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Kevin J. Yarema Associate Professor of Biomedical Engineering The Johns Hopkins School of Medicine Carbohydrate Engineering ISBN 978-3-527-30632-9 - Wiley-VCH.

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Presentation on theme: "Kevin J. Yarema Associate Professor of Biomedical Engineering The Johns Hopkins School of Medicine Carbohydrate Engineering ISBN 978-3-527-30632-9 - Wiley-VCH."— Presentation transcript:

1 Kevin J. Yarema Associate Professor of Biomedical Engineering The Johns Hopkins School of Medicine Carbohydrate Engineering ISBN Wiley-VCH Introduction to Glycobiology (ME ) Phone:

2 An Overview of Todays Lecture First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Organ TransplantationMetabolic Oligosaccharide Engineering

3 From Google: First – What is Carbohydrate Engineering?

4 For pdfs of the introduction, or any chapter, me at First – What is Carbohydrate Engineering?

5 Basically, in 2005 we didnt really know very precisely What about now, in 2014? Lets define glycoengineering (a subcategory of carbohydrate engineering) as: 1)(primarily) The manipulation of glycans 2)(secondarily) for biomedical purposes* * But, is this even possible? Biologics (i.e. therapeutic glycoproteins) Xenotransplantation

6 The Promise of Sugar-based Therapies The term Glycobiology was coined in 1988 A flurry of clinical translation and commercialization efforts ensued (1990s) Glycans were implicated in many (most!) complex diseases Arthritis Cancer metastasis Immune disorders (Kawasaki Disease) Degenerative muscle disease Duchenne Muscular Dystrophy Arthritis

7 Commercialization and translational efforts were slow to be realized: Difficulties Ensued The Bittersweet Promise of Glycobiology Nature Biotechnology, 2001 (doi: /nbt ) The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 2005 (doi: /nrc1649) Uh oh!!

8 Time for an Analogy - Electric Cars A great idea 25 years ago that didnt work out* (*at the time) *Until today

9 OK – What is being Covered Up by Car Analogies? A Southern Mystery (from The Scientist, July 1, 2008) In 2004, strange things were happening when people living in the Southern United States received Erbitux (aka Cetuximab), an (mAb) anticancer drug. After Erbitux was approved, the first three patients that oncologist Bert O'Neil treated at the University of North Carolina, Chapel Hill, had severe anaphylactic reactions. One fell out of their chair," passing out as blood pressure plummeted. "It alarmed us. "I was quite upset," says research oncologist Christine Chung, when her patient with head and neck cancer had a severe reaction to the drug. "This was a young man and a last ditch effort" to gain a little more time for this patient.... Uh oh!!

10 What Happened? The affected patients had IgE antibodies against galactose-α-1,3- galactose ( -Gal), which triggered anaphylaxis when they were given the drug. -Gal The Southern Mystery angle: Lone Star Tick bites IgE against -Gal What happened? (in more detail) Unlike most other monoclonal antibodies, cetuximab is produced in the mouse cell line SP2/0, which expresses the gene for -1,3- galactosyltransferase. -Gal

11 Commercialization and translational efforts were slow to be realized: Pitfalls Along the Way are Being Overcome The Bittersweet Promise of Glycobiology Nature Biotechnology, 2001 (doi: /nbt ) The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 2005 (doi: /nrc1649) By 2008 we had learned a valuable first do no harm lesson -Gal

12 The Solution – Use a Safe Cell Line for mAb Production A variant of cetuximab, CHO-C225, which is produced in Chinese hamster ovary (CHO) cell lines that do not produce -1,3- galactosyltransferase and, for this reason, has a pattern of glycosylation that differs from that of cetuximab was found to be safe to administer to patients with IgE antibodies against -Gal. The solution – use a safe cell line for mAb production: Patnaik & Stanley (Methods in Enzymology, 2006): For example, Dr. Pamela Stanleys lab has developed a library of CHO mutants allowing desired glycoforms to be dialed in (or out... ):

