Transcript Dr. A. K. Mukhopadhyay

Amal K. Mukhopadhyay, Niels Grabe, Karsten Neuber *
AgeLab Pharma GmbH, Hamburg, Germany
*Dermatology Clinic, University Hospital, Eppendorf, Hamburg
Recombinant DNA Technology and Functional
Genomics have given us an unprecedented
possibility to diagnose, and sometimes even to
treat many diseases which were so far
categorised as of idiopathic origin.
In the post-human genome era, with nearly the entire
human genome sequence being revealed, the proteomics
technology has moved to a centre stage in the drug
discovery processes.
Growth in Pharma companies has been driven
by the technologies emerging from biotech industries
Moving bottlenecks in the discovery value chain
Target
identification
Target
validation
Adapted from Recap.com, L.E.K. analysis
Lead
Identification
Lead
optimisation
Preclinical
developments
"Life would be infinitely happier if we could only be born at the age
of eighty and gradually approach eighteen."
-- Mark Twain, Autobiography with Letters
Mega-Trend Aging
Predicted changes in the burden of disease from 1990 to 2020
1990
Lower Respiratory infection
Diarrheal diseases
Perintalassociated conditions
Unipolar major depression
Ischaemic heart diseaeses
Cerebrovascular diseases
Tuberculosis
Measles
Road traffic accidents
Congenital anomalies
1
2
3
4
5
6
7
8
9
10
2020
1
2
3
4
5
6
7
8
9
10
Ischaemic heart diseaeses
Unipolar major depression
Road traffic accidents
Cerebrovascular diseases
Chronic obstr. lung disease
Lower respiratory tract inf.
Tuberculosis
War
Diarrheal diseases
HIV
Common Types of Cancers ( Rheinland-Pfalz)
Non melanoma
skin cancer
Prostate
Respiratory
tract / Lung
Breast
Non melanoma
skin cancer
Colon
Colon
Uterus
Bladder
Rectum
Rectum
Ovary
Stomach
stomach
Kidney/Urinary
Tract
Melanoma
Non-Hogkin
Lymphoma
Ten most common cancers in men
Melanoma
Bladder
Stomach
Respiratory tract /
Lungs
Ten most common cancers in women
AgeLab: Organspecific Ageing with a focus
on skin
Section:
Aged skin
Section
Young skin
Molecular analysis of ageing process
Alterungsprozessen
Samples
Young
Proteinprofile
Old
Difference
Sick
Kd
Y
220
Increased
97
66
Decreased
7
20
45
40 41
11
30
8 9
34
14
35
38
17
22
37
15
36
32
10
28
31
29
20,1
30
27
12
1
39
26
24
18
19
23
33
14,3
21
16
25
Kd
O
220
Increased
97
66
Decreased
7
20
45
21
40 41
16
8 9
11
30
14
34
17
35
18
22
15
37
38
36
33
19
23
32
10
28
31
20,1
29
30
27
12
24
39
1
14,3
26
25
Examples:
Decreased in Ageing :
Spot 2 and 3 , pI 7.6 and 7.8; Kd 20 ; identified as a CRP-1
Decreased in ageing
Spot 7, pI 6.3 / 32kD) : identified as an
Enzyme/Dehydrogenase
Increased in aged prostate :
Spot 17: pI 6.8 / 30 kD ;
Different members of a protein family
Decreased in ageing : Spots 10 (pI6.0; 25 kD) , 11 (pI5,5 ; 30
kD) , 12 (pI 5.8 ; 18 kD):
Variants of the same proteins ( Enzyme / Reductase )
Spots 14,15,19
DIGE - experimental
workflow
Mix samples
2D sample preparation
2D Clean-Up Kit
2D Quant Kit
Sample A
control
Sample B
treated
Cy3
Cy5
Pooled
Standard
DIGE Cy-dye labelling
Cy2
2D gel co-separation
IPGphor, Ettan DALT six
Image acquisition
Thyphoon 9400
Differential In-gel Analysis (DIA)
DeCyder Software
Cy2/Cy5 co-detection &
normalisation
Cy2/Cy3 co-detection & normalisation
3D protein profile
DeCyder differential in gel (DIA)
analysis
Result of full automatic
spot detection:
- 2153 spots detected
- 270 spots decreased*
- 170 spots increased*
* at least 2-fold change of max spot
volume; no spots excluded by
filtering
DeCyder DIA analysis Example 1
Spot 658 increased, volume ratio 3.93
DeCyder DIA analysis Example 2
Spot 714 increased, volume ratio 6.02
DeCyder DIA analysis Example 3
Spot 1685 increased, volume ratio 7.28
The ProteomeWorks „ Workflow“
SWISS-PROT
TrEMBL,etc.
HTML Links
IMAGING
SAMPLE
PREP
2-D GELS
OR
BLOTS
PDQUEST
SPOT
CUTTER
PROTEIN
DIGEST
STATION
ProteinLynx
PepSeq
MASS SPEC
MALDI-TOF
MS/MS
ESI-MS
Marker-Proteins for Aging
6 years
39 years
81 years
34 years
78 years
M1
M2
6 years
Systems-Biology
Bioinformatics based diagnostics
Regulatory
Network of
Cells
Protein-Chip
New Diagnostic procedure
• Minimal-invasive Micro-biopsy for
sample collection
• Suitable for epidermal diseases
- Dermatitis
- Eczema
- Epidermal skincancer
- Skin cancer precursor cell
abnormalities.
AgeLabs diagnostic Protein-Chip
- Chip is coated with specific
antibodies against identified
markers
- AgeLab has identified more than
50 different potential marker
proteins.
–Apoptosis regulatory proteins (13)
–Cell cycle regulatory proteins (9)
–Nitric oxide (NO) signaltransduction
regulatory Proteins (5)
–Intermediate Filament-Proteins (13)
–Chaperones (7)
–Tumor Suppressors (8)
Chip-Substrat
Protein Chip Development
• Tissue sample was labelled with
fluorescent dye and after incubation, the
chip was scanned in a fluorescence
scanner.
Early detection of epidermal skin
cancer
•
•
•
Currently punch-biopsy followed by histology is the only
procedure for diagnosis of actinic keratoses, a precancerous stage. The procedure is painful, leaves scar
and the outcome is unfortunately not absolutely
dependable.
With the help of AgeLab's procedure which is free from
any scar development, a diagnostic procedure is being
offered for the first time which is patients-friendly and
dependable results can be expected.
Early diagnosis of pre-cancerous stage in skin becomes
possible with the help of the protein chip based microdiagnostic procedure developed by AgeLab.
Euro-India Biotech-Conference,
Hamburg, September, 2004
Contact email:
[email protected]
Expression of Thanks
University of Hamburg
Prof. F.A. Leidenberger
AgeLab GmbH:
Dr. Matthias Koenig
Mr. Björn. Ehlers,
Ms. Monika Kistler
Special thanks are due to
Amersham Biotech, Freiburg
and Bio Agency, Hamburg