Taken together, structural genomics, proteomics, and functional genomics provide a pathway that links genome sequencing with drug discovery. New tools and technology improve the speed and efficiency of that route.

by Peter Gwynne and Gary Heebner

The successful sequencing of the human genome and the subsequent sequencings of almost 150 other organisms’ genomes have set the scene for a new phase of life science. The postgenomic era is characterized by huge amounts of data, occasional surprises, and the identification of increasing numbers of proteins. Many of the new molecules exist in low abundance in the cell or have unknown functions. But as the research shifts from structural genomics to proteomics to functional genomics, life scientists can gain better understanding of how these molecules work together to provide a homeostatic environment for organisms.

The research promises dividends for drug discovery. Francis Collins, director of the National Human Genome Research Institute, has pointed out that pharmaceutical firms currently target fewer than 500 human gene products. “Today,” says Patrick Gilles, manager of R&D for Invitrogen Corporation, “there are perhaps thousands that could be used as targets. New information gained through functional genomic studies will contribute to effective therapeutic design and efficient screening of and validation of small molecule drug candidates.” Doug Storts, platform manager, genetic analysis at Promega Corporation, agrees. “Genomics opens up the potential for new targets,” he says. “The protein, especially in the context of the cell, gives a much clearer picture of what is of value in drug discovery and what is second tier.”

Pharmaceutical firms have moved rapidly to take advantage of the information provided by genomic research. “We’re using genomics technologies all along the value chain to support and improve our decision-making process,” explains Melvyn Hollis, vice president and global head of functional genomics at Aventis.

Assembling the Pieces
Functional genomics, which involves establishing the relationship between genes and their proteins, represents the climax of a progression that started with structural genomics, or high throughput determination of proteins’ structures, and moved on to isolating and studying the structures of proteins, otherwise known as proteomics. In fact all three facets of postgenomic research, which combine under the broad moniker of “systems biology,” contribute to the goal of assembling all the pieces of the genetic puzzle. “I see all three fields continuing to ramp up,” says Stanley Fields, professor of genome sciences at the University of Washington. “They can provide clues to very large numbers of uncharacterized proteins and genes. They can even provide clues to the function of proteins we know a lot about.”

Emerging tools and technologies facilitate the progress of these “omics” disciplines. “DNA and protein arrays, mass spectrometry, yeast two-hybrid, RNA interference, and increased use of robotics are all advances that are moving the fields along very quickly,” says Fields, whose own research uses the yeast two-hybrid system to study protein interactions.

Genome sequencing remains a significant contributor to advances across the spectrum of life science. “We see a lot of hype about new sequencing technologies,” says Kevin McKernan, vice president and co-chief scientific officer at Agencourt Bioscience Corporation. “Until these mature, SAGE sequencing and digital karyotyping will be critical for cancer profiling. Someone with a DNA sequencer can look at a lot of things.”

New Tool for ‘Omics’
A significant new tool for broad exploration of “omics” fields has emerged recently. Several companies have introduced or announced plans to market chips, otherwise known as microarrays, that contain the entire human genome. Applied Biosystems set the ball rolling in July, when it announced that it will offer a whole genome chip by the end of this year. Shortly after that, NimbleGen Systems reported that it was running tests for customers with its own version of a full human genome chip. Then in October Affymetrix, the traditional leader in the microarray market, reported that it was accepting orders for its own whole-genome chip. And Agilent Technologies quickly followed up by announcing that it had started to ship its chip to users.

The reality of human genome chips isn’t new. But until recently no firm had placed the entire human genome on a single chip; life scientists had to rely on two-chip and five-chip systems. The single-chip versions offer two advantages over those predecessors. First, they cost less; Affymetrix, for example, plans to charge between $300 and $500 apiece for its single chips, slightly more than half the cost of its two-chip set of the human genome. The single chips also promise more convenience in use than the multiple-chip versions. Researchers will use the various types of single chip in their efforts to discover mechanisms of disease.

Genetics in Reverse
Jeff Harford, genomics team leader for LI-COR, points out another hot technology: reverse genetics. “Everyone’s talking about RNA interference [RNAi] as targeted reverse genetics that shows a great deal of potential,” he says. Equally significant is targeting induced local lesions in genomes, or “tilling.” This process, developed at the Fred Hutchinson Cancer Research Center, “gives you an allelic series of point mutations,” Harford explains. “It is a very high throughput technology that allows you to screen over two million bases of information in a single day.”

