkaloids, cocaine, and reserpine, for example, led to the discovery and characterization of monoamine neurotransmitter systems; opiate alkaloids such as morphine led to endogenous opioid systems; nicotine, muscarine, and cholinesterase inhibitors led to cholinergic systems; and caffeine and related substances led to purinergic systems.

Neuropharmacology also played a fundamental role in the delineation of the numerous receptor subtypes through which neurotransmitters elicit biologic responses. The early idea that one neurotransmitter acts on only one receptor was replaced decades ago with the recognition that for each neurotransmitter there are multiple receptors. This discovery led to the development of synthetic drugs with increasing selectivity for individual types of receptors, and the evolution of these neuropharmacologic agents has represented important advances in clinical medicine. These advances include the use of selective β₁-adrenergic antagonists for cardiovascular disease, selective β₂-adrenergic agonists for asthma, μ-opioid antagonists for opiate overdose, and 5HT_1D-serotonin agonists for migraine, to name just a few examples.

As well, the identification of multiple receptor subtypes for neurotransmitters contributed to the recognition of complex postreceptor signal transduction cascades through which receptors ultimately produce their biologic responses. From G proteins to second messengers to protein phosphorylation pathways to regulation of gene expression, studies of the effects of drugs on the nervous system have provided crucial windows onto the functioning of intracellular signaling. For instance, investigation of the mechanisms by which organic nitrates cause vasodilation in the treatment of angina led to the discovery of nitric oxide as a critical signaling molecule, and studies of aspirin and related nonsteroidal anti-inflammatory drugs (NSAIDs) led to the discovery of a host of signaling molecules derived from arachidonic acid, including prostaglandins and leukotrienes.

Drugs serve as prototypical external or environmental factors in determining how the brain adapts or maladapts over time in response to repeated perturbations. Many adaptations that occur in response to repeated drug exposure are models for adaptations to other external exposures, including stress and experience.

The ability of a drug to produce an effect on an organism is dependent on many of its properties, from its absorption to its stability to its elimination. To briefly summarize these processes, the first factor to be considered is the route of administration, which can determine how rapidly a drug reaches its target organ and which organs it affects. Oral administration typically results in a relatively slow onset of action. Parenteral describes all other routes of administration, including subcutaneous (under the skin), intraperitoneal (into the peritoneal–abdominal cavity), intravenous (into the venous system), intracerebroventricular (into the cerebral ventricular system), intrathecal (into spinal fluid), and intracerebral (into the brain parenchyma) delivery. The bioavailability of a drug determines how much of the drug that is administered actually reaches its target. Bioavailability can be influenced by absorption of the drug from the gut if administered orally. It also can be affected by binding of the drug to plasma proteins, which makes the drug unavailable to bind to its target. Moreover, it can be influenced by a drug’s ability to penetrate the blood–brain barrier if the drug acts on the brain (Chapter 2), or its ability to permeate cell membranes if the drug acts on intracellular proteins.

Drug action also depends on the stability of the drug once it is absorbed, that is, how rapidly it is metabolized to inactive congeners or eliminated from the body through urine, bile, or exhaled air. Some drugs (prodrugs) must be converted into active metabolites before they can exert their biologic effects.

Each of these factors, which can be categorized as pharmacokinetic considerations, is a critical determinant of drug action and influences both the clinical use of drugs and the process of developing new agents. However, these pharmacokinetic properties are not discussed in detail in this book because they are not, strictly speaking, related to the underlying mechanisms of drug action—the pharmacodynamic features that are the primary concern of these chapters. As an introduction to this topic, a brief description of the process by which a drug interacts with its initial protein target follows. Pharmacogenetics, which describes the influence of an individual’s genes in determining the response to a given drug, is also critical, but still in the earliest phases of understanding (see below).

Drug Binding

Neuropharmacology is changing rapidly in response to the molecular revolution. In previous decades, neuropharmacology focused on the synapse and, more particularly, on the effects of drugs on neurotransmitters or neurotransmitter receptors. The action of drugs on synaptic targets remains an important field of investigation. The initial target of a drug generally determines the particular cells and neural circuits on which the drug acts and at the same time the potential efficacy and side effects of the pharmacologic agent. However, the molecular revolution has made it clear that the initial binding of a drug to its target—for example, the binding of a drug to a neurotransmitter receptor—is only the beginning of a signaling cascade that affects the behavior of cells and ultimately complex circuits.

