Microarray

better drugs by
MICROARRAY
Microarray analysis allows the elucidation of biological processes and pathways
on a genomic scale.This revolutionary technology promises an unprecedented
view of drug action and side reaction, the ability to identify drug targets and
optimise lead compounds, the capacity to predict patient responsiveness
vis-à-vis patient genetics in advance of clinical trials, and an opportunity to
minimise side-effects and risk factors following drug approval. A new era of
safer and more efficacious personalised pharmaceuticals leading to a disease-
free world are anticipated in light of this sweeping technological advance.

M
icroarrays are revolutionary analytical based on nylon filters and radioisotopes. The By Dr Mark Schena
devices that allow biological explo- experimental advantages of microarrays suggested
ration on a genomic scale (Schena et immediate applications in drug discovery and
al. 1995). Amplified complementary DNA (cDNA) development5, and this prediction has been borne
sequences1, oligonucleotides2, proteins3, tissues4 out by more than 200 drug-oriented scientific pub-
and other biochemical targets are attached to pla- lications in the past five years (see
nar substrates at discrete locations. Specific bind- http://arrayit.com/e-library). Microarrays have
ing interactions between discrete target molecules been used to examine tumour promoting sub-
on the substrate and fluorescent probe molecules in stances6, inflammatory disease states7, yeast drug
solution provide quantitative gene expression and targets8, antibacterial drugs9, liver toxins in the
genotyping information, as well as detailed meas- mouse10 and a myriad of other drug-related
urements of protein-protein interactions, small processes. A closer look at the drug discovery
molecule affinities and many other biochemical process in the context of microarray technology
processes (Figure 1). Microarrays, similar to reveals key areas that can be improved by microar-
microprocessors, use parallelism, miniaturisation ray analysis.
and automation as the three conceptual corner- Essentially all of the drugs that comprise the
stones. Key advances in biochemistry including the >$100 billion per year worldwide pharmaceutical
discovery of the double helix and DNA polymerase industry derive their efficacy from a simple bio-
in the 1950s, the development of recombinant logical paradigm: proteins execute the biological
DNA technology and the polymerase chain reac- function of genes, and altering protein function
tion (PCR) in the 1970s and 1980s respectively, can ameliorate disease symptoms by inhibiting cell
and the recent completion of the human genome signalling pathways, slowing the replication of
sequence are expediting the universal adoption of infectious bacteria and viruses, and promoting the
microarrays as analytical tools (Figure 2). division of blood cells and other cell types and
The speed, precision, affordability and efficiency processes that are medically beneficial. The vast
of microarray analysis offer order of magnitude majority of drugs are small molecules that bind
improvements over traditional molecular assays directly to cellular proteins and alter their activity,

