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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)



2 21 June 2011

The drug discovery process



3 21 June 2011

Gene Transcript

Protein MetaboliteHuman cell

Which are drug targets?



4 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.Venter J.C. et al., Science, 2001, 291:1304-1351



5 21 June 2011

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



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



7 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 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.)



8 21 June 2011

General workflow of ProteomicsGeneral workflow of Proteomics

sample

collection

cells

body fluids

tissues

protein

identification

mass spectrometry

protein

separation

chromatography

electrophoresis

...

bioinformatics



9 21 June 2011

Overview of Proteomic strategies

Chandramouli K. & Qian P.Y., Human Genomics and Proteomics, 2009, 1-22



10 21 June 2011

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)



11 21 June 2011

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



12 21 June 2011

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



13 21 June 2011

Proteomics by isotope-coded affinitytags (ICAT)

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

quantify



14 21 June 2011

Proteomics by ProteinChips array

Reddy G. & Dalmasso E.A., J. Biomed. Biotech., 2003, 4:237-241

chromatographic

surface

hydrophobic

anionic

IMAC

normal phase

cationic



15 21 June 2011

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



16 21 June 2011

Protein identification by matching protein

database

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



17 21 June 2011

#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



18 21 June 2011

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



19 21 June 2011

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.)



20 21 June 2011

Drug target/biomarker identification

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



21 21 June 2011

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



22 21 June 2011

Lead selection and drug efficacy study

Jeffery D.A. & Bogyo M., Curr. Opion. Biol., 2003, 14:87-95



23 21 June 2011

Study of drug toxicity mechanism

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



24 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.Summeren, A.V. et al., Toxicol. Sci., 2011, 120:109-122



25 21 June 2011

Drug screening with toxicology

Collins B.C. et al., Expert Opin. Drug. Metab. Toxicol., 2007, 3:689-704

Expressionprofilingexperiments



26 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.



27 21 June 2011

• 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



28 21 June 2011

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



29 21 June 2011

Thank you for your attention !

trial lecture cyh

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