PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. The Post-Genomic Era by Wim D’Haeze, Ph.D. Not so long ago, it was considered as a major contribution if someone had identified and functionally characterized a given eukaryotic protein as part of a research project that aimed to honor the researcher with a doctoral title. Often, this type of detailed studies provided the scientific community with an in-depth knowledge of the role that a particular eukaryotic protein plays in a biological system. Notwithstanding the scientifically very productive output, it is clear that such an approach was labor-intensive and relatively low efficient when one would like to answer questions at a cellular or systems level. Thus, it was time to design and engineer novel approaches, and to welcome considerable changes in the molecular life sciences. And as we all know, changes became apparent. One of the more abrupt changes included the advances in DNA sequencing technologies that made it possible to efficiently and accurately sequence stunting numbers of basepairs on a daily basis. The sequence of entire genomes of various organisms including pathogenic bacteria (e.g. Bacillus subtilis and Mycobacterium tuberculosis), plants (e.g. Arabidopsis thaliana and Oryza sativa), animals (e.g. chicken) and human became available to the scientific communities. This provided us with a wealth of information that could for instance be used to study the structure of the genome, and to enhance our knowledge of evolutionary aspects (e.g. genetic islands, horizontal gene transfer, genetic regions with a different GC content). In addition, and maybe even more interesting, similarity searches against databases dramatically increased the number of genes that encode for proteins with a putatively known and described function which gives a better idea about the biochemical pathways and mechanisms within a given organism. The obvious questions generated by this success were how to find our way in this plethora of genetic information, and how to fish for those gene products that might be suitable drug targets. For instance, proteins that allow the pathogenic bacterium M. tuberculosis to escape the host immune system are of particular interest and good candidates to be used for the screening of drugs that may inhibit this mechanism in order to ameliorate tuberculosis. Although this seems an obvious and rather simple question to answer, it is not that obvious to identify those genes in a pool of thousands of genes. This led to the development of the so-called high throughput approaches including but not limited to high throughput screening (HTS). High throughput micro-array-mediated approaches in which literally all open reading frames of for instance M. tuberculosis can be spotted on a chip are used to specifically identify those the expression of which is up- or down-regulated upon internalization in human macrophages or when the bacterium is exposed to given environmental conditions that mimic those at certain locations in human such as a decreased environmental pH. Genes of interest can easily be identified because the entire genome sequence is available, and the corresponding proteins can then be studied in more detail at the functional level and be used in high throughput screens using compound libraries. This may lead to the identification of putative drugs that affect the activity of the protein under investigation. It is appropriate to mention that HTS is becoming a common and frequently used approach in modern life science laboratories, and numerous examples that illustrate its usefulness are available. HTS is used to screen for interactions between carbohydrates and pathogens, which provides insights into the roles that specific carbohydrates play during the infection process. This may lead to novel tools for disease diagnosis and eventually prevention. The glycomics approach that is currently being used to screen for carbohydrates that induce the immune response against unique pathogens or disease-specific lipopolysaccharides or (lipo)oligosaccharides may help to discover carbohydrate-based vaccines against diseases such as malaria, bacterial infections, and cancer. In addition, a recently developed ligand to visualize amyloid aggregates in Alzheimer’s disease mice models in vivo will soon be used in high throughput screens. Using this ligand, which is able to cross the blood brain barrier, near-infrared fluorescence imaging and HTS, it will be possible to visualize amyloid aggregates in the brains of Alzheimer’s disease mice models that were exposed to candidate drugs as a function of time. Because this amyloid aggregate specific ligand allows to gather quantitative information on plaque levels in Alzheimer disease model mice, one can monitor the effect of a putative drug on the formation of amyloid aggregates in vivo as a function of time without killing the mice and performing histological approaches on imbedded tissues. More recently, a considerable advance has been made in the capacity of high throughput screens. At the ultra-high throughput level, it is now possible to perform 100,000 assays per day instead of a 10,000 per year. HTS is also evolving to include more and accurate target identification and validation, in addition to better compound