Kinase Screening and Profiling: Methods and Protocols

228 Pages·2016·7.057 MB·English

by  Hicham Zegzouti, Said A. Goueli (eds.)

Methods in
Molecular Biology 1360
Hicham Zegzouti
Said A. Goueli Editors
Kinase
Screening
and Profi ling
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
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Kinase Screening and Profiling
Methods and Protocols
Edited by
Hicham Zegzouti and Said A. Goueli
Promega Corporation , Madison , Wisconsin , USA
Editors
Hicham Zegzouti Said A. G oueli
Promega Corporation Promega Corporation
Madison, WI, USA Madison , W I, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-3072-2 ISBN 978-1-4939-3073-9 (eBook)
DOI 10.1007/978-1-4939-3073-9
Library of Congress Control Number: 2015951783
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Prefa ce
Protein kinases play a central role in cellular physiology and diverse biological processes and
thus alterations in their expression or activity have considerable consequences. Based on the
critical role they play in regulation of normal and abnormal cellular growth and differentia-
tion, these enzymes prove to be very valuable therapeutic targets. It has been estimated that
over 60% of current discovery programs in pharmaceutical companies are focused on pro-
tein kinase inhibitors. As of 2015 there are 30 small molecule kinase inhibitors approved for
human oncology and for autoimmune diseases and over 150 are currently in clinical trials.
These drugs have grossed over $30 billion and projected to reach over $40 billion in 2
years. It is worth noting that the majority of these clinical trials target only 42 protein
kinases and about 50% of these inhibitors target kinases which already have approved drugs.
Furthermore, over 100 kinases have unknown function and 50% of all kinases are largely
uncharacterized. Thus there is signifi cant hope for breakthrough therapies directed towards
previously untargeted kinases.
Although so much success has been achieved with developing novel therapeutics for pro-
tein kinases in multiple disorders, many hurdles and obstacles had to be overcome at the early
stages of considering protein kinases as validated drug target. These include the high concen-
tration of cellular ATP for developing ATP competitive inhibitors, structural similarities for
developing selective inhibitors against specifi c kinase, cellular permeability of the drugs, and
above all cellular toxicities as kinases are involved in multiple functions and play intricate role
in several signaling pathways. Incorrect or incomplete assessment of cellular selectivity sub-
stantially increases the risk of unexpected toxicities at later stages of drug development.
In addressing these issues and with the expectations of expanding the therapeutic
potential of targeting as many kinases as possible, we believe a volume devoted to the
screening and profi ling of protein kinase inhibitors or activators will be timely. The volume
will cover current technologies that are in practice at various academic and industrial
research laboratories. It covers several facets of the drug discovery processes starting with
target identifi cation, assay development, and screening chemical libraries for hit identifi ca-
tion and lead optimization. The screening strategies have been mainly done through bio-
chemical assays with emerging trend for cellular kinase screening. The profi ling part of the
volume discusses several strategies and techniques that are required to minimize off-target
hits and minimize cellular toxicities that are caused by liability kinases. We have been fortu-
nate to have a diverse list of experienced authors whose main focus is on the kinases as drug
target with different strategies in reaching that goal. We hope this volume will benefi t sci-
entists and researchers who are interested in this fi eld of drug discovery.
Madison, WI, USA H icham Z egzouti
S aid A. G oueli
v