Drug Discovery World Winter 2002/3 43

Microarrays

References
1 Schena, M, Shalon, D, Davis,
RW and Brown, PO (1995). A B
Quantitative monitoring of Probe
gene expression patterns with molecules
a complementary DNA
microarray. Science 270, 467-
470. Specific binding
2 Lockhart, DJ, Dong, H, Byrne,
MC, Follettie, MT, Gallo, MV,
Chee, MS, Mittmann, M,Wang,
C, Kobayashi, M, Horton, H,
Brown, EL. Expression
monitoring by hybridization to
high-density oligonucleotide
arrays. Nat. Biotechnol.
14:1675–1680, 1996.
3 MacBeath, G, Schreiber, SL.
Microscopic Planar substrate
Printing proteins as
target elements
microarrays for high-
throughput function
determination. Science
289:1760-1763, 2000.
4 Mucci, NR,Akdas, G, Manely, S,
Rubin, MA. Neuroendocrine
expression in metastatic prostate Figure 1
cancer: evaluation of high Microarray technology. A Schematic illustration of microarray analysis showing the microscopic target elements (ovals)
throughput tissue microarrays to configured in rows and columns on a planar substrate (rectangle). Specific binding interactions between target elements
detect heterogeneous protein and cognate probe molecules in solution (coloured diamonds) provide quantitative gene expression data for many
expression. Hum Pathol 31:406- genes in parallel. B Photograph of an actual 25,000 element (25K) DNA microarray used for gene expression profiling.
414, 2000. Image provided by Paul Haje,TeleChem/arrayit.com (Sunnyvale, CA)
5 Schena, M. (1996). Genome
analysis with gene expression
microarrays. BioEssays 18, 427-
producing changes in cell signalling (Figure 3A). patterns outside of the target pathway can identify
431.
6 Schena, M, Shalon, D, Heller, One prominent class of small molecules alters the side reactions, which could streamline lead com-
R, Chai,A, Brown, PO and RW function of cellular transmembrane receptor pro- pound optimisation and avoid potential toxic side-
Davis (1996). Parallel Human teins, and this drug family includes nearly 40 com- effects in clinical trials and beyond; 5) global
Genome Analysis: Microarray- mercial products with annual sales exceeding $20 expression profiling allows molecular parsing of
Based Expression Monitoring
billion11. Other drugs such as EPOGEN® and the clinical trials population, providing a biochem-
of 1,000 Genes. Proceedings of
the National Academy of NEUPOGEN® are themselves proteins, and ical basis for understanding patient responsiveness
Sciences USA 93, 10614-10619. administration of these recombinant protein drugs and non-responsiveness; 6) genotyping studies
7 Heller, RA, Schena, M, Chai, stimulates red and white blood cell production in prior to clinical trials allow identification of co-
A, Shalon, D, Bedilion,T, patients (Figure 3B) and ameliorates the conse- markers for responsiveness or non-responsiveness;
Gilmore, J,Woolley, DE and
quences of chronic renal failure and chemothera- 7) protein microarrays allow quantitative assess-
Davis, RW (1997). Discovery
and analysis of inflammatory py, respectively. ment of small molecule binding in the context of
disease-related genes using Microarray analysis promises better, safer and the proteome, facilitating target identification on a
cDNA microarrays. more efficacious drugs for 12 key reasons: 1) genomic scale; 8) small molecule microarrays facil-
Proceedings of the National expression profiles can provide quantitative itate massively parallel analysis of small molecule-
Academy of Sciences USA 94,
expression information for every human gene in protein interactions, speeding the traditional
2150-2155.
8 Marton, MJ, DeRisi, JL, every human tissue, and intracellular protein con- process of screening small molecule libraries; 9)
Bennett, HA, Iyer,VR, Meyer, centration bears directly on the drug concentration canine, rabbit, rat and primate microarrays can be
MR, Roberts, CJ, Stoughton, R, required to alter the biochemical activity of cellular used to enhance traditional animal trials; 10) tissue
Burchard, J, Slade, D, Dai, H, proteins; 2) treating cells with modest levels of a microarray (TMA) and laser capture microdissec-
Bassett, DE Jr, Hartwell, LH,
small molecule can produce a gene expression ‘fin- tion (LCM) technologies can pinpoint responsive
Brown, PO, Friend, SH. Drug
target validation and gerprint’, providing clues to the protein and path- tissues and cell types upstream of drug discovery
identification of secondary way targeted by the drug; 3) expression profiles and development; 11) next-generation screening
drug target effects using DNA produced by small molecule treatment can be (NGS) technology provides a myriad of affordable
microarrays. Nat Med 4:1293- superimposed over the patterns of gene expression microarray-based genotyping platforms, including
1301, 1998.
seen in disease tissues, speeding the identification those that allow identification of single nucleotide
Continued on page 46 of lead compounds; 4) changes in gene expression polymorphisms (SNPs) that bear on drug action;

44 Drug Discovery World Winter 2002/3

 Microarray

and 12) digital microarray data can be integrated terns of gene expression altered in disease states,
easily into pharmaceutical databases. allowing the researcher to ‘superimpose’ the two
Microarray expression data can be gathered types of data and identify promising lead com-
quickly for thousands of genes and tissues, com- pounds by the use of a computer. One such scenario
piled into large databases and queried by would be to test a lead compound that inhibits a
researchers to obtain expression levels for putative cellular pathway against a disease that activates the
drug targets. Other factors being equal, greater same pathway or, reciprocally, to use a lead com-
expression levels (protein concentration) require a pound that activates a cellular pathway against a
greater drug concentration to alter protein func- disease that represses the corresponding pathway.
tion, and hence bear directly on the potential effec- This ‘microarray-to-lead’ approach could speed
tiveness of a drug target11. Knowing the expres- drug development by streamlining or circumventing
sion levels for all the genes (proteins) in a pathway the traditional labour-intensive processes of target
has valuable implications for drug target selection identification and screening. The interplay of
in advance of small molecule screening. steroid hormones, altered gene expression patterns
Expression profiles can be gathered for cells and and breast cancer exemplify conceptually how such
tissues treated with pharmacological doses of small studies might be performed14.
molecules and recombinant proteins, and in many Pharmaceuticals have a myriad of beneficial
cases such treatments produce stereotyped expres- effects, but some drugs also manifest one or more
sion patterns resulting from drug-dependent alter- side-effects including dizziness, headache, nausea,
ations of protein function5,7,12. The genes and cel- elevated blood pressure, tremors, hair loss, numb-
lular pathways affected by drug treatment provide ness, dry mouth and a host of others. In many
clues to the proteins bound by the drug, and allow cases, the molecular basis of the side-effects is poor-
the identification of genes that reside downstream ly understood, but the predominant view is that
of the drug targets. Low doses of drug and short such effects probably arise by the unwanted binding
treatment intervals increase the likelihood of elicit- of a drug to one or more non-target proteins inside
ing physiologically relevant changes rather than the cell. Microarray-based expression profiling on a
general cellular toxicity and improve the chances genomic scale could be used to identify drug-
of identifying primary response genes. Gene-drug induced changes in gene expression that fall outside
relationships established by expression profiling the target pathway, yielding the identity of non-tar-
can be assembled into large databases for data gets and the pathways they control. Lead com-
mining, modelling and drug discovery13. pounds and currently marketed drugs could be
Gene-drug relationships can be compared to pat- optimised using an iterative procedure involving

