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Glycomics2004-CrKa
1. Drug Discovery for Novel Glycomics-derived Targets:
Tools for the glycobiologist.
Craig Karr
Cambridge Healthtech Institute
Glycomics - Carbohydrates in Drug Discovery
Cambridge, MA
April 26th-27th, 2004
*Work supported by Millennium Pharmaceuticals, Inc.
and Northeastern University, Department of Biology
Craig Karr April 26-27th, 2004 CHI - Glycomics
2. Drug Discovery for Novel Glycomics-derived Targets:
Tools for the glycobiologist.
1. Target-by-class Approach
2. Parallel Protein Expression & Purification Efforts
3. Rapid Deorphaning using Glycoarrays
4. Immediate Transition to generic HTS Platform
5. Potency & Selectivity within Target Class
6. Pre-Clinical ADME/Tox, DMPK, and Safety
7. Clinical Trials
8. Marketed Therapeutic
Craig Karr April 26-27th, 2004 CHI - Glycomics
3. Traditional Approach to Drug Discovery:
*~50-100 targets advanced per FDA-approved Drug
*Each target requires significant time, cost, and employee resources
Target Proprietary Public Academic 1 Target
Discovery Platform(s) Domain Collaboration Validated
Protein Attempt 1: Attempt 2: Attempt 3: 1 Target
Expression E.coli Insect Cells Mammalian Sourced
Substrate ID HTS Assay HTS Potency & 1 Target
& Assay Dev. Config. Screening Selectivity Screened
Lead Lead Physico-chem ADME/Tox 1 Preclinical
Optimization Confirmation Properties Profiling Candidate
Craig Karr April 26-27th, 2004 CHI - Glycomics
4. High Throughput Biochemistry Approach to Drug Discovery:
*Established processes & platforms increases efficiency, reduces costs
*Target-by-Class approach facilitates chemogenomics & selectivity efforts
Target Targets
-by-Class Parallel Target Discovery Efforts
Validated
Protein Targets
Parallel Expression Efforts
Expression Sourced
Substrate ID Targets
Parallel Screening Efforts
& Assay Dev. Screened
Lead Lead Physico-chem ADME/Tox Preclinical
Optimization Confirmation Properties Profiling Candidate
Craig Karr April 26-27th, 2004 CHI - Glycomics
5. Development of a deorphaning platform for the
rapid progression of glycomics-derived
“XDP-Sugar : polypeptide glycosyltransferases”
Validation using novel glycosyltransferases;
human ppGalNAc-T14 (unpublished)
human gpGalNAc-T15, (unpublished)
Craig Karr April 26-27th, 2004 CHI - Glycomics
10. Rapid Acceptor Profiling for High-throughput Biochemistry
Peptide arrays compatible with all peptide modifying enzymes
Custom Peptide Arrays
• ~900 physiologically-relevant
peptides represented in arrays
• direct link to biochem pathways
• 1 µl assays saves enzyme
• simple wash protocol
• phosphorimaging-compatible
• size similar to a credit card
Craig Karr April 26-27th, 2004 CHI - Glycomics
11. Development of a deorphaning platform for the
rapid progression of glycomics-derived
“XXP-Sugar : oligosaccharide glycosyltransferases”
Validation using known glycosyltransferases;
human β3GlcNAc-T2 (published)
Bovine β4Gal-T (published)
Porcine α3GalT (published)
parasite β3GalNAc-T (J Microbiol. In press)
Craig Karr April 26-27th, 2004 CHI - Glycomics
12. Important Roles for Human β3GlcNAc-T’s
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
RER Cis Golgi
αGase-1 αGase-2 αMase αMase-1
P
P
Dolichol
Inflammation: leukocyte-selectin binding
Medial Golgi Oncology: metastasis
Fuc-T
αMase-2
Obesity: ? SLeX
GlcNAc-T1 GlcNAc-T2
Trans Golgi * *
Gal-T’s
Gal-T’s
Gal-T Sialyl-T iGn-T GlcNAc-T’s GlcNAc-T’s
- GlcNAc - Gal - Man - Fuc - Glc - Sialic acid
Craig Karr April 26-27th, 2004 CHI - Glycomics
13. Published Role for Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Incorrectly Predicted Activity by BLAST analysis = β3Gal-T
Published in vitro & in vivo β3GlcNAc-T2 (syn. -T1) Activity:
β3Gn-T2
EC 2.4. 1.149
UDP-GlcNAc UDP
Terminal Poly-LacNAc
Hennet et al., 1998 J Biol Chem;
Zhou et al., 1999 PNAS;