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x
1 HTRF Kinase Assay Development and Methods
in Inhibitor Characterization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Yong Jia , M ari M anuia , and Jose J uarez
2 A pplication of Eukaryotic Elongation Factor-2 Kinase (eEF-2K)
for Cancer Therapy: Expression, Purification, and High-Throughput
Inhibitor Screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Clint D. J . T avares , Ashwini K. D evkota , K evin N. Dalby ,
and Eun Jeong C ho
3 R ecombinant Kinase Production and Fragment Screening
by NMR Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Byeonggu Han and H ee-Chul A hn
4 Bioluminescence Methods for Assaying Kinases in Quantitative
High-Throughput Screening (qHTS) Format Applied to Yes1
Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase. . . . . . . . . . . . . . . . 47
Mindy I . D avis , D ouglas S. A uld , and James Inglese
5 U sing Bioluminescent Kinase Profiling Strips to Identify
Kinase Inhibitor Selectivity and Promiscuity . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Hicham Z egzouti , J acquelyn H ennek , and Said A . Goueli
6 Measuring Activity of Phosphoinositide Lipid Kinases
Using a Bioluminescent ADP-Detecting Assay . . . . . . . . . . . . . . . . . . . . . . . . 7 5
Andrew W. Tai and Jolanta Vidugiriene
7 A High-Throughput Radiometric Kinase Assay. . . . . . . . . . . . . . . . . . . . . . . . 8 7
Krisna C . Duong-Ly and Jeffrey R . P eterson
8 A High-Content Assay to Screen for Modulators of EGFR Function. . . . . . . . 9 7
Christophe Antczak and Hakim D jaballah
9 Monitoring Protein Kinase Expression and Phosphorylation
in Cell Lysates with Antibody Microarrays. . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Hong Z hang , Xiaoqing S hi , and Steven P elech
10 F rom Enzyme to Whole Blood: Sequential Screening
Procedure for Identification and Evaluation of p38 MAPK Inhibitors . . . . . . . 1 23
Silke M . Bauer , J akub M . K ubiak , Ulrich Rothbauer , and Stefan Laufer
11 Genetically Encoded Fluorescent Indicators to Visualize
Protein Phosphorylation in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 49
Moritoshi S ato and Yoshio Umezawa
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viii Contents
12 Characterization of an Engineered Src Kinase to Study
Src Signaling and Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 57
Leanna R. G entry , Andrei V. Karginov , Klaus M. Hahn ,
and C hanning J . D er
13 Screening One-Bead-One-Compound Peptide Libraries
for Optimal Kinase Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 69
Thi B. Trinh and D ehua Pei
14 D etermination of the Substrate Specificity of Protein Kinases
with Peptide Micro- and Macroarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Shenshen Lai , Dirk F .H. W inkler , Hong Z hang ,
and Steven P elech
15 R apid Identification of Protein Kinase Phosphorylation
Site Motifs Using Combinatorial Peptide Libraries . . . . . . . . . . . . . . . . . . . . . 2 03
Chad J. M iller and B enjamin E . T urk
I ndex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 17
Contributors
HEE-CHUL AHN • Department of Pharmacy, D ongguk University-Seoul, Goyang ,
Gyeonggi, R epublic of Korea
CHRISTOPHE ANTCZAK • HTS Core Facility, Memorial Sloan Kettering Cancer Center,
New York, NY, USA; Center for Proteomic Chemistry , N ovartis Institutes for Biomedical
Research , C ambridge, M A , U SA
DOUGLAS S. A ULD • Center for Proteomic Chemistry , N ovartis Institutes for Biomedical
Research , C ambridge, M A , U SA
SILKE M. BAUER • Department of Pharmaceutical and Medicinal Chemistry,
Institute of Pharmacy, E berhard-Karls-University Tuebingen , Tuebingen, G ermany
EUN JEONG CHO • Texas Screening Alliance for Cancer Therapeutics, and College of
Pharmacy , T he University of Texas at Austin , Austin, T X , U SA
KEVIN N . D ALBY • Division of Medicinal Chemistry and Texas Screening Alliance for
Cancer Therapeutics, T he University of Texas at Austin , Austin, T X , U SA
MINDY I. DAVIS • Division of Preclinical Innovation, National Center for Advancing
Translational Sciences, National Institutes of Health , Rockville, M D , U SA
CHANNING J . DER • Department of Pharmacology , U niversity of North Carolina at Chapel
Hill , C hapel Hill, NC, U SA
ASHWINI K. DEVKOTA • Texas Screening Alliance for Cancer Therapeutics,
The University of Texas at Austin , A ustin, TX, U SA
HAKIM DJABALLAH • HTS Core Facility, Memorial Sloan Kettering Cancer Center,
New York, NY, USA; Institut Pasteur Korea , S eongnam-si, G yeonggi-do, S outh Korea
KRISNA C. D UONG-LY • Cancer Biology Program, Fox Chase Cancer Center , P hiladelphia,
PA, U SA
LEANNA R. GENTRY • Department of Pharmacology, University of North Carolina
at Chapel Hill , Chapel Hill, NC, U SA
SAID A. GOUELI • Promega Corporation, Research and Development , Madison, W I, U SA ;
University of Wisconsin School of Medicine and Public Health , M adison, W I, USA
KLAUS M. HAHN • Department of Pharmacology , U niversity of North Carolina
at Chapel Hill , C hapel Hill, N C, USA
BYEONGGU HAN • Department of Pharmacy, D ongguk University-Seoul, Goyang ,
Gyeonggi, R epublic of Korea
JACQUELYN HENNEK • Promega Corporation, Research and Development Department ,
Madison, W I , U SA
JAMES INGLESE • Division of Preclinical Innovation, National Center for Advancing
Translational Sciences, N ational Institutes of Health , R ockville, M D , U SA
YONG JIA • Genomics Institute of the Novartis Research Foundation , San Diego, C A , U SA
JOSE JUAREZ • Genomics Institute of Novartis Research Foundation , San Diego, C A , USA
ANDREI V. KARGINOV • Department of Pharmacology , U niversity of Illinois at Chicago ,
Chicago, I L, U SA
ix