Pharmacological models

Nowadays, ligand and receptor interactions can be studied with a large variety of experimental systems and techniques (Figure 67). The information from each system is often complementary.

For a long time, physiological experiments constituted the sole approach for testing ligand-receptor interactions. Because of the indirect nature of the results (i.e. a 'distant' response is measured), information about the ligand-receptor interaction could be biased by nature of the experimental system. The positive side of this is that physiological experiments in intact organs or even in vivo are rather close to the clinical reality. The negative side of physiological experiments is that they do not provide clear-cut information about ligand-receptor interactions. Hence, they only constitute marginal tools for the purpose of receptor classification and identification. Indeed, EC50 values and intrinsic activities (a) of agonists are easy to measure, but tissue dependent. KD values and intrinsic efficacies (e) describe the

Figure 67 Pharmacological approaches and relevance of the provided information.

agonist-receptor interactions more accurately. Yet they are difficult to obtain (at least in the absence of radioligand binding studies). Schild regressions of shifted dose-response curves provide an accurate determination of antagonist Kj values, and they may represent the most useful physiological tool for pharmacological receptor classification.

Radioligand binding studies provide direct information about agonist and antagonist affinities for receptors. They also allow detection of the co-existence of receptor subclasses in a given tissue and, in certain instances (G protein-coupled receptors), even discrimination between agonists and antagonists (Figure 44). Because of its simplicity and accuracy, the radioligand binding approach is a very useful tool for the identification, classification and discovery of receptors, as well as for investigating the affinity and specificity of new potential drugs for receptors of interest. However, ra-dioligand binding experiments provide only crude information about the physiological actions and the therapeutic benefit of the investigated drugs.

Receptors from animal sources have long been used as templates for predicting drug activity on human receptors, and, in general, they are sufficiently good for this purpose. However, it has also been found that slight differences between human and animal receptors can have profound effects on drug activity. It is known that there are differences in affinity that result from relatively small amino acid sequence differences (even a single amino acid) between human and animal receptors. This is especially true for non-peptide antagonists for peptide receptors where it appears that evolution has produced mutations that have not altered binding of natural peptides, but do produce differences for foreign non-peptide ligands.

Possible species-related differences in drug action could be avoided by using human blood cells or on post-mortem obtained tissues. However, this material is sometimes hard to obtain and, because of post-mortem delays, of unequal quality. It has now become possible to circumvent the use of human tissues by using tumor cell lines containing the desired receptor or by transfecting tumour cell lines (e.g. Chinese Hamster ovary cells) with human DNA coding for the desired receptor, and to use the expressed receptors for screening tests. Additional advantages of such systems include:

• Radioligand binding and functional experiments (i.e. the measurement of receptor-evoked responses) can be measured under the same experimental conditions so that the experimental data are directly comparable. The receptors can be investigated by the binding of both agonist and antagonist radioligands as well as by the measurement of various functional responses (e.g. angiotensin II-mediated inositol phospholipid hydrolysis, transient rise in the cytosolic calcium concentration and extracellular acidification by CHO cells expressing the human AT1 receptor) (Figure 68).

• The non-transfected, wild type cells can be used as negative control.

Release of acidic metabolites Cytosolic calcium transients

Figure 68 In Wtro-measurable responses upon exposing angiotensin II (A II) to CHO cells expressing the human AT1 receptor.

Figure 68 In Wtro-measurable responses upon exposing angiotensin II (A II) to CHO cells expressing the human AT1 receptor.

• Cells can be transfected with genes coding for mutated receptors to identify amino acids that are crucial, e.g. for the binding of a given agonist or antagonist.

• Cells can be transfected with genes coding for two or more receptors to investigate any synergism or opposing effects.

The choice of cell line into which a G protein-coupled receptor is best transfected depends on the subsequent studies to be performed. In general, it is best for a host cell line to have a reasonably rapid growth rate and high transfection efficiency. Obviously, it is important to ascertain that the appropriate G proteins and effectors are endogenously expressed in the host cell line. It must also be ascertained that the cDNA to be transfected is not already expressed endogenously in the host cell line.

The majority of cells in a typical transfection experiment will express exogenous DNA transiently (Figure 69). Hence, transfected DNA will be lost from the host cell after a number of cell divisions. In a small proportion of transfected cells, the exogenous DNA will be randomly integrated into the chromosomal DNA of the recipient. If this takes place, the exogenous DNA has become a stable element of the genome of the host cell and this cell is now stably transfected (Figure 69). The number of stable transfectants is dependent on the efficiency with which the cells initially take up the exogenous DNA as well as on the frequency at which stable integration of the exogenous DNA into the chromosomal DNA occurs. With the incorporation of a selectable marker in the exogenous DNA, it is possible to select for cells that have this DNA integrated into their own chromosomal DNA. Finally, receptor vector

Transient transfection

Transient transfection

Figure 69 Stable and transient transfection of host cells with cDNA.

it is important to consider that the expression of integrated exogenous DNAs is subject to the local environment at the site of integration, e.g. strong expression can be obtained from even the weakest of promoters if integration occurs near a strong enhancer sequence.

