Proteomics in Drug Design and Discovery: Trial Lecture

Proteomics in drug design and discovery
Trial lecture
Yunhan Chu
Department of Chemistry, Norwegian University of Science and Technology (NTNU)
21 June 2011
The drug discovery process
21 June 2011
Which are drug targets?

Gene Transcript
Protein
Human cell
Metabolite
21 June 2011
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.
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Venter J.C. et al., Science, 2001, 291:1304-1351
21 June 2011
Why Proteomics?
Genomics Transcriptomics Proteomics
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. DNA tells what possibly, RNA what probably and Proteins what actually happens.
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Proteomics
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.
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.
Wasinger V.C., Electrophoresis, 1995, 7:10901094 He Q.Y. & Chiu J.F., J. Cell. Biochem., 2003, 89:868-886
21 June 2011
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 proteomics
To define a proteins role in a cellular process on the basis of specific functional groups, protein-protein/ligand interactions, and novel pathways.
Functional (pathways are cascades of specific protein interactions that are necessary to activate distinct cellular functions.)
Expression
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
21 June 2011
General workflow of Proteomics
bioinformatics
sample collection cells body fluids tissues
protein separation chromatography electrophoresis ...
protein identification mass spectrometry
21 June 2011
Overview of Proteomic strategies
Chandramouli K. & Qian P.Y., Human Genomics and Proteomics, 2009, 1-22 21 June 2011
Proteomics by two-dimensional gel electrophoresis (2DGE)

Isolated cell / tissue
*
Enrich fraction of interest using affinity chromatography
12 14 16
*
2DGE
Isoelectric point (pI) molecular weight (MW)
Image analysis
Digestion
MALDI-MS
200 400 600 80010001200
m/z
Protein identification
21 June 2011
Ryan T.E. & Patterson S.D., Drug Discovery World Winter, 2001/2
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Proteomics by multidimensional protein identification technology (MudPIT)

SCX
RPT elute
Translation of mass spectra to amino acid sequence
Motoyama A., Anal. Chem., 2008, 80:7187-7193
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21 June 2011
Proteomics by activity-based probes (ABPs)

Label/tag Linker Reactive group
Han S.Y. & Kim S.H., Arch. Pharm. Chem. Life Sci., 2007, 340:169-177
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Proteomics by isotope-coded affinity tags (ICAT)
quantify
Patterson S.D., Aebersold R.H., Nature Genetics, 2003, 33:311-323
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21 June 2011
Proteomics by ProteinChips array

chromatographic surface
hydrophobic
anionic
cationic
IMAC
normal phase
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Reddy G. & Dalmasso E.A., J. Biomed. Biotech., 2003, 4:237-241 21 June 2011
Protein identification by mass spectrometry

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 peptides
+ _
Ionizer MALDI SELDI ESI
Mass analyzer Time-Of-Flight (TOF) Quadrapole Ion-Trap

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Detector Electron Multiplier (EM)

21 June 2011
Hamacher M. et al. (Eds.), Proteomics in Drug Research, Wiley-VCH, 2006
Protein identification by matching protein database
Domon B. & Aebersold R., Mol. Cell. Proteomics, 2006, 5:1921-1926
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21 June 2011
Proteomic technologies Pros

Technology Can detect 1000s of proteins at once Circumvents the proteome coverage problems Easily automated 2DGE MudPIT ICAT Protein chips ABPs
# #
Detects protein activity

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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21 June 2011
Proteomic technologies Cons

Technology 2DGE MudPIT ICAT Protein chips ABPs Cons Cannot detect proteins that are very small, large, acidic or basic, poorly soluble and of low abundance; Difficult to automate Does not detect abundance, activity, or interactions Does not detect post-translational modifications or interactions Require the cloning of proteins Probes are needed for all protein families, hence proteomic-scale coverage is difficult to ascertain
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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21 June 2011
Application of Proteomics in drug discovery