When a drug binds to a protein, it affects the functioning of that protein, thereby establishing a form of allosteric regulation. A drug can conceivably bind to any site on a protein. A simple site may involve just a few contiguous amino acid residues in a protein’s primary structure, while a relatively complex site may involve discontinuous residues from the protein’s primary structure that are brought near each other by the protein’s secondary and tertiary structures. Ultimately, the three-dimensional shape, or conformation, of a binding site and the electrostatic charges distributed across the site must complement the shape and charge of the drug. The interaction of a drug with its binding site can influence the intrinsic activity of the target protein, for example, the catalytic activity of an enzyme or the conductance of an ion channel, or it can influence the ability of the protein to interact with some other molecule, such as the ability of a receptor to bind to its neurotransmitter.

In classic studies of drug mechanisms of action, a mechanism is defined by a drug’s ability to bind to an unknown receptor in tissue homogenates or on tissue sections. In these studies the drug, termed the ligand, is radiolabeled and incubated with a tissue preparation, which is washed extensively to remove loosely bound drug. A radioactive atom must be added to the drug without altering its ligand binding properties, a process that can be exceedingly difficult. The resulting ligand binding should be specific; that is, the ligand must bind to its specific target protein, which must be distinguished from binding to other proteins or even to the wall of a plastic test tube. In many cases, binding is stereoselective, or specific for only one stereoisomer of a drug. Binding also should be saturable. A limited amount of ligand binding occurs in the preparation because the amount of the specific target is limited. (A tissue preparation contains a finite amount of an individual receptor protein compared with a test tube wall, which is theoretically infinite.) Additionally, binding should attain a steady state. Time, temperature, and other conditions of incubation should enable the ligand binding to achieve a state of equilibrium.

The extent to which a ligand binds to a tissue preparation is a function of the concentration of the ligand 1–1. The total binding comprises two components: (1) specific binding, which is saturable, and (2) nonspecific binding, which is not saturable. In the ideal situation, in which binding to a specific receptor site is competitive and fully reversible in the steady state, specific binding can be defined as the fraction of total binding that can be displaced by incubating the radiolabeled ligand–tissue mixture with a large excess of unlabeled ligand. Conversely, the nondisplaceable radioactive portion of the preparation is considered nonspecific binding.

There are several discrepancies, however, between ideal and actual conditions. Not all binding to target proteins is truly reversible; the affinity of some ligand–receptor interactions is so high that resulting complexes are not readily dissociable. Moreover, artifactual sites may be present and may show striking apparent specificity. While the ideal situation assumes that the tissue preparation contains just one specific target, in actuality many drugs can bind specifically to many related subtypes of a protein target; for example, serotonin binds to numerous subtypes of serotonin receptors. Consequently, the resulting binding curves can be quite complicated and difficult to interpret.

The specific binding of a ligand to a tissue preparation is quantified according to two properties: the affinity of the binding, which is expressed as a dissociation constant (K_d), and the total amount of the binding (B_max) 1–2. These terms are analogous to those used in studies of enzyme kinetics—for example, the Michaelis–Menten equation—in which K_a is the activation constant for an enzyme and its cofactor and V_max is the maximum catalytic activity of the enzyme. The K_d is defined as the concentration of ligand at which half of the specific binding sites are occupied; larger K_d values (eg, 100 nM vs 1 nM) reflect lower affinities of the drug. When ligand binding is plotted as a function of the log of drug concentration, a sigmoidal curve is obtained 1–2B. Ligand binding data are often transformed mathematically to yield a Scatchard plot, in which the ratio of bound ligand to free ligand is plotted as a function of bound ligand 1–3. Because it is difficult to measure the amount of free (unbound) ligand, total ligand minus bound ligand is used. The shape of Scatchard plots provides an indication of the number of binding sites in a tissue preparation, as well as the K_d and B_max values for each site.

Another method for studying ligand–target interactions makes use of competition curves. These curves describe the ability of a drug to compete with a radioligand in binding to a tissue preparation 1–4. The drug concentration at which half of the radioligand binding is displaced (K_i) is a measure of the affinity of the drug for a binding site in the context of a specific radioligand. Historically, such competition studies have played an important role in defining many subtypes of neurotransmitter receptors. In general, such pharmacologic distinctions of receptors accurately predicted broad categories of receptor proteins, which subsequently were identified with greater precision by means of molecular cloning techniques, as discussed later in this chapter. In ideal situations, the competing drug and the radioligand bind to the same site of the target protein; such binding is termed competitive. In more complicated situations, the drug and radioligand bind to different sites on the same protein; in such cases, the binding is noncompetitive and results in far more complicated competition curves. These assessments of ligand binding, which have traditionally been performed on tissue homogenates or membrane fractions, can also be performed on brain sections, a process termed receptor autoradiography.

