18 PharmaGenomics July/August 2002

The Promise of Protein Microarrays

t this early stage of the21st century, peopleseem to be feeling asense of indifference to

technological advances. After all,these advances seem to come atsuch a rapid pace and in so manydifferent areas of life that we canbarely keep up with them. Whenthe person on the street considerstechnology, she or he probablythinks more about enhancementsthan breakthroughs. The familiar-ity of having a computer on thedesktop, with continual upgradesthat themselves dwarf the com-puting power of the systems oftwo or three decades ago, has onlyamplified that feeling.

Molecular and cell biologists,however, are like the explorers ofold — more attuned to break-throughs than to enhancements.Technology for these scientistsoften is about frontiers. And thefrontiers they explore have majorimplications for the pharmaceuti-cal component of health care.

One relatively new technology— protein microarrays — is anideal example of a new frontier.Ken Howard, a reporter for theonline GenomeWeb, calls it “thenext big little thing.” (1) Proteinmicroarray technology is, in part,an outgrowth of the humangenome project. Thanks to the

successful sequencing of thehuman genome, as well as the se-quencing of genomes of other or-ganisms, scientists today have ac-cess to a catalog of all humangenes and the genes of manyplants, animals and microorgan-isms. This genetic information al-lows one to determine the proteinmakeup of cells, which is criticalbecause the protein is the“worker” substance of a cell.

Development of Microarray-based AnalyticsWhat’s so special about this newtechnology? Protein microarraytechnology allows the simultane-ous analysis of thousands of mole-cular parameters with a single ex-periment. (2) In one example, re-searchers were able to print a verylarge number of protein spots ona single glass slide and then usethe slide to identify protein-pro-tein and drug-protein interac-tions. (3) The implications for re-searchers are tremendous, as KenHoward was told: “Instead ofspending 12 hours in a cold room[analyzing one protein], you cananalyze 10,000 proteins in a singlechip in 10 hours. It allows you todo things much more quickly andwith greater precision.”

Microarrays are produced inone of two ways. In some cases,

on-chip synthesis is used. In oth-ers, researchers employ an arrayerbased on contact printing or inkjet technology. Proteins can be de-posited as microspots in specifiedpositions on the chip.

To be fair, while the applicationof the technology to proteinanalysis might be recent, the mi-croarray as an idea is hardly new.Roger Ekins and his colleaguesfirst espoused the fundamentaltheoretical concepts beginning inthe late 1980s. (4) They deter-mined that microspot ligand-binding assays were sensitive fromboth a theoretical analysis point ofview, as well as from practical ex-periments.

Ekins’ studies during the early1990s led to a sensitive microar-ray-based analytical technology.Analytes, such as thyroid stimulat-ing hormone (TSH) or HepatitisB surface antigen, were detecteddown to femtomolar concentra-tions. The key, Ekins and his col-leagues found, was the ability tocontrol the concentration of cap-ture molecules in a small area,called a “microspot.”

Researchers at the Natural andMedicinal Sciences Institute(NMI) at the University of Tübin-gen in Germany summarized twoimportant principles for the highsensitivity obtainable in a mi-croarray format: that the bindingreaction occurs at the highest pos-sible target concentration and thatthe capture-molecule-target com-plex localized in a microspot gen-erates a higher signal density (incomparison to a macrospot). (5)The researchers provide multipleexamples of the considerable util-

Industry WatchIndustry Watch

A

Anthony J.Sinskey1,3,Stan N.Finkelstein1,2

and Scott M.Cooper1

1Program on thePharmaceuticalIndustry (POPI),2MIT Sloan Schoolof Managementand 3MITDepartment ofBiology, Cambridge,MA 02139

Despite some formidable technical challenges, proteinmicroarray and other biochip technologies are among themost important tools for the analysis of cell physiology as a set of linked networks.

20 PharmaGenomics July/August 2002

Industry WatchIndustry Watch

ity of protein microarray technol-ogy in studying different kinds ofprotein interactions, and suggestthat “further developments andoptimization of array productionand array performance combinedwith high-throughput generationof protein targets and ligands willextend the number of applicationsof protein microarrays dramati-cally.”

The intellectual appeal of pro-tein microarray technology is ob-vious: it affords researchers a wayto address questions at variouslevels by studying the interactionsbetween protein-protein, antigen-antibody, enzyme-substrate, pro-tein-DNA and ligand-receptor.

Technical Hurdles RemainToday, the transition of proteinmicroarrays and other biochipsfrom theory to actuality is evi-dence of just how quickly ge-nomics-based medicine is emerg-ing. Yet, some major technicalhurdles must be overcome beforeprotein microarrays will signifi-cantly impact the drug discoveryprocess. That’s where challengesand opportunities arise for thepharmaceutical and diagnostic in-dustries.

The source of capture mole-cules and the difficulties of dealingwith proteins represent the great-est obstacles to widespread use ofprotein microarrays. Proteins are

diverse molecules with varyingphysical/chemical properties, in-cluding charge and post-transla-tional modifications (glycosyla-tion, acetylation or phosphoryla-tion). Also, proteins that havebeen immobilized on a surface inthe form of an array must be keptin a functional state. This hampersthe ability of researchers toachieve high throughput withhigh-quality protein productiontechniques — the very achieve-ment needed, according to theNMI researchers, to extend pro-tein microarray applications. Thesolution might lie in the develop-ment of directed protein microar-rays, which are predicated on thebelief that some classes of proteinsmight be more amenable to ma-nipulation than others.