Shiraishi et al., 2001 J Biol Chem,
Craig Karr April 26-27th, 2004 CHI - Glycomics
15. Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Rapid identification of critical non-reducing sugar (Gal), linkage (1,4), & conformation (β)
LacNac-biot selected by cost/benefit analysis
SAM Membrane /
Phosphor image
controls
*109 custom biotinylated-acceptors
Craig Karr April 26-27th, 2004 CHI - Glycomics
16. Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Rapid identification of critical non-reducing sugar (Gal), linkage (1,4), & conformation (β)
Scintillation
Proximity
Assay
Initial Activity vs. Mock:
• 100 ug PVT - SPA beads / well
• PE Biosystems MicroBeta reader
• 10 ng β3GnT2 / well
• 100 uM / 200 nCi UDP-[3H]-GlcNAc
• 100 uM biotin-acceptors
• 10 mM Trizma pH 7.5
• 10 mM MnCl2
• 0.05% BSA
• 10% Glycerol
• 4 h at 22 oC
• 10 ul rxn
Craig Karr April 26-27th, 2004 CHI - Glycomics
17. Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Selected
for Selected
Bulk assays for
based on further
cost/benefit kinetics
analysis analysis
Craig Karr April 26-27th, 2004 CHI - Glycomics
19. Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
β3GnT2 Activity vs Solution Phase Acceptors
(Phosphate-release Assay; Acceptors profiled at 1.0 mM)
1.6
Gal-β,4-GlcNAc
1.4 Gal-β,4-Glc-ph
Gal-β,4-Glc
Galactose
1.2 x Glucose
OD 610 nm
1.0
0.8
0.6
0.4
0.2
0 5 10 15 20 25 30
Time (min)
Craig Karr April 26-27th, 2004 CHI - Glycomics
20. Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Miniaturized profiling possible with polyvalent oligosaccharide micro-arrays
GlycoChip™
GlycoChip™
* GlycoChip is a product of Glycominds, Israel.
Craig Karr April 26-27th, 2004 CHI - Glycomics
21. Acceptor Profiling using Polyvalent MicroArrays
Terminal sugar represented for simplicity. Branched oligosaccharides not indicated.
Rha GalU blank Ara GlcU
Glc GlcNAc Man Neu5Ac Xyl
GlycoChip™
Gal GalNAc Fuc Glc
biotin
* GlycoChip is a product of Glycominds, Israel.
Craig Karr April 26-27th, 2004 CHI - Glycomics
22. Acceptor Profiling using Polyvalent MicroArrays
Terminal sugar represented for simplicity. Branched oligosaccharides not indicated.
Rha GalU blank Ara GlcU
~ 250 pxls Glc GlcNAc Man Neu5Ac Xyl
* Manβ4Glc
GlycoChip™
Gal GalNAc Fuc Glc
* Gal (α/β) * Galβ4(3)Glc(NAc) biotin * Galβ4GlcNAc core
~ 500 pxls ~ 2500 pxls ~ 50 pxls ~ 100 pxls
* GlycoChip is a product of Glycominds, Israel.
Craig Karr April 26-27th, 2004 CHI - Glycomics
23. Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics
24. Acceptor Profiling of Porcine α3GalT
UDP-Gal : terminal-Gal α1,3-galactosyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics
25. Acceptor Profiling of Bovine β4GalT
UDP-Gal : terminal-GlcNAc β1,4-galactosyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics
26. Acceptor Profiling of Giardia β3GalNac-T (CWS)
UDP-GalNAc : GalNAc β1,3-N-acetylgalactosaminyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics
27. Acceptor Profiling using Polyvalent MicroArrays
*Compatible with all oligosaccharide-modifying enzymes
A. Bovine β4Gal-T C. Porcine α3Gal-T
B. Human β3GlcNAc-T2 D. Giardia β3GalNAc-T (CWS)
Craig Karr April 26-27th, 2004 CHI - Glycomics
28. Summary:
• Solution-phase monovalent arrays & solid-phase
polyvalent microarrays are effective tools to
de-orphan and study novel glycosyltransferases.
• This approach is applicable to glycopeptide,
glycolipid, and carbohydrate modifying transferases.
• Standard and custom order biotinylated-acceptors
are now available and are compatible with bench-top
and high-throughput detection systems.
Craig Karr April 26-27th, 2004 CHI - Glycomics
29. Acknowledgements:
Millennium Pharmaceuticals, Inc.
Susan Fish
Tom Parsons
Mike Kuranda
Dejan Bojanic (*Novartis, Cambridge)
Northeastern University
Ed Jarroll
For additional Information contact
Craig_Karr@Yahoo.com
Craig Karr April 26-27th, 2004 CHI - Glycomics