As with any technique, the limitations of ligand binding assays must be appreciated. One of the most critical limitations is that binding assays, and the determination of K_d, K_i, and B_max values, are highly dependent on experimental conditions and therefore must be interpreted with considerable caution. The specific radioligand, the temperature of incubation, the salt and ionic content of the buffer, and the presence of different guanine nucleotides (Chapter 4) are among the many factors that can exert dramatic effects on ligand binding. The cloning of receptors and other target proteins, and the ability to express them on cells without endogenous expression of the target, has made at least some aspects of characterizing the binding properties of drugs more straightforward.

Drug Efficacy

Binding studies describe the physical relationship between a drug and its target but do not directly assess the biologic consequences of this association. Although drug binding and biologic effect are intricately related, they help define two distinct aspects of drug action: potency and efficacy. Potency (affinity, or K_d) describes the strength of the binding between a drug and its target. Efficacy describes the biologic effect exerted on the target by virtue of the drug binding. These properties can be understood by considering the effect of a drug on a neurotransmitter receptor. As previously explained, the drug must physically bind to the receptor, which requires a physical attraction between the two. Subsequently, that binding must elicit a change in the receptor that leads to a biologic response. For a G protein–coupled receptor, drug binding must trigger a conformational change in the receptor that alters its interactions with its G protein α subunit (Chapter 3). For a ligand-gated channel (receptor ionophore), drug binding must trigger a conformational change that opens or closes the pore that is intrinsic to the receptor.

Drugs differ dramatically with respect to their potency and efficacy. Traditionally, two categories of drug have been described: agonist and antagonist. When an agonist binds to a receptor, it mimics the endogenous neurotransmitter by producing the same conformational change and hence the same biologic response. According to this definition, all neurotransmitters are receptor agonists. When an antagonist binds to a receptor, it elicits no such change; thus, an antagonist is inherently inert and exerts a biologic effect only by interfering with an endogenous ligand. For opioid receptors, which are receptors for the endogenous opioid peptides such as the enkephalins (Chapter 7), morphine and naloxone are classic examples of an agonist and antagonist, respectively. The differences in efficacy associated with agonists and antagonists are independent of the affinity with which each binds to its receptor; both can exhibit high or low affinities. How can two molecules that bind to the same receptor site exert such different effects on the receptor? There are two possible explanations. An antagonist may share one moiety that is required for binding to the receptor with an agonist, but may lack a moiety required for efficacy. Alternatively, the antagonist and agonist may bind to overlapping but distinct sites on the receptor.

In addition to the actions of classic agonists and antagonists, an intermediate category of drug efficacy is exemplified by partial agonists. When a drug binds to a receptor and elicits only a partial biologic response, the drug presumably lacks a portion of the molecule required for full biologic effect or binds to a slightly different site on the receptor 1–5. An interesting situation arises when partial agonists possess high potency. At low drug doses, a mild agonist effect is obtained. At high doses, a similarly mild agonist effect is obtained because of limits in the intrinsic efficacy of the molecule. However, at high doses, the drug can antagonize the ability of a full agonist, including the endogenous neurotransmitter, to activate the receptor because its affinity is greater than that of the full agonist. For this reason, partial agonists are sometimes referred to as mixed agonists–antagonists. Partial agonists can be quite useful clinically; for example, buprenorphine is a partial agonist at opioid receptors and is used in the treatment of chronic pain and opiate addiction (Chapters 11 and 16). At low doses, buprenorphine elicits a mild analgesic and rewarding effect. Higher doses not only fail to yield a stronger effect, which limits the abuse liability of this drug, but also antagonize the action of full opioid agonists and thereby discourage abuse of opiates such as morphine.

Inverse agonists achieve efficacy in still another way. When an inverse agonist binds to a receptor, it elicits the biologic response that is the opposite of that associated with an agonist. If an agonist opens an ion channel, an inverse agonist closes the channel. If an agonist facilitates receptor-to-G protein coupling, an inverse agonist attenuates such coupling. The action of an inverse agonist requires some basal activity on the part of the receptor, which means that the receptor is not quiescent in the absence of ligand but instead possesses some level of intrinsic biologic activity, such as channel conductance or G protein coupling. Indeed, most receptors do exhibit such baseline activity.

