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Glycomics2004-CrKa

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Drug Discovery for Novel Glycomics-derived Targets; Tools for the Glycobiologist

Drug Discovery for Novel Glycomics-derived Targets; Tools for the Glycobiologist

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  • 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
  • 6. 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 quot;- Controlquot; quot;+ Controlquot; quot;- Controlquot; gpGalNAc-T15 + EA2 Craig Karr April 26-27th, 2004 CHI - Glycomics
  • 7. 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
  • 8. 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
  • 9. 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
  • 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
  • 14. 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
  • 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
  • 18. 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
  • 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