M e t h o d s i n M o l e c u l a r B i o l o g yTM

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to www.springer.com/series/7651

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Drug Design and Discovery

Methods and Protocols

Edited by

Seetharama D. Satyanarayanajois

Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

EditorSeetharama D. Satyanarayanajois, Ph.D.Department of Basic Pharmaceutical SciencesCollege of PharmacyUniversity of Louisiana at Monroe Monroe, LAUSAjois@ulm.edu

ISSN 1064-3745 e-ISSN 1940-6029ISBN 978-1-61779-011-9 e-ISBN 978-1-61779-012-6DOI 10.1007/978-1-61779-012-6Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2011920804

© Springer Science+Business Media, LLC 2011All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

v

Preface

In a rational drug discovery approach, it is necessary to identify the cause of the disease and its mechanism at the molecular level. Protein molecules that are the basic cause of the disease are identified first. Altering or modifying the protein–protein interaction could lead to therapeutic agents. The state-of-the-art methodology in drug discovery demands rational drug design, which will lower side effects and enhance therapeutic effects. Research in the pharmaceutical sciences and medicinal chemistry has taken an important new direction in the past two decades with a focus on large molecules, especially peptides and proteins, and DNA therapeutics. Protein and peptide drugs are currently the most rapidly expanding class of drugs. At present, more than 130 different proteins or peptides have been approved for clinical use. Whether it is peptide/protein-based drugs or organic molecules, the pro-cess of drug discovery involves several steps. The first is target identification and lead optimization. In the former process, molecules that bind a drug target and modulate the biological activity are identified using an in vitro assay, while in the latter potential drug molecules are optimized with respect to in vitro potency and other important parameters reflecting bioavailability and pharmacokinetic or toxicological properties. When a thera-peutic target has been identified and validated, the next stage in drug discovery is to con-duct high-throughput screening based on target binding or cellular assay to identify a lead compound. Once a lead compound is identified, the compound is modified chemically for higher activity and less toxicity. This involves the synthesis of a large number of analogs of the lead compound and testing them for biological activity. With several hundred protein targets available, screening thousands of compounds for biological activity and toxicity is a tedious and time-consuming process. In recent years, there has been an interest in dis-rupting protein–protein interactions using small molecules and peptides. With this inter-est in protein–protein interactions for targeting drugs, the number of drug targets will increase from hundreds to thousands. Once the drug-like molecule enters preclinical and clinical trials, it becomes an extremely expensive task to study each target. Hence, several methods have been discovered to screen compounds that may have drug-like properties. These methods involve computational, spectroscopic, analytical, and purification meth-ods, cellular assays, and molecular biology methods. In this particular volume of Methods in Molecular Biology, we present 16 chapters related to drug discovery and screening. It is impossible to cover all the methods related to drug discovery in a single volume. Our intent is to give an in-depth view of some protocols that are commonly used in drug discovery laboratories. Some of these techniques may be old and some are relatively new. They include computational docking, quantitative structure–activity relationship (QSAR), peptide synthesis, labeling of peptides and proteins with fluorescent labels, DNA-microarray, zebrafish model for drug screening, and other analytical screening and bio-logical assays that are routinely used during the drug discovery process. With the availability of three-dimensional structures of protein/DNA target molecules, computational meth-ods have played a key role in designing and screening thousands of compounds as possible candidates for druggable molecules. Hence, we have covered computational methods in

vi Preface

detail. Cellular and whole body imaging using fluorescently labeled molecules have gained popularity compared to procedures using radioactively labeled compounds. The method of fluorescent labeling of peptides and proteins is covered in detail in one chapter. Overall, this volume will serve as a laboratory reference for pharmaceutical chemists, medicinal chemists, and pharmacologists as well as for molecular biologists.

I would like to thank my wife, Latha Nagarajarao, for helping me to edit the chapters. Thanks to Dr. John Walker, chief editor of the series, for his advice and to all the authors who contributed to this series for their valuable time and sharing their detailed knowledge.