Very few drugs can be placed in discrete categories—agonist, antagonist, and inverse agonist. Many drugs that are classically described as agonists, such as morphine, are not full agonists but strong partial agonists. Conversely, many drugs that are classically categorized as antagonists—for example, naloxone—are not completely inert and thus can be very weak partial agonists. Moreover, some neurotransmitters show less efficacy than synthetic drugs, which indicates that they also are partial agonists. Consequently, drugs should be thought of as existing on a continuum ranging from full agonist to inert antagonist to full inverse agonist 1–6.

The complex nature of the interactions between drugs and their target receptors can be illustrated by a discussion of the γ-aminobutyric acid receptor (GABA_A)—an important receptor for the neurotransmitter GABA (Chapter 5). This receptor is a heteropentamer that has two main types of subunits, α and β. GABA binds to a site on the β subunit and triggers the opening of a Cl⁻ channel that is intrinsic to the receptor complex. Muscimol (an agonist) and bicuculline (an antagonist) also bind at this site and thus compete with GABA. The α subunit of the GABA_A receptor contains a binding site for a class of synthetic molecules known as benzodiazepines. Agonists that bind at this site, such as diazepam, are antianxiety agents that, when bound to the site, allosterically facilitate the ability of GABA to bind to and activate the GABA_A receptor (Chapters 5 and 15). Antagonists at this site, such as flumazenil, bind to the site but do not affect receptor function. Because this site lacks endogenous ligands, flumazenil is clinically inactive when bound to the GABA_A receptor; however, it can be used to treat diazepam overdose because it displaces diazepam from the binding site. Inverse agonists, such as β-carboline, which intensify anxiety, bind very near the benzodiazepine agonist site and allosterically inhibit the ability of GABA to bind to and activate the receptor. Diazepam and β-carboline are a noncompetitive agonist and inverse agonist, respectively, of the GABA_A receptor, in that they interact with a binding site distinct from the GABA site. They are also considered positive and negative allosteric modulators of the GABA_A receptor. Although this type of complex receptor pharmacology was first described for the GABA_A receptor, a similar level of complexity can characterize drug interaction at virtually any type of receptor.

In more recent years, more complex actions of receptors have contributed to still additional modes of drug action. Specific types of receptors can form complexes with other receptors, with a resulting generation of novel agonist activity for the receptor heteromer 1–7A. For example, heterodimers of μ and δ opioid receptors can be activated or inhibited by drugs that show lower affinities to either receptor type alone. As well, different agonists at a given receptor can direct the receptor to signal via distinct intracellular pathways, a process referred to as ligand-directed or biased signaling 1–7B. Similar ligand–directed differences can be seen with inverse agonists. The discovery of ligand-directed signaling greatly complicates the consideration of the intrinsic efficacy of a ligand, since ligands might vary qualitatively in addition to quantitatively with respect to their downstream actions.

Finally, it must be emphasized that binding sites with a high affinity for drugs do not necessarily have an endogenous ligand. No evidence, for example, supports the existence of an endogenous ligand for the benzodiazepine binding site on the GABA_A receptor. Rather, the discovery of this class of drugs and their binding site is testimony to the power and promise of medicinal chemistry to target distinctive features of proteins that are not exploited by nature. Indeed, our growing knowledge of the many complexities of receptor function and drug action discussed above is contributing to the development of a host of agents with novel pharmacologic and hence clinical activity.

Dose-Dependent Drug Response

That the effect of a drug on a target protein is dependent on the concentration of a drug is implicit in the discussions of drug binding and efficacy presented in the preceding sections. This dose dependency of drug action is one of the principal tenets of neuropharmacology and illustrates the importance of studying the effects of a wide range of drug doses.

One application of dose–response curves is in determining whether a form of treatment—for example, chronic exposure to an antidepressant—increases or decreases the responsiveness of a particular receptor system. Hypothetical cases are illustrated in 1–8, which shows that a reduction in receptor sensitivity in response to treatment is characterized by a rightward or downward shift in the dose–response curve, whereas an increase in receptor sensitivity is characterized by a leftward or upward shift in the dose–response curve.

Dose–response curves also can reveal that the biologic effects of a specific drug may not be a simple (monotonic) function of drug dose. When the effects of a drug are more complex, nonmonotonic, for example, they are represented by an inverted U-shaped curve 1–9. Such drugs elicit a progressively greater biologic response with greater drug dose up to a point, after which higher drug doses begin to produce smaller effects. This shift in effect most likely occurs because the drug begins to act on a different target protein at higher drug doses, and action on the second target opposes the effects of the first. Alternatively, high doses may cause receptor desensitization.