New technology also improves researchers’ ability to make connections between the levels of the molecule and the deteriorating organ. “In genomics, the emerging ability to correlate haplotypes with disease status or susceptibility to disease is important,” says Elliott Dawson, president and founder of BioVentures, Inc. “In proteomics, the expanding application of mass spectrometry to identify proteins and their modifications, such as their states of phosphorylation, and the ability to do so in a high throughput situation represent significant advances.”

Keeping in Touch with Events in Science In today’s multidisciplinary world, researchers frequently need to keep in touch with events in fields different from their own. Since 1996 a website called EurekAlert! has provided a suitable source of such news. “It’s free and a very easy way for people to keep up-to-date with what’s happening in science,” says Cathy O’Malley, senior program associate at EurekAlert! Sponsored by the American Association for the Advancement of Science, which publishes Science, the site provides a forum through which about 500 research institutions, universities, government agencies, and corporations can distribute science related news to reporters and news media. “But about 90 percent of the people who visit are nonjournalists,” O’Malley says. “We know anecdotally that the majority of users are researchers, students, educators, and a lot of retired people. We get a lot of comments from scientists who follow up with us or contact public information officers directly.” Administrators continually update the site to make it more appealing to public visitors. “We’ve just signed up the Riken Center for Developmental Biology as our first Japanese contributor,” O’Malley says. “We’re trying to increase contributions from Australia, Canada, and Europe. We offer in-context modules at which you can find out about specific topics. Currently we feature nanotechnology and bioinformatics, and early next year we’ll launch one on marine biology and another on diseases in the developing world. We’re also developing a multilanguage section to highlight some of our international content.” www.eurekalert.org

High throughput screening itself contributes to advances in omics. “Companies are investing hundreds of millions of dollars per year in the technology, with the idea of quick return on investment,” says Brian Holaway, head of marketing and business development in the protein expression group at Roche Applied Science. “More and more assay kits are being developed for the high throughput platforms.”

Scientists must also deal with the data that those kits produce. “Analytical tools are absolutely essential,” says Scott Kahn, chief science officer of Accelrys, a subsidiary of Pharmacopeia. “Anything you can get to enhance the information and aggregate more information around your point of interest is important.”

A Surge in Sequencing
High throughput DNA sequencing has developed at an astonishing rate. Only a few years ago, scientists regarded sequencing the entire genome of a eukaryotic organism as an insurmountable task. But rapid developments in genomics enabled researchers to tackle and complete that type of project. Automated DNA sequencers from such companies as Amersham Biosciences, Applied Biosystems, and LI-COR, faster and more powerful computers from IBM and Hewlett-Packard, among others, and bioinformatics software from Accelrys, DNAStar, and others have played a large part in the progress made to date by genomics researchers.

According to Harford, LI-COR’s instruments have one key advantage in sequencing technology. “What really sets us apart is long reads; if you need 1,000 bases of information in a single run, we fit into that,” he says. “Our instruments fit very well into plant and animal research facilities. Scientists in these facilities are typically involved in phylogenetics, mapping, gene expression, and/or reverse genetics research. They’re using AFLP and microsatellites for mapping and phylogenetics, cDNA AFLP for gene expression, and tilling for genomewide reverse genetics.”

Agencourt has its own offerings in sequencing. “We are very well known for a range of services, from basic sequencing to very large-scale projects like whole genome efforts and library development,” says Paul McEwan, vice president and co-chief scientific officer. “If customers come to us at the very beginning of small projects, we can stay with them as the projects grow. We can take on functional genomics work, SNP discovery, and proteomics. We’ve developed high throughput systems for many of the core genomic technologies, so that scientists who don’t have a factory full of equipment can leverage ours.” One typical project is an effort in digital karyotyping carried out in collaboration with Johns Hopkins University.

The company also offers an automated purification service, licensed from the Whitehead Institute and called SPRI, for laboratories that carry out high throughput sequencing. “This is a magnetic bead approach to purifying various kinds of DNA,” McKernan says. “We’ve applied it to purifying RNA, blood, and plasmids from bacteria so that nobody is required to do manual purification.”

Proteins in Cells
Genome sequencing opened the way for researchers to explore how families of proteins behave in cells. Advances in high throughput sample handling have enabled scientists to isolate and characterize large numbers of proteins simultaneously and hence to gain a better understanding of the ways in which proteins interact with each other and control cellular function.

Vendors have responded rapidly to researchers’ need to work with larger numbers of samples and smaller sample volumes – in microwell plates, for example. Kits for protein isolation, separation, and characterization from Amersham Biosciences, Bio-Rad Laboratories, PerkinElmer, Promega, Sigma-Aldrich, and other companies have enabled even scientists who lack strong backgrounds in protein chemistry to enter the world of proteomics. “We have a variety of purification systems, including MagneHis, a magnetic system for purification of His-tagged proteins that can be automated for use in a 96-well format,” says Craig Smith, Promega’s platform manager for proteomics.