Figure 2
Microarray technology
timeline. Key discoveries in
Watson and Crick Berg and co-workers Universal biochemistry have fuelled the
elucidate DNA develop recombinant Schena and co-workers adoption of genesis and proliferation of
structure in DNA technology at develop microarray microarray microarray technology
Cambridge Stanford technology at Stanford technology

1953 1959 1972 1983 1995 2001 2005

Kornberg and Mullis and Ventor, Lander and

co-workers co-workers co-workers
discover develop PCR determine human
polymerase in at Cetus genome sequence
St Louis as worldwide
consortium

Drug Discovery World Winter 2002/3 45

Microarrays

Continued from page 44

9 Wilson, M, DeRisi, J, A
Kristensen, HH, Imboden, P,
Rane, S, Brown, PO, Schoolnik,
Normal
GK. Exploring drug-induced
alterations in gene expression
in Mycobacterium tuberculosis
by microarray hybridization.
Proc Natl Acad Sci U S A
+ small
96:12833-12838, 1999.
molecule
10 Reilly,TP, Bourdi, M, Brady,
drug
JN, Pise-Masison, CA,
Radonovich, MF, George, JW,
Pohl, LR. Expression profiling
of acetaminophen liver toxicity
B
in mice using microarray
technology. Biochem Biophys Normal
Res Commun 282:321-328,
2001.
11 Debouck, C, Metcalf, B.The
impact of genomics on drug
discovery. Annu Rev Pharmacol
+ protein
Toxicol 40:193-207, 2000.
drug
12 Debouck, C, Goodfellow,
PN. DNA microarrays in drug
discovery and development.
DNA microarrays in drug
discovery and development.
Nat Genet. 21(1 Suppl):48-50,
1999.
13 Scherf, U, Ross, DT,
Waltham, M, Smith, LH, Lee, JK, Figure 3
Tanabe, L, Kohn, KW, Reinhold, Molecular mechanisms of drug action. A A small molecule drug (red diamond) binds to a specific intracellular protein
WC, Myers,TG, Andrews, DT, (yellow sphere) and alters its function, which inhibits a cellular pathway and ameliorates a disease. B A protein drug
Scudiero, DA, Eisen, MB, (yellow sphere) induces cellular division, beneficially elevating blood cell counts following surgery or chemotherapy
Sausville, EA, Pommier,Y,
Botstein, D, Brown, PO,
Weinstein, JN. A gene structural modification and microarray analysis to (NGS) technology allows thousands of patients
expression database for the
molecular pharmacology of
obtain structural variants that bind strongly to the and multiple genes to be screened in a single
cancer. Nat Genet 24:236-244, target protein but not to non-targets, with binding test17. Screening clinical trials candidates in
2000. specificity being assessed by expression monitoring advance of phase 2 trials might provide a more
14 Gruvberger, S, Ringner, M, during the iterative process. This general concept accurate assessment of drug efficacy and safety, by
Chen,Y, Panavally, S, Saal, LH, has been validated in a number of studies, including excluding patients that are incapable of drug
Borg, A, Ferno, M, Peterson, C,
Meltzer, PS. Estrogen receptor
those that correlate chemical exposure with specif- responsiveness for genetic reasons that have noth-
status in breast cancer is ic changes in gene expression15. ing to do with the efficacy of the drug per se.
associated with remarkably Phase 2 clinical trails are essential for determin- Recent microarray experiments on HER-2/neu
distinct gene expression ing the effectiveness and safety of a drug, but such expression in breast cancer patients underscore
patterns. Cancer Res 61:5979- trials rarely consider the genetics of the patients the heterogeneity of the patient population and
5984, 2001.
15 Hamadeh, HK, Bushel, PR,
that the drug is designed to treat. The availability the importance of elucidating the genetic basis of
Jayadev, S, DiSorbo, O, Bennett, of a complete human genome sequence and the heterogeneity18.
L, Li, L,Tennant, R, Stoll, R, affordable microarray genotyping technology, The burgeoning field of protein microarray tech-
Barrett, JC, Paules, RS, make it feasible to identify markers that might nology allows researchers to study protein-protein
Blanchard, K, Afshari, CA. determine responsiveness or non-responsiveness in interactions, protein-substrate specificity, protein-
Prediction of compound
signature using high density
the clinical trials population. One reasonable sce- drug binding, and other biochemical reactions in a
gene expression profiling. nario is that non-responsiveness tracks with the microarray format3. Protein microarray platforms
Toxicol Sci 67:232-240, 2002. presence of a single nucleotide polymorphism provide a major technological advance over tradi-
(SNP) in the target protein, which reduces drug tional and cumbersome protein assays that use
binding affinity and efficacy. SNP and other columns, filter discs and microplates and microar-
sequence variants are readily identified by rays are expected to replace most traditional bio-
Continued on page 48 microarray16, and next generation screening chemical assays in the near future. Microarrays con-