Monroe, LA Seetharama D. Satyanarayanajois

vii

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vContributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Virtual Screening for Lead Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Yat T. Tang and Garland R. Marshall

2 Computer-Aided Drug Discovery and Development . . . . . . . . . . . . . . . . . . . . . . . 23Shuxing Zhang

3 Using Active Site Mapping and Receptor-Based Pharmacophore Tools: Prelude to Docking and De Novo/Fragment-Based Ligand Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Ashutosh Tripathi, J. Andrew Surface, and Glen E. Kellogg

4 Methods for Evaluation of Structural and Biological Properties of Antiinvasive Natural Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55Mudit Mudit, Mohammad Khanfar, Girish V. Shah, and Khalid A. El Sayed

5 Solid-Phase Peptide Synthesis Using Microwave Irradiation . . . . . . . . . . . . . . . . . 73Justin K. Murray, Jennifer Aral, and Les P. Miranda

6 Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Jatinder S. Josan, Channa R.De Silva, Byunghee Yoo, Ronald M. Lynch, Mark D. Pagel, Josef Vagner, and Victor J. Hruby

7 DNA-Directed Assembly Microarray for Protein and Small Molecule Inhibitor Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Ng Jin Kiat, Fritz Simeon, Too Heng Phon, and Parayil Kumaran Ajikumar

8 Selection of Peptide Ligands for Human Placental Transcytosis Systems Using In Vitro Phage Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Saleem Basha, Shruthi Vaidhyanathan, and Giovanni M. Pauletti

9 Optimization of the Tetrazolium Dye (MTT) Colorimetric Assay for Cellular Growth and Viability. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157Paul W. Sylvester

10 Imaging NF-kB Signaling in Mice for Screening Anticancer Drugs . . . . . . . . . . . . 169Delira Robbins and Yunfeng Zhao

11 Evaluation of Anticancer Agents Using Flow Cytometry Analysis of Cancer Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179Vineet Gupta, Qian-Jin Zhang, and Yong-Yu Liu

12 Chemical Screening with Zebrafish Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193Hanbing Zhong and Shuo Lin

viii Contents

13 iTRAQ™ Labeling Coupled with LC-MALDI Mass Spectrometry for Monitoring Temporal Response of Colorectal Cancer Cells to Butyrate Treatment . . . . . . . . . . . . . . . . . . . . . . . . . 207Hwee Tong Tan, Teck Kwang Lim, Maxey C.M. Chung, and Qingsong Lin

14 Heterotypic Cell Adhesion Assay for the Study of Cell Adhesion Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225Seetharama D. Satyanarayanajois, Sharon Ronald, and Jining Liu

15 Evaluation of Antibacterial Activity of Proteins and Peptides Using a Specific Animal Model for Wound Healing. . . . . . . . . . . . . . . . . . . . . . . . 245Ramar Perumal Samy, Maung Maung Thwin, Vincent T.K. Chow, Ho Bow, and Ponnampalam Gopalakrishnakone

16 In Vitro Immunogenicity Risk Assessment of Therapeutic Proteins in Preclinical Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267Puneet Gaitonde and Sathy V. Balu-Iyer

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

ix

Contributors

Parayil Kumaran ajiKumar • Singapore-Massachusetts Institute of Technology Alliance (SMA), National University of Singapore, SingaporeDepartment of Chemical Engineering, Massachusetts Institute of Technology,

Cambridge, MA, USAjennifer aral • Peptide Research & Discovery, Amgen, Thousand Oaks, CA, USASathy V. Balu-iyer • Department of Pharmaceutical Sciences,

SUNY – University, Buffalo, Amherst, NY, USASaleem BaSha • James L . Winkle College of Pharmacy, University of Cincinnati,

Cincinnati, OH, USAho Bow • Department of Microbiology, National University of Singapore,

SingaporeVincent t.K. chow • Department of Microbiology,

National University of Singapore, Singaporemaxey c.m. chung • Department of Biochemistry, Yong Loo Lin School

of Medicine, National University of Singapore, SingaporeDepartment of Biological Sciences, Faculty of Science,