It must be assumed that the genetic material introduced into the surrogate cell can find its way to the appropriate locus, be translated correctly and the resulting product processed as in native systems. In general, there is considerable evidence that non-standard translational events may affect the nature of expression products. Expression of multi-unit receptors can be especially difficult because of the potential for incorrect assembly. Although this is usually not a problem with GPCRs, there are cases in which alternative splicing of pre-mRNA or post-translational changes (glycosylation, palmitoylation, terminal amino acid acylation, carboxy-ter-minal amidation, sulfation, methylation) account for differences in G protein coupling and ligand affinity. Moreover, whereas the stoichiometry between receptors and G proteins is fixed in natural systems, recombinant systems deal with two new potential phenomena:

• Constitutive receptor activity (receptor-mediated increase in basal activity; i.e. without agonist present) (see also Section 4.11).

• Increased receptor promiscuity with respect to activation of different types of G proteins (see also Section 4.5).

Quantitative measurements have also been considerably developed during the past few years. Thanks to recent advances in chemical techniques (combinatorial chemistry) thousands of structurally related substances can theoretically be made in a single day. This demands high throughput screening techniques for their testing. At present, this is routinely done by radioligands, but this technique only provides information about whether and how well a drug is recognized by a given receptor, not about its ability to stimulate that receptor. The search for agonist activity is now greatly facilitated due to the development of receptor-independent assays (Figure 70):

• Binding of [35S]GTPyS to cell membranes allows the detection of agonists for nearly all GPCRs by a single assay. This is based on the faculty of agonists to promote the exchange of G protein-bound GDP by GTP. [35S]GTPyS acts like GTP and stimulates dissociation of the G protein into the [35S]GTPyS-bound Ga subunit and the Py complex (see Section 4.5). However, unlike GTP, [35S]GTPyS is relatively resistant to hydrolysis by the endogenous GTP-ase activity of Ga so that it remains bound.

• Based on the observation that the a subunits of the mammalian G proteins G15 and G16 are able to be activated by a much wider range of receptors as compared to the other G proteins and that the C-terminus of the a subunit is most important for receptor recognition, attempts are now made to transfect cells with chimaeric G proteins. This strategy involves co-expression of the receptor with chimeric

Drug Receptor Interacts

Binding

End Organ Response

Melanocytes Microphysiometry

Ext race Hula Space

Ligan d

Effect

Effect i

Drug Receptor Interacts

Binding

End Organ Response

Melanocytes Microphysiometry

Ext race Hula Space

Ligan d

Effect

Effect i

Figure 70 G protein-coupled receptor-triggered cascade of biochemical events in the cytosol. (1) Ligand-receptor binding, (2) G protein activation, (3) second messenger generation, (4) second messenger-triggered events (and detection with reporter systems), (5) the observable end-organ responses. Reproduced from Kenakin, T. (1996) The classification of seven transmembrane receptors in recombinant expression systems. Pharmacological Reviews, 48, 413-463, with permission from the American Society for Pharmacology and Experimental Theraputics.

Figure 70 G protein-coupled receptor-triggered cascade of biochemical events in the cytosol. (1) Ligand-receptor binding, (2) G protein activation, (3) second messenger generation, (4) second messenger-triggered events (and detection with reporter systems), (5) the observable end-organ responses. Reproduced from Kenakin, T. (1996) The classification of seven transmembrane receptors in recombinant expression systems. Pharmacological Reviews, 48, 413-463, with permission from the American Society for Pharmacology and Experimental Theraputics.

G proteins that preserve the receptor-coupling domain for the GPCR of interest, but have been fused with a domain that interacts with a different effector protein. This should enable the detection of agonists for nearly all GPCRs by a single assay.

• A rapidly expanding technology is in the field of 'reporters' of cytosolic second messengers such as cAMP. There are several types of reporters.

- Introduction of reporter genes whose expression is affected by the second messenger. Receptor activation should result in an increased transcription of the gene and expression of the gene product can be quantitated some hours later. For example, the luciferase gene can be set under transcriptional control of a regulatory DNA sequence responsive to cAMP. The intracellular level of the luciferase enzyme can be quantitated by measuring its activity (bioluminiscence reaction with luciferin as substrate).

- Introduction of reporter proteins that signal the elevation of the second messenger directly in the cytosol. For example, cells can be transfected with the gene coding for the calcium-binding protein, aequorin. The cytosolic calcium concentration can then be sensed by fluorescence measurements.

• The melanophore system allows a rapid evaluation of the effects of drugs on receptors that regulate cAMP. When melanophores respond to light or are stimulated by factors that elevate cAMP, they respond by dispersing their melano-somes throughout the cytoplasm and the cells appear dark. In contrast, signals that result in a decrease in cAMP levels, result in melanosome aggregation to the cell centre and the cells appear light.

• Finally, it has also been proposed that the measurement of extracellular acidification constitutes a universal assay system for receptor stimulation. The concept relates to the fact that the rate of cellular metabolism is directly linked to hydrogen ion extrusion by the cell, and this can be measured by an increase in the pH in the medium surrounding the cell.

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