13 A Simpler Solution? – Just Eliminate the Sugar(s)? First – How? Second – Would it work?

14 Optimizing antibody–FcR interactions. An important strategy to obtain a stronger anti-tumor ADCC reaction is to optimize the interaction of the antibody Fc-portion with activating FcRs. This can be achieved by blocking the inhibitory FcγRIIB in vivo with monoclonal antibodies, or by modifying the primary amino acid sequence (amino acid [AA] modifications) or the sugar moiety of the antibody to obtain selective or enhanced binding to activating FcRs. Nimmerjahn, F., and Ravetch, J. V. (2007) Antibodies, Fc receptors and cancer. Curr Opin Immunol 19, Antibody-dependent cellular cytotoxicity (ADCC) is an emerging cancer treatment. During ADCC, antibodies bound to tumor cells recruit innate immune effector cells that express cellular receptors (Fc receptors [FcRs]) specific for the constant region of the antibody, thereby triggering phagocytosis and the release of inflammatory mediators and cytotoxic substances Just Eliminate the Sugar(s)? – No, they are Critical for Activity

15 Bad for ADCC Required for IVIg Intravenous immunoglobulin (IVIg) therapy is used to treat a wide range of autoimmune conditions and consists of pooled immunoglobulin G (IgG) from healthy donors. The immunosuppressive effects of IVIg are, in part, attributed to terminal α2,6-linked sialic acid residues on the N-linked glycans of the IgG Fc (fragment crystallizable) domain. Sugars Determine Antibody Activity More about the sugars in a few minutes, but lets first learn more about IVIg therapy

16 IVIg therapy is used to treat a wide range of conditions; FDA approved: IVIg (Intravenous Immunoglobin) Therapy: A Quick Overview Allogeneic bone marrow transplant Chronic lymphocytic leukemia Common variable immunodeficiency Idiopathic thrombocytopenic purpura (ITP) Pediatric HIV Primary immunodeficiencies Kawasaki disease Chronic inflammatory demyelinating polyneuropathy Kidney transplant* Kawasaki Disease Idiopathic thrombo- cytopenic purpura (ITP) /PAST_ISSUE/2011/DMB_2011_04/A4252 Autoimmune disease It is a safe (but expensive!) immunosuppressive therapy

17 IVIg Therapy: The Current Market and Projections The Market: $3.6 billion in 2012 Cost is ~ $15,000 per patient 2 g / kg) The market is projected to (at least) double by The Future; over 30-off label uses and clinical trials including: Unexplained recurring miscarriage Autism Alzheimers disease Chronic fatigue syndrome AutismAlzheimers disease

18 A solution: Recombinant Ig (?) The upside: controlled production The downside: Only 1 out of ~10 antibodies is properly glycosylated IVIg Therapy: Challenges and a (Partial) Solution The Problem / Challenge: IVIg is obtained from blood donors A single batch requires pooling 1,000 to 15,000 samples Preparation is cumbersome and prone to contamination There simply is not enough supply to meet projected demand ~10% sialylation (many copies are not active) Sialic acid

19 From IVIg Therapy to Big Picture Implications Optimal and consistent protein glycosylation in mammalian cell culture (Glycobiology, 2009) Obtaining a consistent glycoform profile in (recombinant glycoprotein) production is desired due to regulatory concerns Glycosylation optimization will improve therapeutic efficacy Clearly, any improvements toward the control of this important biochemical pathway will have far- reaching influences on industry IVIg exemplifies the need for glycosylation optimization in biologics

20 Back to IVIg (and rProteins in General*) Sub-optimal glycosylationThe solution Poor glycosylation compromises safety, pharmacokinetics, and activity But getting there is complex!! Post-synthetic modification Cell / genetic engineering Cell culture variables Current solutions Optimal and consistent protein glycosylation in mammalian cell culture (Glycobiology, 2009)

21 Cell / genetic engineering Cell (Genetic Engineering) Modulation of Glycan Production Goal: increase sialylation

22 CMP-Neu5Ac ST6GALNAC6 ST6GALNAC5 ST6GALNAC4 ST6GALNAC3 ST8SIA1 ST8SIA5 ST3GAL1 ST3GAL2 ST3GAL4 ST3GAL5 ST3GAL6 ST6GALNAC3 ST6GALNAC2 ST6GALNAC1 ST6GALNAC4 ST6GAL2 ST8SIA3 ST8SIA4 ST8SIA2 ST3GAL1 ST3GAL2 ST3GAL3 ST3GAL4 ST3GAL6 CMP CMPNT a2,8- a2,3- a2,6- a2,3- a2,8- NEU1 NEU2 NEU3 NEU4 Sialoglycoconjugate production Glycan recycling ST6GAL1 ST8SIA6 Golgi KL OK, That Sounds Easy Enough, Lets GE in a ST! Goal: increase sialylation But, which one?

