Biochips as Pathways to Drug Discovery - A. Carmen, G. Hardiman (CRC, 2007) WW

420 Pages·2007·14.29 MB·English

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Drug Discovery Series

Series Editor

Andrew Carmen

Johnson & Johnson PRD, LLC
San Diego, California, U.S.A.

1.
Virtual Screening in Drug Discovery, edited by Juan Alvarez
and Brian Shoichet

2.
Industrialization of Drug Discovery: From Target Selection Through
Lead Optimization, edited by Jeffrey S. Handen, Ph.D.

3.
Phage Display in Biotechnology and Drug Discovery, edited by
Sachdev S. Sidhu

4. G Protein-Coupled Receptors in Drug Discovery, edited by
Kenneth H. Lundstrom and Mark L. Chiu

5. Handbook of Assay Development in Drug Discovery, edited by
Lisa K. Minor

6.
In Silico Technologies in Drug Target Identification and Validation,
edited by Darryl León and Scott Markel

7. Biochips as Pathways to Drug Discovery, edited by Andrew Carmen
and Gary Hardiman

DK3227_C000.fm Page ii Monday, September 18, 2006 1:48 PM

Drug Discovery Series/7

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Library of Congress Cataloging-in-Publication Data

Biochips as pathways to drug discovery / edited by Andrew Carmen and Gary
Hardiman.
p. cm.
Includes bibliographical references.
ISBN 1-57444-450-6 (alk. paper)
1. Biochips. 2. Drug development. I. Carmen, Andrew. II. Hardiman, Gary,
1966-

R857.B5B54 2006
615’.19--dc22
2006045577

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Preface

In the summer of 1982 as a biochemistry undergraduate at Cornell University I had
the good fortune to pursue a research project in a prestigious fly (

Drosophila
melanogaster

) laboratory. The then very young John Lis took a chance on me,
offering the opportunity to do undergraduate research in his laboratory. It was an
exciting time even though I was on the periphery, gaining experience with

Drosophila

,
molecular biology technique, and the excitement of seeing the first “blue” flies from
the heterologous-controlled expression of

β

-galactosidase driven by the HSP70
promoter, a cover photo in

Cell

back in 1983. But the upshot was that I wanted to
know what controlled genes, what caused the chromosomal puffing, and what were
the chromatin (and epigenetic) factors.
As a graduate student and McKnight Scholar at the University of California at
Davis, I continued my interest in transcription. Through interactions with Peter Yau
(now at University of Illinois, Champaign-Urbana) and Morton Bradbury, an interest
was spawned in me in the acetylation of histones and other epigenetic factors.
Naturally, I moved on to Mike Grunstein’s laboratory at the University of California
at Los Angeles (UCLA). I knew that epigenetic factors, and certainly histones, were
key elements in transcription. My personal “holy grail” at the time was the elusive
histone acetyltransferase. Being somewhat naive in regard to biochemistry at the
time, I thought purification would be no problem. To my shock and dismay, I could
purify it and follow its activity, but it fell apart, no matter how fast I worked or what
I tried. Fortunately for me, I decided to see if I could find a histone deacetylase
activity in my extracts. Fortuitously, again, I had done a great job at purifying a
fairly stable histone deacetylase complex in some of my “acetyltransferase” extracts.
Not much of it, but it was relatively stable; some quick math determined it was
doable for protein sequence, if purified from approximately 5 kg of yeast. As luck
would have it, Thomas Sutherland at UCLA ran a fermentor facility that would
allow this to happen. Thus, I was enabled to purify the relatively scarce yeast HDA
complex.
Of course, this was only the setup for a bigger problem: Now you have the
enzyme, and good lord, the yeast genome was just sequenced, but you have four
other genes similar to HDA1, including RPD3, HOS1, HOS2, and HOS3. We had
some work to interpret this complexity. The fractionated enzyme activities would
disappear with their corresponding deletion, but how do you tell what they were
actually doing in the cell? Single deletions didn’t seem to have much effect. Fortu-
nately for me, I was in the laboratory concurrent to Andreas Hecht, (now at Max
Planck Freiburg) and Stephen Rundlett, who were developing chromatin IP cross-
linking techniques coupled with PCR for determination of targeting of protein factors
involved in gene-silencing. We decided to take two approaches: see if the deacetylase
proteins could be found associated directly or if a telltale trail of histone acetylation
could be seen in a gene-specific manner. In order to do this we developed a full set

