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

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DK3227_C000.fm Page i Monday, September 18, 2006 1:48 PM 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 CRC Press is an imprint of the Taylor & Francis Group, an informa business Boca Raton London New York DK3227_C000.fm Page iii Monday, September 18, 2006 1:48 PM CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-57444-450-6 (Hardcover) International Standard Book Number-13: 978-1-57444-450-6 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the conse- quences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www. copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. 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 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com DK3227_C000.fm Page iv Monday, September 18, 2006 1:48 PM 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 DK3227_C000.fm Page v Monday, September 18, 2006 1:48 PM 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 DK3227_C000.fm Page vi Monday, September 18, 2006 1:48 PM 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. DK3227_C000.fm Page vii Monday, September 18, 2006 1:48 PM 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. DK3227_C000.fm Page viii Monday, September 18, 2006 1:48 PM 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. DK3227_C000.fm Page ix Monday, September 18, 2006 1:48 PM

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