23 One reason for uncertainties is the complex, non-template-based biosynthetic routes for glycans Question: How to determine the specific gene(s) responsible? Solution: Use an engineering (computational modeling) approach! Genetically Engineering Glycosylation is NOT Easy Keeping in mind that glycosylation is actually 10x-fold more complex....

24 Cell / genetic engineering OK, Lets Try Something Else – Cell Culture Variables Cell culture variables e.g., NH 3 or CMP-sialic acid

25 CMP-Neu5Ac ST6GALNAC6 ST6GALNAC5 ST6GALNAC4 ST6GALNAC3 ST8SIA1 ST8SIA5 ST3GAL1 ST3GAL2 ST3GAL4 ST3GAL5 ST3GAL6 ST6GALNAC3 ST6GALNAC2 ST6GALNAC1 ST6GALNAC4 ST6GAL2 ST8SIA3 ST8SIA4 ST8SIA2 ST3GAL1 ST3GAL2 ST3GAL3 ST3GAL4 ST3GAL6 CMP CMPNT a2,8- a2,3- a2,6- a2,3- a2,8- NEU1 NEU2 NEU3 NEU4 Sialoglycoconjugate production Glycan recycling ST6GAL1 ST8SIA6 Golgi KL Going Back to the Sialyltransferase (ST) Schematic Goal: increase sialylation CMP-Neu5Ac generally has been presumed NOT to regulate ST activity But that paradigm is being disproved...

26 rProteinGlycoengineered rProtein Glycoengineering holds promise to improve safety/efficacy of rProteins SimpleLow costVersatileEffectiveApproach Post-synthetic modification X X ? Cell culture variables ? ?? ? ? Cell/genetic engineering X X ? Goal: increase sialylation Checking Back in on Glycoengineering Options e.g., media supplementation to increase CMP-sialic acid levels

27 ManNAc is the Feedstock for Sialic Acid Production X Low sialic acid = Poor activity Increased SA = improved activity ManNAc Natural ManNAc N/A 5-10% increase in sialylation The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins Goal: increase sialylation

28 ManNAc is the Feedstock for Sialic Acid Production X Natural ManNAc N/A 5-10% increase in sialylation The application of N-acetylmannosamine to the mammalian cell culture production of recombinant human glycoproteins Ballpark estimates for a 15,000 L/30,000 g bioreactor run $ million ManNAc IVIg therapy costs ~ $15,000 per patient 2 g / kg) Therefore, the value of IVIg is ~ $100 / g And 30,000 g would be worth ~$3,000,000 Most mAb therapies require a dose of 2-20 mg / kg) Therefore, a bioreactor run would be worth $300- 3,000 millIion (i.e., up to $3 billion!)

29 Towards a Solution: 2 nd Generation ManNAc Analogs Ac 4 ManNAc Natural ManNAc X Low sialic acid = Poor activityIncreased SA – improved activity N/A Ac 4 ManNAc N/A X 10-25% increase in SA Refer to notes for references for Ac 4 ManNAc efficiency and cytotoxicity Goal: increase sialylation

30 Towards a Solution – Separating Flux & Toxicity Natural ManNAc X N/A Ac 4 ManNAc N/A X Ac 4 ManNAc Bu 4 ManNAc Complex activities (higher flux, enhanced toxicity) Refs 1-3 (in notes) 3,4,6-O-Bu 3 ManNAc Whole molecule activities (a platform for drug development) Refs 4-9 1,3,4-O-Bu 3 ManNAc The Solution (next slides) Refs 4, 10, 11