The analysis of full dose–response curves is necessary to determine reliably whether a particular treatment causes an increase or decrease in drug responsiveness. 1–10 shows a leftward shift in the dose–response curve for a drug whose biologic effects are an inverted U-shaped function of drug concentration. Without an analysis of the full dose–response curve, an investigator may incorrectly interpret effects of the drug; for example, depending on the concentration of drug used to activate the receptor, a shift in the curve may indicate a reduction, an increase, or a lack of change in drug response.

Drug Interaction With Nonreceptor Proteins

Although most principles of drug action have been ascertained from studies of neurotransmitter and hormone receptors, the same general principles apply to interactions between drugs and nonreceptor proteins. A drug binds to a specific site on a protein, which can be determined by means of ligand binding assays. Drug binding influences the function of a protein by either facilitating or inhibiting that protein’s normal functioning, including its interactions with other macromolecules. Some drugs create a new function for the protein to which they bind; examples include FK506 and related drugs that bind immunophilins (Chapter 4). When such drugs bind to immunophilin proteins, the proteins become potent inhibitors of calcineurin, a protein phosphatase.

The conditions under which two proteins interact are conceptually similar to those for drug–target interactions. Protein–protein interactions have emerged as a central theme of cell regulation (Chapter 4). The binding of proteins such as transcription factors to specific sequences of DNA, which is a key mechanism of gene regulation in development and in neural plasticity in the adult, also operates according to principles like those of drug–target interactions.

Neuropharmacology originally was a phenomenologic science. An investigator administered a drug to an animal or a cell preparation and examined the response. There were two major drawbacks to this black box approach 1–11. First, it did not elucidate the mechanisms of drug action and thus did not enable investigators to relate the initial action of a drug on its protein target to the clinical effects of the drug. Earlier in this chapter, it was pointed out that the actions of fluoxetine extend beyond inhibiting serotonin transporters or increasing serotonin function. To understand fully how fluoxetine works it is necessary to determine its action on the overall workings of the brain, from its effects on molecules and cells to its effects on neural circuits and ultimately on behavior.

Second, traditional neuropharmacology depended on ligand binding studies to identify the protein targets of a drug and to understand its actions on brain function. Such protein targets were typically defined by potency series, which compared the ability of ligands to interact with different binding sites. However, many of the neurotransmitter receptors that were originally identified by ligand binding studies proved to be misleading once it became possible to identify individual proteins by molecular means with great precision. Whereas pharmacologic studies identified 3 major subtypes of serotonin receptors, we now know that 14 subtypes exist, with some subtypes misclassified by ligand binding experiments (Chapter 6).

Neuropharmacologists are currently using the penetrating tools of molecular biology and cellular physiology to extend their experimental repertoire. New research is aimed at defining the action of a drug on its cloned protein target in precise molecular terms and analyzing the protein target in various functional states, for example, in phosphorylated versus dephosphorylated states. Ultimately, investigators delineate the crystal structure of these proteins before and after they are bound to a drug to better understand the ways in which a drug alters the shape and surface charges of a protein.

Neuropharmacologists are using these new approaches to identify and validate novel targets for drug development, so that novel drugs can be synthesized to interact with those targets. Combinatorial chemistry is enabling the development of a large number of drugs with unique and diverse chemical structures. It incorporates knowledge of drug–target interactions at the molecular level, or structure–activity relationships, to determine what types of chemical moieties can be added to a drug to alter its actions on a protein target. Much of this initial characterization is carried out virtually, by exploring theoretical interactions between chemical structures of small molecules and three-dimensional structures of protein targets. High-throughput screening then allows the testing of a large number of chemical agents, which are promising by such chemi-informatic analyses, to discover their actual abilities to influence cloned proteins of interest. Finally, exploratory research is under way to use higher-throughput behavioral analyses to facilitate the discovery of novel classes of compound that exert interesting, and potentially clinically useful, behavioral effects in laboratory animals. Together, these tools are enabling researchers to develop any desired types of drugs—for example, agonists, antagonists, weak partial agonists, or inverse agonists—for any targeted proteins or behavioral end point.