What else is needed? Forstarters, rapid, high-quality isola-tion techniques are critical, thespecific assay questions have to bedesigned, and scientists need reli-able ways to measure formation,as well as dissociation, of protein-protein interactions. Then, also,are the fundamental biology ques-tions, such as how protein do-mains interact to transmit andcontrol biological information. Inaddition, cell signaling pathways,in principle, can be characterizedrapidly from the initial stages ofreceptor-ligand binding to the cas-cade of biochemical activities re-sulting from the first step. An ex-citing possibility is the ability tounravel the complex interactionsthat occur on a cell when morethan one receptor-ligand bindingoccurs. This will bring greater easeto unraveling feedback, as well asfeed forward loops. Finally, manycell types can be analyzed simulta-neously, increasing the ability togenerate knowledge in a broaderbiological base.

Post-translational modificationand cellular compartmentaliza-tion of proteins also present chal-lenges, as they affect activity, speci-ficity and stability. Proteins havevariable half-lives, meaning strate-

gies for analyzing multi-compo-nent proteins are anything butstraightforward. However, solu-tions to these challenges will leadto molecular portraits of normalversus diseased states of cells, al-lowing for more effective inter-vention into the complex cellularnetworks.

Despite all these technical chal-lenges, protein microarrays holdthe promise of becoming one ofthe most important tools for theanalysis of cell physiology as a setof linked networks. The technol-ogy has the potential to lead to asystematic approach that is capa-ble of assessing rapidly and in par-allel the state of each componentin the network. Then, perturba-tions via gene replacement, anti-sense technology or — more con-sequentially — small moleculeswith protein microarrays fromdiseased versus normal cells willlead to new small-molecule drugcandidates. That is why the tech-nology will be so central to the fu-ture of healthcare.

The NMI researchers offer anoutstanding example of what thefuture holds in medical research,where,“protein microarrays willaccelerate immune diagnosticssignificantly by analyzing in paral-lel all relevant diagnostic parame-ters of interest.” They hypothesizethe “analysis of multiple tumormarkers from a minimumamount of biopsy material” andsuggest that “new possibilities forpatient monitoring during diseasetreatment and therapy will be de-veloped based on this emergingtechnology.” (6)

The Biochip BusinessAll this speaks to a burgeoningmarket for protein microarrays.Frost & Sullivan, a New York-based consulting firm specializingin emerging high-technologymarkets, projected in June 2001that the biochip industry will sur-pass the $3.3 billion revenuemark by 2004, with the largestshare garnered by the microarray

The intellectual appeal of protein

microarray technology is that it

affords researchers a way to

address questions at various levels

by studying the interactions

between protein-protein, antigen-

antibody, enzyme-substrate,

protein-DNA and ligand-receptor.

PharmaGenomics July/August 2002 21

segment. (7) The firm “antici-pates that diagnostic applicationswill gradually begin to appearover the forecast period, first inclinical labs and physician officesand, gradually, beyond the fore-cast period, in private homes,”with the market for biochipproducts reaching tens of billionsof dollars annually by 2010.

According to another report,more than 100 companies partici-pated in the microarray and pro-tein chip markets in 2000, andthat number is growing by morethan 35% each year. (8)

For the pharmaceutical indus-try, this new technology is emerg-ing at a critical juncture. In a pe-riod of great increase in the num-ber of top pharmaceutical prod-ucts scheduled to go off patent,tremendous growth in the de-mand for drugs, especially newdrugs, and slowing growth anddeclining R&D productivity, theindustry needs an increase in po-tential drug targets. The industrytoday is spending more money todevelop fewer products. Proteinmicroarray technology is one ofseveral biochip technologies thatcan help shift the trend back toone of continuing innovation.

In today’s pharmaceutical lab,business challenges are reducingdrug development time, speedingup the identification of new andvalidated drug targets and devel-oping high-quality informationearly on in the discovery process.Protein microarray technologymeasures up to all three of these

needs. Its integration with othertechnology platforms will be criti-cal to maximizing the value ofprotein microarrays. The technol-ogy also should be applied as partof emerging strategies for person-alized medicine — one of themajor promises of pharmacoge-nomics.

References1. Ken Howard, “Protein Microar-

rays May Just Be the Next Big(Little) Thing,” Feb. 21, 2002, atwww.genomeweb.com.

2. H. Zlu, et al., Science 293,2101-2105 (2001).

3. G. MacBeath and S.L. Schreiber,Science 289(5485), 1760-1763(2000).

4. See, for example, R.P. Ekins, J.Pharm. Biomed. Anal. 7, 155-168 (1989); R. Ekins, et al.,Ann. Biol. Clin. (Paris) 48, 655-666 (1990); R. Ekins and F.Chu, Ann. Biol. Clin. (Paris) 50,337-353 (1992).

5. M.F. Templin, D. Stoll, M.Schrenk, et al., TRENDS inBiotechnology 20:4, 160-166(April 2002).

6. Ibid.7. Frost & Sullivan, June 13, 2001.8. Genetic Engineering News (De-

cember 2000), atwww.genengnews.com. PG

The pharmaceutical industry is spending

more money to develop fewer products.

Protein microarray technology is one of

several biochip technologies that can help

shift the trend back to one of continuing

innovation.