31 A Closer Look - Simplicity 1,3,4-O-Bu 3 ManNAc Substitution of n-butyrate for acetate increases transmembrane uptake into cells The amphipathic nature of the molecule maximizes uptake The 1,3,4 pattern of butanoylation minimizes toxicity

32 A Closer Look - Cost 1,3,4-O-Bu 3 ManNAc Ballpark estimates for a 15,000 L/30,000 g bioreactor run $24-120K Ac 4 ManNAc $ million ManNAc1,3,4-O-Bu 3 ManNAc $6-75K

33 A Closer Look - Versatility 1,3,4-O-Bu 3 ManNAc 1,3,4-O-Bu 3 Glc/GalNAc adds sialic acid adds Branches (Refs 1,2) 1,3,4-O-Bu 3 ManN(R) R = >25 functional groups adds chemical FGs (Refs 3,4)

34 Illustrating Versatility (and Effectiveness) with EPO Even better w/ non-natural sialic acid Erythropoietin (EPO) ($9 billion market) Serum ½ life: 14 SAs = 8.5 hours ~22 SAs = 25.3 hours Optimally sialylated EPO has longer serum half-life 1,3,4-O-Bu 3 Glc/GalNAc 1,3,4-O-Bu 3 ManNAc Increased sialic acid Increased branching 1,3,4-O-Bu 3 ManN(R) R = >25 functional groups (Refs 1-3)

35 A Closer Look - Effectiveness 1,3,4-O-Bu 3 ManNAc ~75% (global) increase in sialylation > 80 proteins from a glycoproteomics analysis of SW1990 cells Relative S.A. in treated cells (fold increase cf. controls) i.e., ~175% is the average Individual glyco- proteins experience a considerably larger increase in S.A. Metabolic flux increases glycoprotein sialylation...(2012)

36 Effectiveness – The Implications 1,3,4-O-Bu 3 ManNAc ~75% (global) increase in S.A. 1,3,4-O-Bu 3 ManNAc is never harmful wrt sialylation For example, Immunoglobin G, which is ~10% sialyated 1,3,4-O-Bu 3 ManNAc is most effective for proteins with very low starting levels of sialic acid Goal: increase sialylation

37 To Summarize Recombinant Protein Glycoengineering Sub-optimal glycosylationThe solution Poor glycosylation compromises safety, pharmacokinetics, and activity But getting there is complex!! Post-synthetic modification Cell / genetic engineering Cell culture variables Current solutions Optimal and consistent protein glycosylation in mammalian cell culture (Glycobiology, 2009)

38 An Overview of Todays Lecture First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Organ TransplantationMetabolic Oligosaccharide Engineering Next

39 Question: where to get replacements for diseased and worn out hearts? About 600,000 people die of heart disease in the United States every year–thats 1 in every 4 deaths Cardiovascular Disease – USAs #1 Killer

40 One Option – Tissue Engineering Tissue engineering: the creation of new tissues or organs in the laboratory to replace diseased, worn out, or injured body parts

41 A Second Option – Xenotransplantion Baby Fae – recipient of a baboon heart (ca. 1984) Ultimately unsuccessful, spawned a backlash based (in part) on ethical concerns

42 Xenotransplantation (i.e., transplants from other species) is being pursued because of a dire shortage of human donors (and ethical concerns with using primates) The creatures outside looked from pig to man, and from man to pig, and from pig to man again; but already it was impossible to say which was which. George Orwell, Animal Farm Todays scientists are breeding pigs and harvesting their organs for xenotransplants. Pigs are excellent source animals because they are easily bred and typically have large litters of piglets that grow very rapidly, forage for themselves, and reproduce rather quickly. More importantly, pig organs are physiologically and anatomically similar to human organs. A dissected pig whose organs will be used for a xenotransplant. Pigs seem like a good choice to be organ donors – were already eating them, and theyre quite similar to us! Xenotransplants – Some Background Info

43 (From Nature Biotechnology, March 2002 Volume 20 Number 3 pp ) 1 What is the cause of hyperacute rejection? Xenotransplants – Overcoming Hyperacute Rejection

44 Hyperacute rejection (HAR) is caused by binding of large amounts of antibody, consisting predominantly of anti- - 1,3-Gal, to graft blood vessels, activating large amounts of complement. The role of -1,3-Gal in hyperacute and acute vascular rejection Hyperacute Rejection Results from -Gal