The company also focuses on detection. “We have long been a leader in bioluminescent detection technologies,” says research fellow Keith Wood. “Now we offer luminescent chemistry that allows us to look at the physiology of living cells without destroying the cells. We’re developing a whole range of assays that involve luminescence detection, including proteases, kinases, and P450 assays. In some cases, luminescent detection can be up to two logs more sensitive than fluorescence based methods.”

Another firm, Agencourt, offers several services for scientists who work on proteomics. “We can do protein-protein interactions,” explains McEwan. “We have licensed a system from Harvard University for bacterial two-hybrid work that we can leverage to study whole genome protein-protein interactions.”

Studies of Gene Expression
Scientists who specialize in functional genomics comb through the thousands of genes and even more proteins in an organism to find targets for new drugs and then figure out how to affect those targets with compounds that will, they hope, become blockbuster drugs. To study gene function, researchers can insert DNA sequences into living cells, where the sequences can be read and expressed as proteins. To build a library of DNA clones, researchers can turn to companies that provide the tools to produce their own libraries or buy large numbers of clones from firms such as ATCC, Invitrogen, and OriGene.

Invitrogen and Stratagene, meanwhile, provide kits and reagents for gene expression studies. “Microarray technology is a key tool, because with it you can determine expression levels of thousands of genes simultaneously, and therefore analyze gene regulation of whole pathways. Our Genicon resonance light scattering [RLS] technology, based on microscopic nanoparticles that scatter light at different intensities and wavelengths, offers tenfold greater sensitivity than fluorescent methods,” explains Todd Petersen, director of R&D at Invitrogen. “Right behind that is the emergence of RNAi technology. It has a specificity, effectiveness, and persistence in generating gene knockdowns that accelerate understanding of how genes operate. A suite of BLOCK-IT technologies exists, including gateway vectors, that allows high throughput cloning in any mammalian cell type. This permits researchers to move rapidly from gene isolation and characterization to functional studies.”

Handling large numbers of DNA and protein samples requires laboratory automation. Invitrogen, PanVera, and Roche Applied Science have developed complete biochemical and cellular assay kits designed for high throughput systems. Roche offers a series of high throughput biochemical assays for PCR and RT-PCR that complement its well-established LightCycler System. Another product, the LightTyper, “can be considered as an extension of the LightCycler,” says Steven Bye, product manager for Roche’s protein expression group. “It does PCR, product characterization, and mutation detection via melting curve analysis. Software that recognizes melting curves enables it to automatically identify genotypes.” Potential applications of the instrument include understanding single nucleotide polymorphisms.

Dealing with Disparate Data …
Studying relationships between a gene, its protein counterparts, and a specific disease process requires scientists to commingle huge amounts of data from different locations and in disparate formats. Bioinformatics tools and techniques often provide the only way to see the subtle relationships hidden deep within these huge sets of data. A recent analysis of the SARS genome typifies the value of those products. “When we combined the tools of genomics and proteomics, we found data that we wouldn’t have got independently,” says Kahn of Accelrys.

Several suppliers, including DNAStar, have developed suites of informatics tools to help bring together data in a more meaningful platform. Accelrys delivers computational science software that analyzes and solves research problems, as well as informatics tools that help scientists to manage and mine their data.

The firm has recently released two new products to complement its GCG Wisconsin Package, a tool that molecular biologists worldwide use for sequence analysis. “Discovery Studio Gene is a desktop package for sequence analysis that connects to the Wisconsin package and to a knowledge management system that allows scientists to store the results and pass them along,” Kahn says. “And Discovery Studio AtlasStore merges the genomic, proteomic, and cheminformatic worlds so that researchers can look up information in all three. It bridges the three communities.”

Pharmaceutical companies must bridge communities of scientists in much the same way as they seek to identify new proteins critical to the disease process and to understand how drugs can regulate a biochemical or signal transduction pathway to block the action of a target molecule. Several pharmas, among them Aventis and Merck, have started to design and develop small molecule drugs that inhibit specific proteins within signal transduction pathways. The research targets intracellular proteins thought to be critical to the disease process.

… and Different Disciplines
Aventis exemplifies that approach. “We have a variety of different disciplines,” says Hollis. “We have strong proteomics with protein scientists and biophysicists. We have molecular biologists, who traditionally contribute strongly to genomics. We have cell biologists. We also have a functional genomics unit dedicated to human genetics. Within functional genomics we have access to a full array of technology platforms and expertise, including informatics, automation, transcription profiling microarrays, mass spectrometry, and high throughput detection of genes and proteins in normal and disease-relevant human tissue samples.”

