Chemical library or/and Natural product
Figure 1 The current discovery scheme used in the pharmaceutical industry. For the discovery of new crop protection products, companies use, in addition to biochemical tests, assays directly with plants, insects, or plant diseases. *ln case of natural products, this step would involve (re-)isolation of active ingredient and structural elucidation; the active structure may also be modified using traditional or combinatorial chemistry. **Follow-up assays include tests for specificity, initial efficacy studies in cell culture or animal models, as well as ADME (absorption, distribution, metabolism, and excretion tests).
Once a disease-specific target is identified by basic research or —more often—by genomic approaches, it is used to develop a biochemical assay for high-throughput-screening (HTS). The term HTS was created in the early 1990s to describe the screening of not only a few thousand but hundreds of thousands of chemical compounds in a short time.
In addition to new technologies for screening, the advent of combinatorial chemistry was a major breakthrough a decade ago (11-13). A large number of discrete compounds are being synthesized today using automated synthesis machines. However, not every chemical class can be synthesized using this approach, and additional chemical diversity is needed to increase the chance for discovery of novel chemical entities. Natural products are ideally suited to fill such "holes" in the diversity chemical space and also to serve as scaffolds for creation of natural compound-like libraries (7,8,14) (see also other chapters in this book).
There have been many technological breakthroughs, but the fundamental change is most likely the step from the very research-based approach to the "industrialization of discovery" (10,15-22). The genomic efforts offer a good example of this new paradigm. Although sequencing of the human and other genomes is a result of the synergy between innovations in automation and informatics, it is mainly a breakthrough in process management. The basic principle is that basic research is no longer used to discover, for example, the function of a certain gene but standardized technologies are used to analyze the sequence and function of all genes in the genome in a systematic, "industrial" process. Mass sequencing is done today in factorylike setups with dozens of to several hundred sequencers run in a 24-hour, 7-days-a-week fashion (15) (examples of companies pursuing this approach are Incyte Pharmaceuticals, Human Genome Sciences, Millennium, and recently PE Celara). It was expected that the human genome would be fully sequenced in the year 2000 and that by 2002 over 60 microbial genomes and several metazoan species would be available in company or public databases. This factory-like or industrialized process is now also applied to the next steps in drug discovery.
The development of combinatorial chemistry and HTS has moved the bottleneck in drug discovery to the next step: identification of important target genes causally involved in a certain disease. Again, high-throughput technologies are being developed to discover genes of interest in pharmaceutical companies as well as specialized biotechnology companies. One example of such a functional genomics approach has been published by CuraGen Corporation (New Haven, CT) in collaboration with the University of Washington, Seattle, and others (18). They have used the yeast two-hybrid system to discover protein-protein interaction in yeast. Most of the approximately 6000 yeast genes were expressed separately in two haploid yeast strains. One protein is fused to a DNA-binding domain of a transcription factor; the other is fused to the activator region of the same transcription factor. Upon mating, both proteins are expressed in the diploid yeast and only yeast where such a protein-protein interaction takes place will grow. Although this technology is well known, it had not been applied in such a systematic way for a entire genome. A total of 957 interactions were discovered involving 1004 yeast proteins. However, the fact that certain proteins interact in the yeast two-hybrid system does not necessarily mean that this interaction is relevant to the in vivo situation. Expression profiling during the yeast life cycle and study of the subcellular localization of the proteins will rule out some protein-protein interactions as not relevant to the in vivo situation (18).
The systematic study of the function of human genes is also under way using yeast, mice, zebra fish, Caenorhabditis elegans, Drosophila me-lanogaster, or other model species. In addition to analyzing phenotypes after overexpression or reduced expression of genes, expression profiling in dis eased versus healthy tissue will help to identify new potential disease targets. In many cases such studies are performed by specialized companies and data are made available on a subscription-fee basis (see, e.g., www.exelixis.com, www.paradigmgenetics.com,www.curagen.com).
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