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

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

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

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

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human ppGalNAc-T14 (unpublished)
human gpGalNAc-T15, (unpublished)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

Demonstration of Glycosyltransferase Acceptor Specificity
• ppGalNAc-T14 = glycosylates peptide backbone
• gpGalNAc-T15 = glycosylates GalNAc-modified peptide
• EA2 = naked peptide,
• gp-EA2 = synthetic glycopeptide (GalNAc-modified)
• streptavidin-SPA assay format, Leadseeker imaging system
30
25
20
15
10
5
0
ppGalNAc-T14 + gp- ppGalNAc-T14 + EA2 gpGalNAc-T15 + EA2 ppGalNAc-T14 +
EA2 \"- Control\" \"+ Control\" \"- Control\" gpGalNAc-T15 + EA2
Craig Karr April 26-27th, 2004 CHI - Glycomics

Rapid Acceptor Profiling for High-throughput Biochemistry
• Ligands – Custom biotinylated-(glyco)peptides, 2 µM / 40 pmol per well
• Substrates – 3H, 200 nCi / 2 µM / 40 pmol per well
• Enzymes – 100 ng recombinant human ppGalNAc-T’s per well
• Conditions – 23oC, pH 7.5, 10 mM MnCl2, 0.01% Brij35, 0.01% BSA
• Format – 200 µg LeadSeeker-SPA™ beads; Amersham Bioscience
• Detection – LeadSeeker™ CCD Imager; Amersham Bioscience
• Results – >40-fold signal-to-background; motif-dependent activity detected
ppGalNAc-T14 gpGalNAc-T15
1 gpGalNAc-T15 (novel)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16 ppGalNAc-T14 (novel)
Time Time
(min) 0 30 80 128 180 240 (min) 0 30 80 128 180 240
Craig Karr April 26-27th, 2004 CHI - Glycomics

Rapid Acceptor Profiling for High-throughput Biochemistry
ppGalNAc-T14 (Novel) vs. (glyco)peptide Array
RSA (%) Glycosylation Motif
200 189 100.0 red-EA2
186 98.6 Muc7
180
175 92.8 Muc5ac
160 170 89.7 EA2
140
120
100 28.0 EPO-T
23.6 Muc2
80
60 13.1 Muc1a
55 11.8 r-Muc2
47
40
27 0.9 Zonadhesin
20 25 0.7 mG-Muc
3 0.5 gp-Muc5ac
0 0.4 mod-HIV
0 30 60 90 120 150 0.2 gp-EA2
Craig Karr April 26-27th, 2004 CHI - Glycomics

Rapid Acceptor Profiling for High-throughput Biochemistry
gpGalNAc-T15 (Novel) vs. (glyco)peptide Array
RSA (%)
110
100.0 gp-EA2
100 98
90
80
70 3.8 gp-Muc5ac
60 <0.2 red-EA2
50 <0.2 Muc7
<0.2 Muc5ac
40 <0.2 EA2
<0.2 EPO-T
30 <0.2 Muc2
20 <0.2 Muc1a
<0.2 r-Muc2
10 <0.2 Zonadhesin
2.6 <0.2 mG-Muc
0
<0.2 mod-HIV
0 30 60 90 120 150
Craig Karr April 26-27th, 2004 CHI - Glycomics

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

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

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

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

Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
1 1 Glc-α-sp-biotin
Proof-of-principle
2 2 Glc-β-sp-biotin
(phosphorimage of SAM-captured acceptors)
3 3 Gal- α -sp-biotin
4 4 Fuc- α -sp-biotin
5 5 GlcNAc- β -sp-biotin
6 6 GalNAc- α -sp-biotin
7 7 GalNAc- β -sp-biotin
8 8 Neu5Ac- α -sp-biotin
9 9 Gal- β,3-Gal- β -sp-biotin
* 10 10 Gal- β,3-GlcNAc- β -sp-biotin
Initial Activity vs. Mock:
* 11 11 Gal- β,4-GlcNAc- β -sp-biotin • SAM capture membrane
12 12 Gal- β,3-GalNAc- α -sp-biotin • Phosphorimage detection
• 50 uM UDP-[14C]-GlcNAc
13 13 Fuc- α,3-GlcNAc- β -sp-biotin • 10 mM Trizma pH 7.5
14 14 Fuc- α,4-GlcNAc- β -sp-biotin • 10 mM MnCl2
• 0.2% Triton X-100
15 15 Gal- β,3-GalNAc- β -sp-biotin
• 0.05% BSA
16 16 GlcNAc- β,4-GlcNAc- β -sp-biotin • 20 pmol biotinylated acceptor / spot
Craig Karr April 26-27th, 2004 CHI - Glycomics

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

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

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

Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
β3GnT2 vs Solution Phase Acceptors
(Scintillation Proximity Assay; LeadSeeker™ CCD Imaging; Amersham)
60
(Galβ 1-4GlcNAcβ )2-3,6-GalNAcα -sp-biot
Galβ 1-4GlcNAcβ -sp-biot
50
Galβ 1-4GlcNAcβ 1-6GalNAcα -sp-biot
40 Galβ 1-4GlcNAcβ 1-6(Galβ 1-3)GalNAcα -
30 Galβ 1-4GlcNAcβ 1-3GalNAcα -sp-biot
Galβ 1-4Glcβ -sp-biot
20
GalNAcβ 1-4GlcNAcβ -sp-biot
10 Galβ 1-4(6-O-Su)GlcNAcβ -sp-biot
Galβ 1-3GlcNAcβ -sp-biot
Galβ -sp-biot
0 Glcβ -sp-biot
0 30 60 90 120
Craig Karr April 26-27th, 2004 CHI - Glycomics

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

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

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

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

Acceptor Profiling of Human β3GlcNAc-T2
UDP-GlcNAc : terminal-galactose β1,3-N-acetylglucosaminyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics

Acceptor Profiling of Porcine α3GalT
UDP-Gal : terminal-Gal α1,3-galactosyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics

Acceptor Profiling of Bovine β4GalT
UDP-Gal : terminal-GlcNAc β1,4-galactosyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics

Acceptor Profiling of Giardia β3GalNac-T (CWS)
UDP-GalNAc : GalNAc β1,3-N-acetylgalactosaminyl transferase
Craig Karr April 26-27th, 2004 CHI - Glycomics

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

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

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