Affinity and Efficacy

The following sections are included:

• Dedication
• Contents
• Acknowledgments

Most of us have personal experience with drug action, for we occasionally medicate ourselves with over-the-counter drugs and treatment by a physician often comes in the form of a prescription. This widespread use of drugs has led to common knowledge of their actions, and their molecular mechanisms have steadily come to light as reported in scientific journals and increasingly in lay publications. The ultimate explanation as to why drugs work often receives little attention, however. Simply stated, many drugs stimulate, inhibit or modulate natural signaling pathways…

Ligand-gated ion channels mediate the rapid transfer of information across nerve synapses in the brain and peripheral nervous system. They are also located in extrasynaptic regions of neurons as well as on glia and non-neuronal tissue. These receptors are composed of four to five protein subunits that span the plasma membrane and circumscribe a central ion pore. When the neurotransmitter binds to its pocket on the extracellular domain of the receptor it causes the channel to open, allowing specific ions to flow in or out of the postsynaptic cell depending upon their electrochemical gradients and the selectivity of the channel. Channels that conduct an influx of sodium or calcium depolarize the cell, whereas those that conduct an influx of Cl⁻ hyperpolarize the cell or make it more difficult to be depolarized. These channel currents instantaneously generate electrotonic currents in the region of the postsynaptic membrane. The sum of the depolarizing and hyperpolarizing currents ultimately determines the excitability of the cell. Without this rapid trans-synaptic communication, the information processing capabilities of neural networks would be severely limited…

Ligand-gated ion channels provide a unique opportunity to study affinity and efficacy. This is because the receptor is an ion channel, and its activation state can be measured directly using electrophysiological methods. The measured current can be analyzed to determine how occupancy of a single receptor increases the probability of channel opening or how occupancy of the receptor population causes a graded increase in the current response of the cell. From this approach, we should be able to develop a quantitative measure of affinity and efficacy…

If a drug binds to a ligand-gated ion channel and has no effect, it is still possible to estimate its affinity by measuring how it affects the ionic current elicited by an agonist that binds to the same site. This type of interaction is defined as competitive, and the method for its analysis is the same regardless of whether the two drugs interact at the primary orthosteric site or a secondary allosteric site. In this chapter, we will consider competitive interactions from the perspective of the orthosteric site…

Allosterism is a flexible mechanism of drug action that can be tailored to modify endogenous signaling with precision. This is accomplished through allosteric modulation of the binding of the endogenous ligand by the binding of a drug to another site on the receptor. This secondary drug site is defined as the allosteric site, and conformational changes in this site are associated with complementary changes in the conformation of the orthosteric site. Thus, both sites are allosterically linked, which provides the mechanism for the reciprocal modulation in ligand binding…

In his great book, Origin of Species, Darwin acknowledged that the evolution of an exquisite structure like the eye might seem too implausible so he addressed the issue in a section entitled, “Organs of extreme perfection and complication”. Apparently it is not that implausible because contemporary biologists deduce that eyes must have evolved a minimum of 40 times to explain their distribution throughout the animal kingdom. These organs differ vastly in their ingenious solution to the problem of catching photons and converting them into an electrical signal. But at the heart of the eye in many diverse species is the remarkable light-detecting protein, rhodopsin. It is present in the eyes of vertebrates, arthropods and mollusks, as well as in other light-detecting structures, like melanocytes, pineal glands and the eye spots of algae. Rhodopsin triggers a different signaling pathway in vertebrates than in arthropods and mollusks, but the first activation step and second transduction mechanism follow the same pattern in these groups. This general mechanism also accounts for signal transduction in a large class of proteins known as G protein-coupled receptors. In this chapter, I describe the elements of receptor-G protein signaling beginning with phototransduction in the mammalian retina.

