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1 21 June 2011
Proteomics in drug design and discovery
Trial lecture
Yunhan Chu
Department of Chemistry,
Norwegian University of Science and Technology (NTNU)
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2 21 June 2011
The drug discovery process
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Gene Transcript
Protein MetaboliteHuman cell
Which are drug targets?
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From Genes to Proteins
The Human Genome Project postulates the use of genome information to
identify and validate new drug targets based on the analysis of DNA or RNA.Venter J.C. et al., Science, 2001, 291:1304-1351
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Why Proteomics?
DNA tells what possibly,
RNA what probably and
Proteins what actually happens.
• Protein alternation cannot be fully deduced from DNA.
• RNA expression does not always reflect protein levels:
transitional control, degradation, turnover.
• Body fluids are not suitable for RNA expression analysis.
• Proteins are the physiological/pathological active key players.
Genomics Transcriptomics Proteomics
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6 21 June 2011
Proteomics
Proteomics is a research field that involves large scale identification,
characterization, and quantitation of proteins expressed in a cell,
tissue, or organism under given conditions such as drug treatment.
The term “proteomics” was first coined by Wasinger V.C. in 1995,
defined as the study of proteome. A proteome is the entire protein
complement expressed by a genome.
Wasinger V.C., Electrophoresis, 1995, 7:1090–1094He Q.Y. & Chiu J.F., J. Cell. Biochem., 2003, 89:868-886
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Classification of Proteomics
Expression proteomics
To identify proteins present in a biological sample or the proteins that are
differentially expressed between samples such as diseased vs. normal tissues.
Functional proteomicsTo define a protein’s role in a cellular process on the basis of specific functional
groups, protein-protein/ligand interactions, and novel pathways.
Structural proteomics
To determine tertiary structures of proteins, mainly using X-ray crystallography
and computational biology.He Q.Y. & Chiu J.F., J. Cell. Biochem., 2003, 89:868-886
Expression
Functional (pathways are cascades ofspecific protein interactionsthat are necessary to activatedistinct cellular functions.)
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General workflow of ProteomicsGeneral workflow of Proteomics
sample
collection
cells
body fluids
tissues
protein
identification
mass spectrometry
protein
separation
chromatography
electrophoresis
...
bioinformatics
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Overview of Proteomic strategies
Chandramouli K. & Qian P.Y., Human Genomics and Proteomics, 2009, 1-22
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Isolated cell / tissue
Enrich fraction of interest usingaffinity chromatography
Image analysis Digestion
MALDI-MS
Protein
identification
2DGE
Proteomics by two-dimensional gelelectrophoresis (2DGE)
Ryan T.E. & Patterson S.D., Drug Discovery World Winter , 2001/2
200 400 600 80010001200m/z m/z
12 14 16
*
*
Isoelectric point (pI)
molecular weight (MW)
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Motoyama A., Anal. Chem., 2008, 80:7187-7193
Proteomics by multidimensional protein
identification technology (MudPIT)
SCX
RPT
Translation of mass spectra
to amino acid sequence
elute
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Proteomics by activity-based probes (ABPs)
Han S.Y. & Kim S.H., Arch. Pharm. Chem. Life Sci., 2007, 340:169-177
Linker Reactive groupLabel/tag
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Proteomics by isotope-coded affinitytags (ICAT)
Patterson S.D., Aebersold R.H., Nature Genetics, 2003, 33:311-323
quantify
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Proteomics by ProteinChips array
Reddy G. & Dalmasso E.A., J. Biomed. Biotech., 2003, 4:237-241
chromatographic
surface
hydrophobic
anionic
IMAC
normal phase
cationic
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Ionizer
peptides
+
_
Mass analyzer Detector
• MALDI
• SELDI
• ESI
• Time-Of-Flight (TOF)
• Quadrapole
• Ion-Trap
• Electron
Multiplier
(EM)
Different mass spectrometry (MS)• Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS
• Surface enhanced laser desorption ionization time of flight (SELDI-TOF) MS
• Electro-spray ionization (ESI) tandem MS/MS
Protein identification by mass spectrometry