46 Drug Discovery World Winter 2002/3

 Microarray

taining the complete set of 25,000-35,000 proteins

expressed in human cells would allow comprehen-
sive assessment of drug binding in a single experi-
ment, and such tools are on the immediate horizon.
Small molecule microarrays19 containing thou-
sands of different drug targets allow small mole-
cules to be screened against a protein target in a
microscale format. Drug microarrays obviate the
need for a separate well for each compound as in
traditional microplate assays, which greatly min-
imises reagent consumption and improves
throughput. Ten microlitres of drug solution is suf-
ficient to manufacture >20,000 drug microarrays,
emphasising the compact size of the microarray
format. Continued technological advance in small
molecule synthesis and coupling chemistry suggest
that ‘drug chips’ will become routine tools in drug
discovery in the near future. Recent advances using
reflective ‘mirror’ substrates increase fluorescent
signals, reduce background, and offer 2-10-fold
increases in signal-to-noise ratio (Figure 4), further
improving the performance of microarray assays. Figure 4
Microarrays can be manufactured using gene Mirror substrate technology. Identical microarray samples were printed, hybridised and
scanned using an optically flat glass substrate (A), or an optically flat glass substrate coated
and protein sequences from organisms other than
with a highly reflective (mirror) backing, providing a five-fold increase in signal-to-noise ratio.
human, including canine, rabbit, mouse, rat, chim- Substrates and fluorescent labelling kits were provided courtesy of TeleChem/arrayit.com
panzee and others, and model studies in these (Sunnyvale, CA), and fluorescent scanning was achieved using a ScanArray Express from
organisms are showing great promise for toxicity PerkinElmer (Billerica, MA).The space bars correspond to 0.25mm (250µm)
testing in pre-clinical trials and for understanding
basic disease mechanisms20,21.
The advent of tissue microarrays4 and laser cap- and LCM technologies, and it is now possible to
ture microdissection-based microarrays22 allow think about drug action at the level of single cells.
gene expression analysis at single cell resolution, Segments of patient DNA can be printed at high
which is extremely valuable because many drugs density and hybridised in parallel with fluorescent
are thought to act on a subset of the cells present oligonucleotides to determine patient genotypes.
in a particular tissue. Lead compound identifica- NGS technology exploits multicolour fluorescence
tion and optimisation will make good use of TMA (Figure 5), and allows thousands of patients to be

Figure 5
Two colour fluorescence. Multicolour labelling and detection using Cy3 and Cy5 depicted as a red-green composite image can be used to increase the
precision of genomic and proteomic microarray assays including next generation screening. Scanning and data analysis was performed using a ScanArray
Express microarray scanner from PerkinElmer (Billerica, MA). Image provided by Dr Robin Stears,TeleChem/arrayit.com (Sunnyvale, CA).The scale bar
corresponds to 0.25mm (250µm)