When a drug binds to and activates a GPCR, the potential outcomes are numerous. For, depending on the location of its receptor, the drug could cause diuresis, hypotension, pain relief, motivation, alertness, appetite suppression, a reduction in airway resistance in the lung, and many other effects. Each of these is the result of a train of events beginning with the initial receptor activation and ending with the final response. One could also measure the different steps that lead up to the final response, which may include things like, adenylate cyclase activity, phospholipase Cβ activity, Ca²⁺ mobilization, activation of ion channels, recruitment of arrestin, phosphorylation of proteins, activation of contractile filaments, and modulation of the firing rate of a neuron in a neuronal network. Any of these responses, whether proximal or distal in the signaling pathway, could be analyzed to glean information about the initial agonist-receptor interaction and how this interaction can be quantified in terms of the two components, affinity and efficacy…

G protein-coupled receptors number about a thousand, and the functions they mediate throughout the body are limitless. At any one receptor, agonist activation elicits a train of biochemical events that culminate in a physiological response. Each event represents a potentially measurable response. For example, when β₂ adrenoceptors are activated in tracheal smooth muscle, one could measure changes in adenylate cyclase activity, Ca²⁺ -activated potassium channel currents, Ca²⁺ influx, smooth-muscle contraction, airway resistance and many others. The β₂-adrenoceptor could also be expressed in a cell line, and an endogenous signaling pathway can be measured (e.g., cAMP accumulation). Alternatively, a recombinant receptor and reporter system (CREB/luciferase) could be introduced into the cell and its signal can be monitored. Thus, the output from receptor activation is diverse, but the input for a given agonist-receptor pair consists of only two components, affinity and efficacy. The goal of this chapter is to explain how to estimate these components from the output…

Most G protein-coupled receptors are silent in their native environment unless activated by an agonist. But when expressed at high density in heterologous cells, they often elicit small signaling responses in the absence of agonist, like changes in adenylate cyclase activity, stimulation in phosphoinositide hydrolysis, or stimulation in [³⁵S]GTPγS binding, depending upon their preference for G proteins. Given that these proteins evolved to function as molecular switches that flip between active and inactive states, it is not surprising that, at any given instant, a very small fraction of the receptor population might briefly switch into the active state in the absence of agonist because of thermal activation. In fact, it would be impossible for a ligand to induce a conformational change in a receptor unless the receptor has some tendency, however small, to undergo the conformational change spontaneously (see below). These spontaneously active receptors could generate a measurable response if the receptor population is sufficiently large or if the downstream signaling pathway is highly amplified. In this chapter, I describe how to measure the activity of orthosteric ligands at receptors exhibiting spontaneous receptor activity (constitutive activity)…

How a drug interacts with the active and inactive states of a receptor ultimately determines its effects the body. An agonist, for example, increases the probability that the receptor will assume the active conformation or remain in this conformation for a longer period of time. At a ligand-gated ion channel, this corresponds to an increase in the frequency and duration of random unitary conductance events. Even though each event is probabilistic, the summation of all of the channel openings in the cell yields an average current that is a highly predictable function of how many receptors are occupied by the agonist. The size of this response is correlated with the size of the receptor population, and its consequences can vary depending upon where the receptor is expressed in the body…

So far we have considered how to analyze the responses that individual drugs elicit through G protein-coupled receptors. In this chapter, I describe how to analyze the response resulting from the interaction between two drugs that compete for the same binding site. This site is usually the locus where the natural neurotransmitter binds (orthosteric site), but the analysis is applicable to competitive interactions at any site on the receptor. When an allosteric agonist binds to a receptor and turns it on, for example, the inhibitory behavior of an allosteric antagonist is often indistinguishable from that of a conventional orthosteric antagonist inhibiting the effects of an endogenous neurotransmitter…

When an endogenous ligand binds to a G protein-coupled receptor, it initiates a signaling cascade that ultimately causes a physiological response. An allosteric drug that binds to a secondary site on the receptor may alter the output of the pathway by modifying the affinity and efficacy components of the orthosteric ligand-receptor complex. In this chapter, I describe how to measure this modulation…

In prior chapters, the interaction of drugs with receptors has been discussed exclusively from the perspective of functional assays. In this chapter, I describe how radioligand binding methods can be used to throw light on this topic. Having an alternative means of measuring drug-receptor interactions can often strengthen or extend conclusions drawn from functional studies…

The following section is included:

• Index