Drug target/biomarker identification
(Biomarkers are usually proteins that have their expression altered in response to a disease condition.)
Provide protein profiles of a cell or a issue that can be used to compare a healthy with a diseased state for protein difference in the search for drug targets.
Studies of drug efficacy and toxicity

Comparative analysis of reference protein profiles of normal or disease states vs. treatment states.
Drug screening / lead selection

The elicited drug- or probe- associated proteins can be predictive markers of activity or toxicity or comparative reference for new drug candidates.
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21 June 2011
Drug target/biomarker identification
Page M.J. et al, DDT, 1999, 4:55-62
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21 June 2011
Example: identification of calreticulin as a marker for bladder cancer
2DE of tissues silver staining bladder cancer tissue healthy urothelium
MALDI-TOF-MS and sequencing calreticulin (CRT)

Western blotting bladder cancer tissue healthy urothelium
Kageyama et al., Clin. Chem., 2004, 50:857-866
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21 June 2011
Lead selection and drug efficacy study
Jeffery D.A. & Bogyo M., Curr. Opion. Biol., 2003, 14:87-95
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21 June 2011
Study of drug toxicity mechanism
Gao Y. et al, Brief. Funct. Genomic. Proteomic., 2009, 8:158-166
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21 June 2011
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.
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Summeren, A.V. et al., Toxicol. Sci.,June 120:109-122 21 2011, 2011
Drug screening with toxicology
Expression profiling experiments
Collins B.C. et al., Expert Opin. Drug. Metab. Toxicol., 2007, 3:689-704
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21 June 2011
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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21 June 2011
Future directions of Proteomics

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
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21 June 2011
Future directions of Proteomics - contd

High-efficiency proteomics for drug discovery: high throughput proteomic screening platforms integrated with genomics, informatics and chemistry.
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Ryan T.E. & Patterson S.D., Drug Discovery World Winter, 2001/2 21 June 2011
Thank you for your attention !
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21 June 2011

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Proteomics in drug design and discovery

The drug discovery process

Which are drug targets?

From Genes to Proteins

General workflow of Proteomics

sample collection cells body fluids tissues

protein separation chromatography electrophoresis ...

protein identification mass spectrometry

Overview of Proteomic strategies

Proteomics by two-dimensional gel electrophoresis (2DGE)

200 400 600 80010001200

Proteomics by multidimensional protein identification technology (MudPIT)

Translation of mass spectra to amino acid sequence

Motoyama A., Anal. Chem., 2008, 80:7187-7193

Proteomics by activity-based probes (ABPs)

Proteomics by isotope-coded affinity tags (ICAT)

Patterson S.D., Aebersold R.H., Nature Genetics, 2003, 33:311-323

Proteomics by ProteinChips array

Protein identification by mass spectrometry

Ionizer MALDI SELDI ESI

Mass analyzer Time-Of-Flight (TOF) Quadrapole Ion-Trap

Detector Electron Multiplier (EM)

Hamacher M. et al. (Eds.), Proteomics in Drug Research, Wiley-VCH, 2006

Protein identification by matching protein database

Domon B. & Aebersold R., Mol. Cell. Proteomics, 2006, 5:1921-1926

Proteomic technologies Pros

Detects protein activity

Proteomic technologies Cons

Application of Proteomics in drug discovery

Studies of drug efficacy and toxicity

Drug screening / lead selection

Drug target/biomarker identification

Page M.J. et al, DDT, 1999, 4:55-62

Example: identification of calreticulin as a marker for bladder cancer

2DE of tissues silver staining bladder cancer tissue healthy urothelium

MALDI-TOF-MS and sequencing calreticulin (CRT)

Kageyama et al., Clin. Chem., 2004, 50:857-866

Lead selection and drug efficacy study

Study of drug toxicity mechanism

Gao Y. et al, Brief. Funct. Genomic. Proteomic., 2009, 8:158-166

Example: drug-induced hepatotoxicity of cyclosporin A in HepG2 cells

Drug screening with toxicology

Expression profiling experiments

Future directions of Proteomics

Future directions of Proteomics - contd

Thank you for your attention !

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