National University of Singapore, Singaporechanna r. De SilVa • Department of Chemistry, University of Arizona,

Tucson, AZ, USAKhaliD a. el SayeD • Department of Basic Pharmaceutical Sciences,

College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USAPuneet gaitonDe • Department of Pharmaceutical Sciences, SUNY – University,

Buffalo, Amherst, NY, USAPonnamPalam goPalaKriShnaKone • Venom and Toxin Research Program,

Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Vineet guPta • Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, LA, USA

Victor j. hruBy • Department of Chemistry, University of Arizona, Tucson, AZ, USA

jatinDer S. joSan • Department of Chemistry, University of Arizona, Tucson, AZ, USA

glen e. Kellogg • Department of Medicinal Chemistry & Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA, USA

mohammaD Khanfar • Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

ng jin Kiat • Singapore-Massachusetts Institute of Technology Alliance (SMA), National University of Singapore, Singapore

x Contributors

tecK Kwang lim • Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore

QingSong lin • Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore

Shuo lin • Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA

jining liu • Estee Lauder Companies Innovation Institute (China), Shanghai, China

yong-yu liu • Department of Basic Pharmaceutical Sciences, University of Louisiana at Monroe, Monroe, LA, USA

ronalD m. lynch • Bio5 Institute, University of Arizona, Tucson, AZ, USA

garlanD r. marShall • Center for Computational Biology, School of Medicine, Washington University in St . Louis, St . Louis, MO, USA

leS P. miranDa • Peptide Research and Discovery, Amgen, Thousand Oaks, CA, USAjuStin K. murray • Peptide Research & Discovery, Amgen, Thousand Oaks,

CA, USAmuDit muDit • Department of Basic Pharmaceutical Sciences, College of Pharmacy,

University of Louisiana at Monroe, Monroe, LA, USA; Department of Pharmaceutical Sciences, D’ Youville College, School of Pharmacy, Buffalo, NY, USA

gioVanni m. Pauletti • James L . Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, USA

marK D. Pagel • Department of Chemistry, University of Arizona, Tucson, AZ, USA

too heng Phon • Singapore-Massachusetts Institute of Technology Alliance (SMA), National University of Singapore, Singapore

Delira roBBinS • Department of Pharmacology, Toxicology, & Neuroscience, Louisiana State University Health Sciences Center in Shreveport, Shreveport, LA, USA

Sharon ronalD • Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

ramar Perumal Samy • Venom and Toxin Research Program, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Seetharama D. SatyanarayanajoiS • Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

giriSh V. Shah • Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

fritz Simeon • Singapore-Massachusetts Institute of Technology Alliance (SMA), National University of Singapore, Singapore

j. anDrew Surface • Department of Medicinal Chemistry & Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA, USA

Paul w. SylVeSter • Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA, USA

xiContributors

hwee tong tan • Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

yat t. tang • Center for Computational Biology, School of Medicine, Washington University in St . Louis, St . Louis, MO, USA

maung maung thwin • Venom and Toxin Research Program, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

aShutoSh triPathi • Department of Medicinal Chemistry & Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA, USA

joSef Vagner • Bio5 Institute, University of Arizona, Tucson, AZ, USAShruthi VaiDhyanathan • James L . Winkle College of Pharmacy,

University of Cincinnati, Cincinnati, OH, USAByunghee yoo • Department of Chemistry, University of Arizona, Tucson, AZ, USAQian-jin zhang • Department of Biology, Xavier University of Louisiana,

New Orleans, LA, USAShuxing zhang • Department of Experimental Therapeutics, M .D . Anderson

Cancer Center, Houston, TX, USAyunfeng zhao • Department of Pharmacology, Toxicology, & Neuroscience,

Louisiana State University Health Sciences Center in Shreveport, Shreveport, LA, USA

hanBing zhong • Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Shenzhen Graduate School of Peking University, Shenzhen University Town Shenzhen, China

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