Drug Discovery

Rational methods of drug discovery in the past have typically used a biochemical approach. Target enzymes were identified — preferably the rate limiting step in a particular pathway — and then characterized and purified. Most often, these experiments were performed using animal tissue. Occasionally, enzymes could be targeted individually if enough information was known about mechanism of action and structure. Similar approaches were

FIGURE 5.6 DNA-microarray analyses can identify relevant clinical subsets of gliomas. A and B show that different subtypes of gliomas have distinct gene-expression profiles. C and D show identification of molecular subsets of microscopically identical glioblastomas. E and F show the detection of clinically relevant, previously undetected subsets of patients with high-grade gliomas that have significantly different survival times; these genes can identify the subset of patients who are most likely to have prolonged survival. (Nature Rev Neurosci, Mischel et al. (2004). Copyright Nature Publishing Group. Reproduced with permission). See Plate 5.6 in Color Plate Section.

FIGURE 5.6 DNA-microarray analyses can identify relevant clinical subsets of gliomas. A and B show that different subtypes of gliomas have distinct gene-expression profiles. C and D show identification of molecular subsets of microscopically identical glioblastomas. E and F show the detection of clinically relevant, previously undetected subsets of patients with high-grade gliomas that have significantly different survival times; these genes can identify the subset of patients who are most likely to have prolonged survival. (Nature Rev Neurosci, Mischel et al. (2004). Copyright Nature Publishing Group. Reproduced with permission). See Plate 5.6 in Color Plate Section.

employed for identification and isolation of individual receptor subtypes as therapeutic targets. Biochemical techniques to identify drug-binding proteins rely on the affinity and specificity of small molecules to their protein targets; this affinity is quite low in some cases, leading to difficulty in specifying the primary binding partners. These often-laborious experimental approaches resulted in many effective therapies for a variety of diseases. Advances in molecular biology, however, have accelerated drug discovery, particularly in the field of oncology.

The new era of molecular biology and gene cloning techniques has enabled rapid advances in the field of drug discovery. Use of animal models and animal tissue has been helpful for initial studies; however, small variations in protein structure and amino acid sequences may render compounds identified in animal experiments ineffective for treating human disease [93]. The availability of human genes as targets through the efforts of the Human Genome Project has opened a new arena for high throughput testing of potential drug targets from human tissues. Gene expression and profiling can be used at all stages of discovery and development of therapeutic agents (Fig. 5.7). Gene sequencing and cloning can also permit production of targets that are difficult to isolate from natural sources. Site-directed mutagenesis can be utilized to test hypotheses regarding

FIGURE 5.7 Various phases in the discovery and development of therapeutic agents and of diagnostic, prognostic and other biomarkers. Microarray expression profiling can be used to help advance all stages of this process (Adapted with permission from Biochem Pharmacol, Clarke et al, 2001) [109].

target-drug interactions. Microarray analysis has now evolved to help both identify and validate potential novel therapeutic targets in a high throughput manner. This has greatly facilitated the testing of new biologic agents and the discovery of known and unpredicted drug targets.

New drug therapies identified with genomic methodologies have the potential to target molecular mechanisms of disease while avoiding side effects that arise from the activation or inhibition of normal molecular mechanisms within the body that do not have a role in the disease process. Selective tissue expression is attractive for use as drug targets, as this may reduce or eliminate unwanted and unexpected side effects.

Methodologies such as the DNA microarrays, which permit measurement of differential levels of gene expression of thousands of genes, may also provide an ideal framework for measuring how a compound effects metabolism, function and regulation on a much larger — e.g., genomic — scale [14,39].

For example, a combined approach of affinity purification, followed by array-based transcription profiling was used to identify candidate proteins of a novel class of anticancer agents [94]. This also permitted evaluation of the transcriptional effects of these agents on the tumor cells, specifically of sets of genes involved in cell metabolism, cell cycle control, and the immune response. This process highlighted a number of genes that were down-regulated in a time-dependent manner when treated with a pharmacologically relevant and clinically achievable drug concentration [94]. This integrated strategy led to the identification of drug-binding targets of biologically active small molecules.

Therapy for brain tumors based on small molecule inhibitors of growth factor receptors or their downstream targets can be individualized based on the results of gene expression patterns. This relies on microarray studies that demonstrate the presence of, and activity of, the intended target. Using yeast as a model system, drug inhibition of target genes can be compared to inactivation of the target by mutation [95]. Mutant strains with the most similar expression profile are then selected and treated to determine the 'ideal drug' with optimal specificity. These methods can be used to identify both co-regulated genes and unexpected target genes or 'off-target' effects. This approach was used to assess the calcineurin signaling pathway, which is inhibited by FK506 or cyclosporine A [95].

Microarrays are also useful in determination of the relationship between structure and activity by comparing active and inactive derivatives of novel drugs [96]. Using the induction of gene expression as a measure of drug function is useful for preclinical investigation of novel therapeutic agents to select newer drugs that have molecular selectivity.

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