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FIGURE 5.2 Amplification inherent in different vantage points along the stimulus-response pathway in cells. Agonists have a rank order of efficacy of 3 > 2 > 1 and a rank order of potency of 2 > 1 > 3. Assays proximal to the agonist-receptor interaction have the least amplification. The product of the initial interaction goes on to activate other processes in the cell. The signal is generally amplified. As this continues, texture with respect to differences in efficacy is lost and the agonists all demonstrate full agonism.

FIGURE 5.2 Amplification inherent in different vantage points along the stimulus-response pathway in cells. Agonists have a rank order of efficacy of 3 > 2 > 1 and a rank order of potency of 2 > 1 > 3. Assays proximal to the agonist-receptor interaction have the least amplification. The product of the initial interaction goes on to activate other processes in the cell. The signal is generally amplified. As this continues, texture with respect to differences in efficacy is lost and the agonists all demonstrate full agonism.

FIGURE 5.3 Different types of functional readouts of agonism. Receptors need not mediate cellular response but may demonstrate behaviors such as internalization into the cytoplasm of the cell (mechanism 1). Receptors can also interact with membrane proteins such as G-proteins (mechanism 2) and produce cytosolic messenger molecules (mechanism 3), which can go on to mediate gene expression (mechanism 4). Receptors can also mediate changes in cellular metabolism (mechanism 5).

FIGURE 5.3 Different types of functional readouts of agonism. Receptors need not mediate cellular response but may demonstrate behaviors such as internalization into the cytoplasm of the cell (mechanism 1). Receptors can also interact with membrane proteins such as G-proteins (mechanism 2) and produce cytosolic messenger molecules (mechanism 3), which can go on to mediate gene expression (mechanism 4). Receptors can also mediate changes in cellular metabolism (mechanism 5).

response pathway the agonism is observed the more amplified the signal. Figure 5.3 illustrates the effects of three agonists at different points along the stimulus-response cascade of a hypothetical cell. At the initial step (i.e., G-protein activation, ion channel opening), all are partial agonists and it can be seen that the order of potency is 2 > 1 > 3 and the order of efficacy is 3 > 2 > 1. If the effects of these agonists were to be observed at a step further in the stimulus-response cascade (i.e., production of second messenger), it can be seen that agonists 2 and 3 are full agonists while agonist 1 is a partial agonist. Their rank order of potency does not change but now there is no distinction between the relative efficacies of agonists 2 and 3. At yet another step in the cascade (namely, end organ response), all are full agonists with the same rank order of potency. The point of this simulation is to note the differences, in terms of the characterization of the agonists (full versus partial agonists, relative orders of efficacy), that occur by simply viewing their effects at different points along the stimulus-response pathway.

Historically, isolated tissues have been used as the primary form of functional assay, but since these usually come from animals the species differences coupled with the fact that human recombinant systems now can be used have made this approach obsolete. Functional assays in whole-cell formats, where end organ response is observed (these will be referred to as group I assays), can be found as specialized cells such as melanophores, yeast cells, or microphysiometry assays. Group II assays record the product of a pharmacological stimulation (for example, an induction of a gene that goes on to produce a traceable product such as light sensitive protein). Second messengers

(such as cyclic AMP, calcium, and inositol triphosphate) can also be monitored directly either in whole-cell or broken-cell formats (group III assays). Finally, membrane assays such as the observation of binding of GTPyS to G-proteins can be used. While this is an assay done in binding mode, it measures the ability of agonists to induce response and thus may also be considered a functional assay. It is worth considering the strengths and shortcomings of all of these approaches:

Group I assays (end organ response) are the most highly amplified and therefore most sensitive assays. This is an advantage in screening for weakly efficacious agonists but has the disadvantage of showing all agonists above a given level of efficacy to be full agonists. Under these circumstances, information about efficacy cannot be discerned from the assay since at least for all the agonists that produce maximal system response no information regarding relative efficacy can be obtained. There are cell culture group I assays. One such approach uses microphysiometry. All cells respond to changes in metabolism by adjustment of internal hydrogen ion concentration. This process is tightly controlled by hydrogen ion pumps that extrude hydrogen ions into the medium surrounding the cell. Therefore, with extremely sensitive monitoring of the pH surrounding cells in culture a sensitive indicator of cellular function can be obtained. Microphysiometry measures the hydrogen ion extrusion of cells to yield a generic readout of cellular function. Agonists can perturb this control of hydrogen ion output. One of the major advantages of this format is that it is generic (i.e., the observed does not depend on the nature of the biochemical coupling mechanisms in the cytosol of the cell). For example, the success of cell transfection

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FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor. (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors. (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and 100 nM. Dose-response curve for the effects seen in panel B.

Time (sec.) Log [calcitonin]

FIGURE 5.4 Microphysiometry responses of HEK 293 cells transfected with human calcitonin receptor. (a) Use of microphysiometry to detect receptor expression. Before transfection with human calcitonin receptor cDNA, HEK cells do not respond to human calcitonin. After transfection, calcitonin produces a metabolic response, thereby indicating successful membrane expression of receptors. (b) Cumulative concentration-response curve to human calcitonin shown in real time. Calcitonin added at the arrows in concentrations of 0.01, 0.1, 1.10, and 100 nM. Dose-response curve for the effects seen in panel B.

experiments can be monitored with microphysiometry. Unless receptors are biochemically tagged, it may be difficult to determine whether the transfection of cDNA for a receptor into a cell actually results in membrane expression of the receptor. On occasion, the cell is unable to process the cDNA to form the complete receptor and it is not expressed on the cell surface. Figure 5.4a shows microphysiometry responses to calcitonin (an agonist for the human calcitonin receptor) before and after transfection of the cells with cDNA for the human calcitonin receptor. The appearance of the calcitonin response indicates that successful membrane expression of the receptor occurred. Another positive feature of this format is the fact that responses can be observed in real time. This allows the observation of steady states and the possibility of obtaining cumulative dose-response curves to agonists (see Figure 5.4b and c).

A specialized cell type that is extremely valuable in drug discovery is the Xenopus laevis melanophore. This is a cell derived from the skin of frogs that controls the dispersion of pigment in response to receptor stimulation. Thus, activation of Gi protein causes the formation of small granules of pigment in the cell rendering them transparent to visible light. In contrast, activation of Gs and Gq protein causes dispersion of the melanin resulting in an opaque cell (loss of transmittance of visible light). Therefore, the activation of receptors can be observed in real time through changes in the transmittance of visible light through a cell monolayer. Figure 5.5 shows the activation of human b-adrenoceptors in melanophores by b-adrenoceptor agonists. It can be seen that activation of Gs protein by the activated b-adrenoceptor leads to an increase in pigmentation of the melanophore. This, in turn, is quantified as a reduced transmittance of visible light to yield graded responses to the agonists. One of the key features of this format is that the responses can be observed in real time. Figure 5.6a shows the reduced transmittance to visible light of melanophores transfected with human calcitonin receptor acitvated with the agonist human calcitonin. Another feature of this format is that the transfected receptors are very efficiently coupled (i.e., agonists are extremely potent in these systems). Figure 5.6b shows the dose-response curve to human calcitonin in transfected melanophores compared to the less efficiently coupled calcium fluorescence assay in human embryonic kidney cells for this same receptor.

Another specialized cell line that has been utilized for functional drug screening are yeast cells. A major

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