identification and optimization procedures. This is made possible in part by the advances in modern ultra-HTS tools and the increase of the number of wells per microtiter plate from 96 to 384 and possibly 1536. Furthermore, the number of proteins that are partially characterized using proteomics approaches is dramatically increasing as well as the number of data points that will be used in the future in high throughput screens. Undoubtedly, these approaches will significantly hasten the process to find new drug targets and suitable drugs to ameliorate human diseases, but the challenge yet remains as to how to integrate these large volumes of data and observations into an accurate and appropriate model of cellular behavior. In other words, it would be extremely interesting and helpful if we were able to monitor the effect of a given drug on the extent and duration of for instance apoptosis in a cancer cell, or to monitor cellular and molecular responses initiated upon infection with or internalization of a given pathogenic bacterium in the absence and presence of a putative drug or vaccine. In order to better study such in vivo events in time, a pretty new approach is rapidly being developed which is called high content screening (HCS). HCS is defined as the simultaneous analysis of various parameters and aspects of a cell considering the cell as the ultimate functional endpoint or unit for any biological stimulus. This approach couples cell-based assays with large-scale robotics, automated image capture, advanced image analysis and allows to investigate changes in cell morphology, to monitor cytotoxicity, and to visualize apoptosis events and cell proliferation as a factor of time and in response to for instance the prolonged application of a candidate drug. Remarkably, such a screen easily generates 10,000 high-resolution images that need to be stored and analyzed in an accurate manner. To my understanding, HCS is a rapidly evolving technical approach for which several companies are designing new equipment. Very advanced cytometers, for instance, are being developed that can sensitively and accurately measure changes in various cellular parameters at a speed of 70,000 cells per second. Cells of interest can be isolated and subsequently imaged in an automated setting. The new equipment allows one to measure subcellular parameters in several cells over time allowing the analysis of the heterogeneity of certain responses rather than restrict our observation to an averaged response. Thus, it is feasible to visualize the effect of a certain drug on cellular processes in vivo as a function of time. This will allow us to estimate the possible side effects of a drug. One will also be able to investigate how a cell behaves after being treated with a drug for a prolonged period of time. High throughput HTS and advanced HCS will be powerful tools, but yet it needs to be considered that those approaches are somehow restricted to the cellular level. What happens at the systems level? It is understandable that considerable attention and effort is directed to the development of methods to rapidly identify a drug that affects the action of a protein or a pathway, but the results still do not sufficiently contribute to our understanding of the function of a given protein of interest nor do they unravel the mode of action of the drug. In my opinion, it seems unlikely that such questions can be resolved using high throughput approaches. A drug may be efficient, but only at a relatively high dose that may render it toxic when used for longer periods of time. In such cases, it is important to design and produce optimal drugs that could be applied in lower doses. However, this requires an in-depth understanding of both the function and structure of the protein, and how the drug interacts with and inhibits the activity of that protein. After all, it might be necessary under those circumstances to go back to the roots to perform fundamental experiments that may take as long as the time required to obtain a doctoral title even in the post-genomic era. ### Wim D’Haeze is a Bio-Engineer in Chemistry and received his Ph.D. in Biotechnology at the Ghent University (Belgium) in June 2001. His doctoral thesis work was focused on the understanding of several early steps of the symbiotic interaction between the Gram-negative soil bacterium Azorhizobium caulinodans and the tropical legume Sesbania rostrata. The initial steps require the production of bacterial compounds including signal molecules and complex surface polysaccharides, that are pivotal for invasion of the plant tissue and the formation of new organ tissues. In the three subsequent years, he performed post-doctoral research at the Complex Carbohydrate Research Center at the University of Georgia (Athens, GA) dealing in part with the structural and functional characterization of azorhizobial extracellular polysaccharides. Currently, Wim D’Haeze is employed at The Scripps Research Institute (La Jolla, CA) as Science Writer and focuses on a new horizon regarding the molecular basis of devastative neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, in order to screen for and develop new therapeutics. E-mail: wim.dhaeze@sbcglobal.net. ### << Previous Next >> [ View All Perspectives ] |
|