O Novel Drugscreening Strategies

The combination of the heterologous expression of cloned DNA, the molecular cloning of new biological targets, and the ability to manipulate gene sequences has created powerful new tools that can be applied to the process of drug discovery and development. In its most straightforward application, the ability to simply express newly identified receptor protein targets offers a novel means of obtaining information that may be difficult, or even impossible, to obtain from more complex native biological systems. There is a reason for this. A newly identified protein can be expressed in isolation. Even for closely related enzyme or receptor subtypes, heterologous expression of the individual subtype can potentially provide data that are specific for the subtype being expressed, whereas the data from native biological systems will reflect the summation of the individual subtypes that may be present.

The potential advantage of heterologous expression is illustrated in Figure 4.9 for the interaction of a drug with multiple binding sites. In panel A, which can represent the data

Figure 4.9 • Convoluted data from binding to multiple receptor subtypes versus classic mass action.

Figure 4.9 • Convoluted data from binding to multiple receptor subtypes versus classic mass action.

obtained from a native biological system, the data are complex, and the curve reflects interactions of the drug with two populations of receptors: one with high affinity, representing 50% of the total receptor population, and one with low affinity, representing the remaining 50%. The individual contributions of these two populations of receptors are indicated in panel B, which could also reflect the data obtained if rDNA encoding these two receptors were expressed individually in a heterologous expression system. Although in some cases, the data, as in panel A, can be analyzed with success, frequently they cannot, especially if more than two subtypes are present or if any one subtype makes up less than 10% of the total receptor population or if the affinities of the drug for the two receptor populations differ by less than 10-fold.

Another important reason for integrating heterologous expression into drug-screening strategies is that data can usually be obtained for the human target protein rather than an animal substitute. This does not mean that organ preparations or animal models will be totally replaced. For the purposes of the identification of lead compounds and the optimization of selectivity, affinity, etc., however, the use of recombinant expression systems provides some obvious advantages.

By combining heterologous expression with novel functional assays, it is possible to increase both specificity and throughput (the number of compounds that can be screened per unit time). For example, reporter genes have been developed that respond to various intracellular second messengers, such as the activation of guanine nucleotide-binding proteins (G proteins), and levels of cyclic adenosine monophosphate (cAMP), or calcium. One approach to the development of novel functional assays involves the use of promoter regions in DNA that control the transcription of genes. This approach is exemplified by the cAMP response element (CRE). This is a specifically defined sequence of DNA that is a binding site for the cAMP response element-binding (CREB) protein. In the unstimulated condition, the binding of CREB to the CRE prevents the transcription and expression of genes that follow it (Fig. 4.10). When CREB

is phosphorylated by c AMP-dependent protein kinase A (PKA), however, its conformation changes, permitting the transcription and expression of the downstream gene. Thus, increases in intracellular cAMP, such as those caused by receptors that activate adenylyl cyclase (e.g., jS-adrenergic, vasopressin, etc.), will stimulate the activity of PKA, which, in turn, results in the phosphorylation of CREB and the activation of gene transcription.

In nature, there are a limited number of genes whose activity is regulated by a CRE. Biologically, however, the expression of almost any gene can be regulated in a cAMP-dependent fashion if it is placed downstream of a CRE, using rDNA techniques. If the products of the expression of the downstream gene can be easily detected, they can serve as reporters for any receptor or enzyme that can modulate the formation of cAMP in the cell. The genes encoding chloramphenicol acetyl transferase (CAT), luciferase, and S-galactosidase are three examples of potential "reporter genes," whose products can be easily detected. Sensitive enzymatic assays have been developed for all of these enzymes; thus, any changes in their transcription will be quickly reflected by changes in enzyme activity. By coex-pressing the reporter gene along with the genes encoding receptors and enzymes that modulate cAMP formation, it is possible to obtain very sensitive functional measures of the activation of the coexpressed enzyme or receptor.

Another example of the use of a reporter gene for high-throughput drug screening is the receptor selection and amplification technology (r-SAT) assay. This assay takes advantage of the fact that the activation of several different classes of receptors can cause cellular proliferation. If genes for such receptors are linked with a reporter gene, such as j-galactosidase, the activity of the reporter will be increased as the number of cells increase as a consequence of receptor activation. Initially, a limitation of this assay was that it only worked with receptors that normally coupled to cellular proliferation; by making a mutation in one of the second-messenger proteins involved with the prolifera-tive response, however, it was possible to get additional

Figure 4.10 • Activation of transcription by a cAMP response element (CRE). CREB is phosphorylated by cAMP-dependent protein kinase.

receptors to work in this assay. This second-messenger protein, Gq, was cloned, and a recombinant chimera was made that included part of another second messenger known as Gi. In native cells, receptors that activate Gi are not known for their stimulation of cell proliferation, but when such receptors are coexpressed in the r-SAT assay with the chimeric Gq, their activity can be measured.

A similar strategy involving chimeric proteins has been used for receptors whose second-messenger signaling pathways are not clearly understood. For example, the development of potential therapeutic agents acting on the hGH receptor has been difficult because of a lack of a good signaling assay. The functional activity of other receptors that are structurally and functionally related to the growth hormone receptor can be measured, however, in a cell proliferation assay. One such receptor that has been cloned is the murine receptor for granulocyte colony-stimulating factor (G-CSF). By making a recombinant chimeric receptor containing the ligand-binding domain of the hGH receptor with the second-messenger-coupling domain of the murein G-CSF receptor, it was possible to stimulate cellular proliferation with hGH.

In addition to providing a useful pharmacological screen for hGH analogs, the construction of this chimeric receptor provides considerable insight into the mechanism of agonist-induced growth hormone receptor activation. The growth hormone-binding domain is clearly localized to the extracellular amino terminus of the receptor, whereas the transmembrane and intracellular domains are implicated in the signal transduction process. It was also determined that successful signal transduction required receptor dimerization by the agonist (i.e., simultaneous interaction of two receptor molecules with one molecule of growth hormone). Based on this information, a mechanism-based strategy was used for the design of potential antagonists. Thus, hGH analogs were prepared, were incapable of producing receptor dimerization, and were found to be potent antagonists.

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