AgeLab Pharma GmbH, Hamburg, Germany *Dermatology Clinic, University Hospital, Eppendorf, Hamburg
Amal K. Mukhopadhyay, Niels Grabe, Karsten Neuber *
Recombinant DNA Technology and Functional Genomics have given us an unprecedented possibility to diagnose, and sometimes even totreat 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 drivenby the technologies emerging from biotech industries
Targetidentification
Targetvalidation
LeadIdentification
Leadoptimisation
Preclinicaldevelopments
Moving bottlenecks in the discovery value chain
Adapted from Recap.com, L.E.K. analysis
"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
Predicted changes in the burden of disease from 1990 to 2020
Lower Respiratory infection 1 1 Ischaemic heart diseaesesDiarrheal diseases 2 2 Unipolar major depressionPerintalassociated conditions 3 3 Road traffic accidentsUnipolar major depression 4 4 Cerebrovascular diseasesIschaemic heart diseaeses 5 5 Chronic obstr. lung diseaseCerebrovascular diseases 6 6 Lower respiratory tract inf.Tuberculosis 7 7 TuberculosisMeasles 8 8 WarRoad traffic accidents 9 9 Diarrheal diseasesCongenital anomalies 10 10 HIV
1990 2020
Non melanoma skin cancer
stomach
Bladder
Colon
Prostate
Respiratory tract / Lung
Rectum
Stomach
Uterus
Breast
Non-Hogkin Lymphoma
Melanoma
Kidney/Urinary Tract
Stomach
Ovary
Non melanoma skin cancer
Colon
Rectum
Melanoma
Bladder
Respiratory tract / Lungs
Ten most common cancers in men Ten most common cancers in women
Common Types of Cancers ( Rheinland-Pfalz)
Molecular analysis of ageing process Alterungsprozessen
Young Old Sick
Protein-profile
Samples
Difference
Kd
220
9766
45
30
20,1
14,3
Increased
Decreased
1
7
8 9
10
11
12
14
15
16
20
21
22
23
24 25
26
27
28
2930
31
3233
34
35
36
37
38
39
1718
19
40 41
Y
Kd
220
9766
45
30
20,1
14,3
Increased
Decreased
1
7
8 9
10
11
12
14
15
16
20
21
22
23
2425
26
27
28
29
30
31
3233
34
35
36
37
38
39
17
18
19
40 41
O
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 anEnzyme/ Dehydrogenase
I ncreased in aged prostate :
Spot 17: pI 6.8 / 30 kD ;
Diff erent members of a protein family
Decreased in ageing : Spots 10 (pI 6.0; 25 kD) , 11 (pI 5,5 ; 30kD) , 12 (pI 5.8 ; 18 kD):
Variants of the same proteins ( Enzyme / Reductase )
Spots 14,15,19
Pooled Standard
2D gel co-separationIPGphor, Ettan DALT six
Image acquisitionThyphoon 9400
Cy2/Cy3 co-detection & normalisation
Cy2/Cy5 co-detection & normalisation
Differential In-gel Analysis (DIA)DeCyder Software
3D protein profile
Cy3 Cy5 Cy2
Mix samples
Sample A control
Sample B treated
DIGE Cy-dye labelling
2D sample preparation2D Clean-Up Kit2D Quant Kit
DIGE - experimental workflow
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
The ProteomeWorks „ Workflow“
IMAGING PDQUEST
2-D GELSOR
BLOTS
SAMPLEPREP
ProteinLynx PepSeq
PROTEINDIGEST
STATION
MASS SPECMALDI-TOF
MS/MSESI-MS
SWISS-PROTTrEMBL,etc.HTML Links
SPOTCUTTER
• Minimal-invasive Micro-biopsy for sample collection
• Suitable for epidermal diseases- Dermatitis- Eczema- Epidermal skincancer- Skin cancer precursor cell abnormalities.
New Diagnostic procedure
- 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
AgeLabs diagnostic Protein-Chip
• Tissue sample was labelled with fluorescent dye and after incubation, the chip was scanned in a fluorescence scanner.
Protein Chip Development
Early detection of epidermal skin cancer
• Currently punch-biopsy followed by histology is the only procedure for diagnosis of actinic keratoses, a pre-cancerous 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 micro-
diagnostic procedure developed by AgeLab.