Identification targets

In forward chemical genetics, having found a compound from the library, a hit , that elicits the desired phenotype, the final step is to identify its bio-molecular point of action. This is usually the biggest hurdle, especially if the target is novel. 

There are two other technologies stiU in development with similar potentials. So-called yeast three hybrid screens are related to the better known two-hybrid screens, where the binding interaction that is being hunted for triggers the assembly of a dimeric transcription factor. The functional transcription factor then stimulates expression of a reporter gene. One half of the transcription factor is fused to a known smaU-molecule receptor (say for a dmg), whilst the other is fused to memhers 

Pharmaceutical companies have to be continuously vigilant and forecast the future directions of drug development and regulatory requirements. They have to use their core technical competencies to deliver a pipeline of products to remain competitive and profitable in the long term. 

Most diseases, except in the case of trauma and infectious diseases, have a genetic connection. Genetic makeup and variations (see single nucleotide polymorphism in Section 11.5) determine a person s individuality and susceptibility to diseases, pathogens, and drug responses. 

The current method of drug discovery commences with the study of how the body functions, in both normal and abnormal cases afflicted with diseases. The aim is to break down the disease process into cellular and molecular levels. An understanding of the status of genes and their associated proteins would help to pinpoint the cause of the disease. Drugs can be tailor-made to attack the epicenter of diseases. In this way, more specific (fewer side effects) and effective (high therapeutic index, see Section 5.2) drugs can be discovered and manufactured to intervene or restore the cellular or molecular dysfunction. 

From the Human Genome Project, we know that there are approximately three billion base pairs that make up the DNA molecule (refer to Appendix 2). Only certain segments of the enormous DNA molecule encode for proteins. These segments are called genes. The estimate is that there are about 30,000 genes that encode proteins. Exhibit 2.1 provides some information about the number of genes and the complexity of life forms. 

Exhibits 2.2,2.3, and 2.4 provide examples of genetic causes of diseases, for example, cancer, sickle cell anemia, and cystic fibrosis. It should be noted that although some of these diseases are the result of mutations in a single gene (including Huntington s disease and Duchenne muscular dystrophy), most are due to the influence of multiple genes. 

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The process of target identification analyzes a complex disease process by dissecting it into its fundamental components. This makes it possible to identify the one that is most integral to the manifestation of the disease. Target identification aims to understand the biological processes related to a disease, and to identify its mechanism and the structure of individual elements of the disease. Commonly these individual elements are receptors, enzymes, etc., which become the target of new drugs. 

Sacks, I. J. et al., 1977, Target Identification Procedure for Plutonium Reprocessing Facilities, LLNL UCRL 79215-Rl, June. 

Jensen AA, Frolund B, Liljefors T et al (2005) Neuronal nicotinic acetylcholine receptors structural revelations, target identifications, and therapeutic inspirations. J Med Chem 48 4705—4745... 

Darnell, J. C., Mostovetsky, O., and Darnell, R. B. (2005). FMRP RNA targets Identification and validation. Genes Brain Behav. 4, 341-349. 

Although a number of cell lines were shown to be sensitive to inhibition by PatA (Low et al., 2005), we selected RKO cells (IC50 of 0.4 nM in cell proliferation assay) to prepare lysates for the isolation and identification of target protein(s). We often select RKO cells for target identification of small molecules using biotin-conjugates, because they appear to be particularly suitable for target protein isolation. 

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