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of specific antibodies directed toward every possible histone acetylation site and
every deacetylase. A large team of graduate students and “postdocs” took on the
daunting task of multiplex PCR, with limited automation. The deacetylases proved
elusive, but their trail of action could be followed. Eventually, we found site-specific
targeting of the enzyme but clearly, though, there was a better way.
It was the dawn of microarray. As Mike Grunstein said to me at the time, “Pat
Brown has it on a Web site. You can build it.” No more of that tedious multiplex
PCR stuff. Not knowing what I was getting into, I agreed to the project, along with
the help of the very skilled Rick Klufas and the late Mike Eng, both highly motivated
UCLA instrumentation facility staff. They were key to the success of the project.
To me, I got my feet wet in microarray and found it attractive enough to use as a
base for my next position. No more linear science, do the whole genome, or any
subset, in one go. Answer questions faster in days that previously took years.
Having spent, arguably, too much time at UCLA “having fun” as a postdoc and
assistant research scientist, I wanted a new challenge. I had spent a number of years
collaborating with Merck scientists and saw that I had more opportunity than I had
been led to believe in pharmaceutical R&D. An actual application and direction,
rather than just pure science, was compelling. The perfect position was found in
La Jolla at the new R.W. Johnson facility, now Johnson & Johnson Pharmaceutical
Research & Development, managing a small genomic operation. There I wouldn’t
just be building the technology, but I could drive it. If it didn’t exist, I had the
resources to define the new direction.
So, it was strive for the new chip to answer the questions that just 10 years ago
were daunting. Find the targets, bind the drugs, optimize them, put them in rats, and
test in people. Sounds simple. The truth is that there are many problems and inefficien-
cies in drug discovery. In a fiercely competitive marketplace, pharmaceutical companies
can not afford to spend excess dollars on developing drugs that will fail to get FDA
approval or will have some profoundly poor characteristics. In this book we present a
comprehensive look at how the industry faces these challenges, in many cases with new
technologies such as biochips to reduce the cost of drug discovery and improve drug
safety. The industry is getting smarter, finding the targets and weeding out potentially
problematic drugs sooner, thus cutting costs. In short order, we may also find that the
one drug for everyone may not be the norm. Pharmacogenomics presents a hope not
only to get better drugs, but to fit the right drugs to the right people. This might also
have implications that we may improve selection for clinical trials. Here, we look at
how these trends will affect the industry and what the outcomes might be on the science
and long-term prospects of these technologies and the companies utilizing them.

Andrew Carmen, Ph.D.

San Diego, California

In 1989 I received my first introduction to molecular biology in the laboratory of
Frank Gannon, then at the National University of Galway in Ireland. As a B.Sc.
honors student in microbiology, I had, like my classmates, to complete a 6-month
laboratory project; mine was on the examination of strains of

E. coli

for the
production of lambda phage extracts. This I dove into with the help of the late

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Riche Powell, and after many late nights, the lack of success in producing successful
extracts was attributed to the