Drug Discovery World Winter 2002/3 47

Microarrays

Continued from page 46

16 Hacia, JG, Fan, JB, Ryder, O,

Jin, L, Edgemon, K, Ghandour, Human genes Therapeutic agents
Microarrays
G, Mayer, RA, Sun, B, Hsie, L, (25-35,000) (106-108)
(information processors)
Robbins, CM, Brody, LC,Wang,
D, Lander, ES, Lipshutz, R,
Fodor, SP, Collins, FS.
Determination of ancestral
alleles for human single-
nucleotide polymorphisms ‘One gene-one drug’
using high-density
oligonucleotide arrays. Nat
Genet 22:164-167, 1999.
17 Schena, M. In Microarray
Analysis, 1st Edition, J.Wiley
and Sons, Hoboken, NJ, pp. Disease-free world
399-401, 2002. (2050)
18 Simon, R, Nocito, A,
Hubscher,T, Bucher, C,
Torhorst, J, Schraml, P,
Bubendorf, L, Mihatsch, MM, Figure 6
Moch, H,Wilber, K, Schotzau, Microarrays are information processors. Microarray assays enable drug discovery by speeding the specific partnering of
A, Kononen, J, Sauter, G. each of the 25-35,000 genes in the human genome with a specific therapeutic agent.This ‘one gene-one drug’ paradigm
Patterns of her-2/neu promises a disease-free world by the year 2050
amplification and
overexpression in primary and
metastatic breast cancer. J Natl
Cancer Inst 93:1141-1146,
screened for multiple disease loci in a single test17. Beadle and Tatum’s pioneering ‘one gene-one
2001. A key feature of the NGS approach is that the cost enzyme’ hypothesis of the 1940s, provides the
19 Kuruvilla, FG, Shamji, AF, of each test is amortised across the hundreds or foundation on which to achieve a disease-free
Sternson, SM, Hergenrother, PJ, thousands of patients represented on the chip, world by 2050. DDW
Schreiber, SL. Dissecting which reduces the genotyping cost by several
glucose signalling with
diversity-oriented synthesis
orders of magnitude compared to conventional
and small-molecule ‘one chip per patient’ microarrays. The capacity to
microarrays. Nature 416:653- screen SNPs and other sequence variants and to
657, 2002. identify viral and bacterial types rapidly and inex-
20 Hoffman, EP, Dressman, D. pensively will improve major aspects of drug devel-
Molecular pathophysiology and
targeted therapeutics for
opment and administration. The digital format of
muscular dystrophy.Trends all microarray data allows seamless integration
Pharmacol Sci 22:465-470, into the massive databases used in the pharmaceu-
2001. tical industry.
21 Bigger, CB, Brasky, KM, Microarrays promise to speed up drug discovery,
Lanford, RE. DNA microarray
analysis of chimpanzee liver
provide safer and more personalised medicines and
during acute resolving hepatitis eradicate disease in an argument that goes some-
C virus infection. J Virol thing like this. Aberrant gene function (mediated
75:7059-7066, 2001. by proteins) causes every human disease and the
22 Salunga, RC, Guo, H, Luo, L, sequence of every disease-causing gene is now
Bittner, A, Joy, KC, Chambers,
JR,Wan, JS, Jackson, MR,
known. Millions of small molecules made available
Erlander, MG. Gene Expression through the combined sources of natural products,
Analysis via cDNA Microarrays organic synthesis and combinatorial chemistry, Dr Mark Schena, the ‘Father of Microarray
of Laser Capture represent a yet-to-be-discovered drug against every Technology’, received his BA from UC Berkeley,
Microdissected Cells from human protein. In some cases, genes, genes prod- his PhD from UCSF and postdoctoral training at
Fixed Tissue. In DNA
Microarrays: A Practical
ucts and derivatives thereof can be used to amelio- Stanford University. Dr Schena wrote the first
Approach, M. Schena (editor), rate disease. Microarray assays allow the examina- paper on microarrays in 1995, the first two tech-
2nd Edition, Oxford University tion of DNA, RNA, proteins, small molecules and nical books for Oxford Press and Eaton
Press, Oxford, UK, pp. 121- tissues in a massively parallel format, allowing Publishing, and has just completed the first text-
137, 2000. partnering between every human gene (protein) book, Microarray Analysis, for J. Wiley & sons.
and a specific therapeutic agent (Figure 6). This He has given more than 80 lectures on microar-
‘one gene-one drug’ hypothesis, reminiscent of rays in 15 countries.

48 Drug Discovery World Winter 2002/3