An interesting question in the field is whether a specific drug is more desirable than a less specific one. The initial era of modern neuropharmacology focused on the search for ever more specific medicinal agents, for instance, moving from antipsychotic drugs that antagonize numerous receptors to more specific agents that antagonize just one receptor type. The expectation was that such “cleaner” drugs would show similar or even greater efficacy without the unwanted side effects of the “dirty” drugs. To date, however, such specific agents have been disappointing clinically, which has led to the realization that drugs with multiple targets may offer some advantages for complex brain diseases. The focus on specific agents is also somewhat semantic: fluoxetine is quite specific in that to a first approximation it antagonizes serotonin transporters only, but in doing so it promotes serotonin action at 14 receptors that are expressed by a large fraction of all of the brain’s 100 billion nerve cells.

Molecular Diversity of the Brain

Traditional neuropharmacology focused on a very narrow subset of cellular proteins, which included neurotransmitter receptors and transporters and proteins involved in the synthesis or degradation of neurotransmitters. Such proteins account for only a few hundred of perhaps hundreds of thousands of distinct gene products that are expressed in the brain once alternative splicing and protein processing are taken into account (Chapter 4).

The concentration on neurotransmitter-based drugs excessively narrowed the scope of neuropharmacology and interfered with the development of drugs based on new mechanisms of action. Focusing on the 100,000 or more proteins expressed in the brain but not based on neurotransmitters is likely to lead to the identification of fundamentally novel classes of neuropharmacologic agents. Over the next decade, functional genomics and proteomics—the processes of sequencing, identifying, and characterizing individual gene products—will provide a template for exploring this vast array of proteins. Large numbers of proteins that regulate receptor sensitivity already have been identified, as have large numbers of modulatory proteins that govern these regulatory proteins. Indeed, it is an exciting prospect that the completion of the various genome projects is allowing us to know the full set of receptors and regulatory proteins expressed by nerve cells. Genomic and proteomic methods are also making it possible to better understand the complex effects of a drug or disease state on target neurons. For example, advanced bioinformatic analysis of global changes in gene expression induced in a given cell type by a drug is making it possible to deduce key regulatory proteins that drive important aspects of drug action. Such gene networks are then used to drive the search for novel drug targets.

Neuropharmacology in the postgenomics era will be involved with the development of drugs aimed at a new set of proteins and the clarification of the mechanisms by which these drugs regulate brain function and behavior. This potential transformation of current treatment of psychiatric and neurologic disorders holds great promise for the future.

Pharmacogenetics

Pharmacogenetics represents the ultimate application of the molecular era to neuropharmacology. It holds the promise of personalized or precision medicine, where an individual’s particular genetic makeup predicts his or her response to a given medication. For instance, it should be possible one day to identify distinct etiologic subtypes of genetically heterogeneous disorders, such as autism, schizophrenia, and epilepsy, to name just a few examples, based on genetic testing. These findings could be combined with brain imaging or other technologies to define pathophysiologic subtypes of the illnesses and to thereby permit treatment with drugs aimed specifically at the underlying biologic abnormalities. Neuron-like cells derived from a patient’s skin biopsy or other peripheral tissue might even be used to study the pathophysiology and treatment of that individual’s illness 1–1.

Pharmacogenetics is in the earliest stages of development, particularly for diseases of the nervous system, but a few examples are currently in clinical practice. Cytochrome P450 enzymes, which are encoded by the CYP gene superfamily, are involved in the metabolism of most drugs. We now know that individuals with particular CYP gene variants metabolize certain drugs much more or less effectively than most people. Such genotypes account for some of the differences in drug responsiveness observed clinically, and have been used to approximate the unusually high or low drug doses required in these individuals.

To date, pharmacogenetics is being applied most widely to the treatment of cancer. Monoclonal antibodies directed against a protein involved in a particular type of cancer, or even specific for an individual’s cancer, are in clinical use. The use of tamoxifen, an estrogen receptor antagonist, in the treatment of breast cancer is reserved for those individuals whose tumors express the estrogen receptor. Likewise, the use of epidermal growth factor (EGF) receptor antagonists, such as gefitinib or erlotinib, for treatment of several types of cancers is increasingly being targeted to individuals whose tumors express mutant forms of the receptor or its signaling proteins. This has recently been applied to glioblastomas, a particularly severe form of brain cancer: only 20% of glioblastomas respond to EGF receptor antagonists and recent work has shown that it is possible to predict those individuals who respond based on EGF receptor function in the tumors.

As large-scale genetic studies of nervous system diseases progress, and specific causative or vulnerability genes are discovered, it should be possible to increasingly match a particular treatment with a particular patient. While this is many years on the horizon, it will truly usher in the molecular era of pharmacotherapeutics.