E. coli

strains not being what they were supposed to
be. Nevertheless, my interest was piqued, and when Frank offered me a postgrad
position in his lab, working on the effects of saltwater adaptation on gene expression
in the Atlantic salmon, this seemed like a good way to spend the next few years. At
that time in late 1989, little of any genome, and in particular the salmon, had been
sequenced by today’s standards. Much of 1989 and 1990 consisted of sampling fish,
at local fish farms and at the National Diagnostics Center in Galway, extracting RNA
and building up a repository of salmon at different developmental stages. By today’s
standards the approach I took, although sound, seems quaint. I generated first-strand
cDNA from these salmon and used this material to screen salmon liver and kidney
cDNA libraries for clones that exhibited differences in their hybridization patterns.
Not surprisingly, the majority of the cDNA clones appeared the same in fresh and
saltwater salmon. I have a great memory, though, of looking at a series of autorads
on a long Irish summer evening and finding a series of cDNAs that clearly had
elevated levels in the saltwater fish compared to the freshwater fish. The next two
years was spent capitalizing on this find and characterizing these cDNAs. Not
surprisingly, many of the cDNAs I had uncovered were what one would expect,
namely genes encoding proteins involved in aerobic metabolism and growth.
In 1993, I decided that after 8 years in the same university, in a location well-
known for lots of rain, I wanted to live in a sunny climate for a couple of years. I
had the opportunity to spend a brief stint in the lab of Frank Talamantes at the
University of California, Santa Cruz in 1992 and, really liking California, I decided
that would be my next move. As luck would have it, I found a postdoc position at
DNAX Research Institute in Palo Alto in October 1993. At that time DNAX was
ramping up its in-house sequencing efforts and applying the high-throughput
approach to novel factor discovery under the direction of Gerard Zurawski and the
late Jacques Chiller. I joined the lab of Fernando Bazan and Rob Kastelein and, with
Fernando Rock, became part of a structural biology group involved in bioinformatics-
based gene discovery, with a strong emphasis on comparative genomics, in particular,
the characterization of novel signaling molecules and pathways in both human, fly,
and nematode systems. This allowed me to first work with DNA microarrays in the
mid 1990s as DNAX had a key interest in the technology. My earliest memory of
DNA microarray data is a 4 mB file Excel file (reasonably large by my mid 1990s
standards) that someone had aptly named “the complete enchilada.”
In 1998, I joined the ill-fated Axys Pharmaceuticals. This, at the time, seemed
like a unique opportunity, the chance to do interesting science in a biotech
setting. Working at Axys afforded me the chance to work with microarrays, in
the context of both oncology and nematode projects, but more importantly it
allowed me to participate in the Molecular Dynamics Early Technology Access
Program, which among other opportunities, introduced me to Andrew, ultimately
leading to this publication. In 1999, after living in four cities in an 18-month
period and my Chrysler Le Baron convertible having traversed the U.S. twice
on two occasions, I slowly began to think hard about what to do next. Another
Boston winter was not an option. Most of my colleagues seemed to be working
for or starting dot-com companies.

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Around this time, when Axys closed its doors in La Jolla in the fall of 1999,
my colleague and friend Antonio Tugores made me aware that the University of
California at San Diego (UCSD) wanted to hire a director to oversee the running of
the UCSD Biomedical Genomics Microarray (BIOGEM) Core Facility. This seemed
like the perfect opportunity to help establish a new genomics facility and assist
diverse researchers in applying this technology to a variety of biological questions.
Being able to optimize the technology, build microarrays that were not commercially
available, and help bring in emerging technologies has been enormously satisfying.
Working on the challenges of biochip technology, particularly dealing with small
sample sizes and applying the technology to the clinical setting, are current interests
of mine.
In this edition, we provide a comprehensive overview of the current state of
biochip technology and the effect biochips are having on biomedical research, in
particular the pharmaceutical industry. Technology platforms are presented and
covered in detail. The clinical and pharmacogenomic relevance of biochips, ChIP-
chip assays, and high-throughput approaches are all reviewed in depth. Chapters are
presented detailing the application of biochips to the study of malaria, toxicogenom-
ics, and SNPs. Intellectual property and market overviews are presented as current
and forward-looking perspectives. The DNA microarray field will thrive in the
coming years, an expansion that will encompass robotics, nucleic acid chemistries,
and informatics. Multidisciplinary approaches will help this field mature and find
its niche in the clinical arena. I trust that you find this book a valuable reference.

Gary Hardiman, Ph.D.

La Jolla, California

Note: This preface expresses the views of the authors and is not intended to express
any views of their respective employers.

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Acknowledgments

I would like to thank all my colleagues who contributed to this work, for without
these innovators we could not have been able to put together this innovative work.
I also thank my employer for allowing me to pursue this endeavor. I thank my wife,
Jeanette, for her support, love, and patience. —

A.C.

This publication contains chapters from world expert scientists, academics, program-
mers, and engineers skilled in the different facets of microarray technology, and I
am grateful to all those who contributed material. Many people behind the scenes
have contributed to the success of this project. Thanks go out to the UCSD Biomedical
Genomics Facility (BIOGEM) and my colleagues at UCSD, in particular professors
Chris Glass, Geoff Rosenfeld, Scott Emr, Bill McGinnis, and Tony Wynshaw-Borris.
I would also like to thank Cyndy Illeman and Deborah Seidle at UCSD Core Bio
Services and the members of BIOGEM past and present, particularly Jennifer Lapira,
Colleen Eckhardt, Ivan Wick, Kristin Stubben, Karin Bacon, Allen Lee, Barbara
Ruggeri, and Roman Sasik. A special thank you goes to Ivan Wick for providing
original images for the book’s cover. I thank my colleagues, friends, and family for
their constant support. Finally, I thank my wife Patricia and daughter Elena for their
love and affection, and their patience every time I disappeared with my laptop to
write or edit. —

G.H.

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