Hamacher M. et al. (Eds.), Proteomics in Drug Research, Wiley-VCH, 2006
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Protein identification by matching protein
database
Domon B. & Aebersold R., Mol. Cell. Proteomics, 2006, 5:1921-1926
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#Easily automated
#Can detect 1000s
of proteins at
once
#Circumvents the
proteome
coverage
problems
Detects protein
activity
Technology Protein chips
#
ABPsICATMudPIT2DGE
Proteomic technologies – Pros
Wang Y. et al., Curr. Comput. Aided Drug Des., 2005, 1:43-52Burbaum J. & Tobal G.M., Curr. Opin. Chem. Biol., 2002, 6:427-433
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Proteomic technologies – Cons
ConsTechnology
Probes are needed for all protein families, hence proteomic-scalecoverage is difficult to ascertain
ABPs
Require the cloning of proteinsProtein chips
Does not detect post-translational modifications or interactionsICAT
Does not detect abundance, activity, or interactionsMudPIT
Cannot detect proteins that are very small, large, acidic or basic,
poorly soluble and of low abundance; Difficult to automate
2DGE
Wang Y. et al., Curr. Comput. Aided Drug Des., 2005, 1:43-52
Burbaum J. & Tobal G.M., Curr. Opin. Chem. Biol., 2002, 6:427-433
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Application of Proteomics in drug discovery
Drug target/biomarker identification
Provide protein profiles of a cell or a issue that can be used to compare ahealthy with a diseased state for protein difference in the search for drug
targets.
Studies of drug efficacy and toxicityComparative analysis of reference protein profiles of normal or disease
states vs. treatment states.
Drug screening / lead selectionThe elicited drug- or probe- associated proteins can be predictive markers of
activity or toxicity or comparative reference for new drug candidates.
(Biomarkers are usually proteins that have their expression altered in response to a disease condition.)
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Drug target/biomarker identification
Page M.J. et al, DDT, 1999, 4:55-62
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healthy urotheliumbladder cancer tissue
MALDI-TOF-MS and sequencing calreticulin (CRT)
Example: identification of calreticulin as a
marker for bladder cancer
Kageyama et al., Clin. Chem., 2004, 50:857-866
2DE of tissues
silver staining
Western blotting
healthy urotheliumbladder cancer tissue
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Lead selection and drug efficacy study
Jeffery D.A. & Bogyo M., Curr. Opion. Biol., 2003, 14:87-95
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Study of drug toxicity mechanism
Gao Y. et al, Brief. Funct. Genomic. Proteomic., 2009, 8:158-166
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Example: drug-induced hepatotoxicity of
cyclosporin A in HepG2 cells
Protein networks generated by shortest path algorithm of MetaCore using the list of
differentially expressed proteins identified by 2DGE/MS analysis.Summeren, A.V. et al., Toxicol. Sci., 2011, 120:109-122
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Drug screening with toxicology
Collins B.C. et al., Expert Opin. Drug. Metab. Toxicol., 2007, 3:689-704
Expressionprofilingexperiments
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Conclusions
• Proteomics are applicable to all areas of drug discovery
from target identification to assessment of drug efficacy
and toxicity.
• The advantages and importance of directly analyzing
proteins make a strong argument for the value of proteomics.
• The shortcomings of throughput and sensitivity
highlight the need for improved automation, enrichment
and detection methods.
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• Gel-free technologies such as MudPIT, iTRAQ, 18O stable
isotope labeling will be more frequently used
• Exploration of proteome prefractionation methods (e.g. Plasma)
for better resolution
• Development of higher sensitive and specific chemical probes
• Using protein microarrays to discover a large collection of
functional or unknown multifunctional proteins
• Support of systematic and efficient analysis of vast proteomics
datasets
Future directions of Proteomics
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Future directions of Proteomics - contd
High-efficiency proteomics for drug discovery: high throughput proteomicscreening platforms integrated with genomics, informatics and chemistry.
Ryan T.E. & Patterson S.D., Drug Discovery World Winter , 2001/2
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