Beyond its own labs, Hollis’s team collaborates closely with the company’s disease groups. “Our main objective is target validation, which is a close partnership between the core functions like functional genomics and the disease experts,” he says. “So we have established molecular biology with a strong technology slant to it.”

BioVentures has developed technologies that link activities at the level of the gene with illness in the organism. “We’re looking at specific human diseases,” Dawson explains. “We’re able to identify various genetic variants, in particular haplotypes that cosegregate with disease and the haplotypes of modified genes that predict the age of onset of disease and the severity of the disease in some cases. We feel that this is of particular interest in being able to make earlier and timelier intervention on behalf of patients.”

The company focuses on the ADMET [absorption, distribution, metabolism, excretion, and toxicology] process. “Failures of drug candidates in ADMET, through lack of efficacy in metabolism or outright toxicology, represent about 50 percent of all drug failures,” Dawson says. “We are developing tools that will determine the variants in drug metabolism pathways that may contribute to adverse effects. We’ve done it first at the genomic level. Now we’re working at the proteomic level.”

The company’s technologies also provide insights into the development of disease. “They enlighten life scientists with respect to the pathways that lead to disease and indicate points of intervention at the molecular level,” Dawson says. “They also help to identify targets for rational drug discovery.”

One point is clear: The postgenomic era virtually demands collaborative science. “We’re working as part of a network of discovery teams,” explains Aventis’s Hollis. “We bring the best genomic technologies to the party. We expect our partners to do the same for their abilities. When it works well, it’s fantastic.”

WEBLINKS ADVERTISERS BioVentures, Inc. research kits and reagents for the study of genetic diversity 615-896-7353 www.bioventures.com Dynal Biotech biomagnetic separation technology for life science research and clinical applications +47 22 06 10 00 www.dynalbiotech.com Gyros [Sweden] miniaturized and integrated laboratory operations using a compact disc platform +46 (0)18 566 300 www.gyros.com Gyros [USA] 732-438-9400 MDL Information Systems, Inc. discovery informatics and software solutions for the life sciences 510-895-1313 www.mdl.com Takara Bio, Inc. kits and reagents for molecular biology research +81 77 543 9254 www.takara-bio.co.jp/english FEATURED COMPANIES Accelrys – a subsidiary of Pharmacopeia bioinformatics software www.accelrys.com Affymetrix human genome chips www.affymetrix.com Agencourt Bioscience Corporation DNA sequencing products and services www.agencourt.com Agilent Technologies, Inc. human genome chips www.agilent.com American Type Culture Collection (ATCC) DNA clones www.atcc.org Amersham Biosciences instruments and reagents for genomics and proteomics research www.amershambiosciences.com Applied Biosystems automated DNA sequencers, human genome chips www.appliedbiosystems.com Aventis pharmaceuticals www.aventis.com Bio-Rad Laboratories instruments and reagents for genomics and proteomics research www.discover.bio-rad.com BioVentures, Inc. kits and reagents for genomics research www.bioventures.com DNAStar bioinformatics software www.dnastar.com EurekAlert! website for scientific news www.eurekalert.org Fred Hutchinson Cancer Research Center nonprofit research institute www.fhcrc.org Harvard University university www.harvard.edu Hewlett-Packard computers and operating systems www.hp.com IBM computers and operating systems www.ibm.com Invitrogen Corporation kits and reagents for genomics and proteomics research www.invitrogen.com Johns Hopkins University university www.jhu.edu LI-COR Biosciences automated DNA sequencers www.licor.com Merck & Company, Inc. pharmaceuticals www.merck.com National Human Genome Research Institute government research center www.genome.gov NimbleGen Systems, Inc. human genome chips www.nimblegen.com OriGene Technologies, Inc. DNA clones www.origene.com PanVera, LLC – an Invitrogen Company high throughput assay kits www.panvera.com PerkinElmer Life and Analytical Sciences instruments and reagents for genomics and proteomics research www.las.perkinelmer.com Promega Corporation kits and reagents for genomics and proteomics research www.promega.com Roche Applied Science kits and reagents for genomics research www.biochem.roche.com Sigma-Aldrich Corporation kits and reagents for genomics and proteomics research www.sigma-aldrich.com Stratagene kits and reagents for gene expression studies www.stratagene.com University of Washington university www.washington.edu Whitehead Institute for Biomedical Research nonprofit, independent research and educational institution www.wi.mit.edu

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