45 Humans and (other) primates do not make -Gal and for that reason avoid HAR ( but for ethical reasons, are not considered to be appropriate sources for large scale organ harvesting and transplantation (by contrast 35,000,000 pigs are already being slaughtered each year in the USA ) Remember that -Gal is a Trisaccharide

46 soluble Gal This seemed like a plausible approach 15 years ago... Yarema & Bertozzi, Current Opinion in Chemical Biology, 1998, 2:49–61 Strategy #1. Can soluble Gal protect against hyperacute rejection? But it has not worked out, for several reasons Strategies to Overcome Hyperacute Rejection

47 Gal X 1,3-galactosyltransferase ( 1,3GT) Three key technologies were required that were falling into place in the 1990s 1.Identification of the 1,3-galactosyltransferase gene (genetics/bioinformatics) 2.homologous recombination of the target genes (molecular/cell biology) 3.adaptation of nuclear transfer technology to pigs (large animal genetics) Strategy #2 – Knockout the 1,3GT Gene

48 Three key technologies were required that were falling into place in the 1990s 1.Identification of the 1,3-galactosyltransferase gene (genetics/bioinformatics) 2.homologous recombination of the target genes (molecular/cell biology) 3.adaptation of nuclear transfer technology to pigs (large animal genetics) Immunogenetics.Immunogenetics. 1995;41(2-3): cDNA sequence and chromosome localization of pig alpha 1,3 galactosyltransferase. Strahan KMStrahan KM, Gu F, Preece AF, Gustavsson I, Andersson L, Gustafsson K.Gu FPreece AFGustavsson IAndersson LGustafsson K Source Division of Cell and Molecular Biology, Institute of Child Health, London, UK. Abstract Human serum contains natural antibodies (NAb), which can bind to endothelial cell surface antigens of other mammals. This is believed to be the major initiating event in the process of hyperacute rejection of pig to primate xenografts. Recent work has implicated galactosyl alpha 1,3 galactosyl beta 1,4 N-acetyl-glucosaminyl carbohydrate epitopes, on the surface of pig endothelial cells, as a major target of human natural antibodies. This epitope is made by a specific galactosyltransferase (alpha 1,3 GT) present in pigs but not in higher primates. We have now cloned and sequenced a full-length pig alpha 1,3 GT cDNA. The predicted 371 amino acid protein sequence shares 85% and 76% identity with previously characterized cattle and mouse alpha 1,3 GT protein sequences, respectively. By using fluorescence and isotopic in situ hybridization, the GGTA1 gene was mapped to the region q2.10-q2.11 of pig chromosome 1, providing further evidence of homology between the subterminal region of pig chromosome 1q and human chromosome 9q, which harbors the locus encoding the AB0 blood group system as well as a human pseudogene homologous to the pig GGTA1 gene The Gal gene was cloned in Strategy #2. Knocking out the -Gal gene Step 1. The 1,3GT Gene was IDd 20 Years Ago

49 Strategy #2. Knocking out the a-Gal epitopeThree key technologies were required that were falling into place in the 1990s 1.Identification of the 1,3-galactosyltransferase gene (genetics/bioinformatics) 2.homologous recombination of the target genes (molecular/cell biology) 3.adaptation of nuclear transfer technology to pigs (large animal genetics) Molecular biology techniques were maturing... (from Nature Biotechnology, March 2002 Volume 20 Number 3 pp ) The Gal gene was knocked out in germ line cells Step 2. A Lot of Really Complex Genetic Manipulation!

50 Strategy #2. Knocking out the -Gal epitope Three key technologies were required that were falling into place in the 1990s 1.Identification of the 1,3-galactosyltransferase gene (genetics/bioinformatics) 2.homologous recombination of the target genes (molecular/cell biology) 3.adaptation of nuclear transfer technology to pigs (large animal genetics) The cloning of large animals was pioneered by Dolly the Sheep Dolly (5 July 1996 – 14 February 2003) Step 3. Moving from Rodents to Large Animals....

51 Figure 3: Five 1,3GT gene knockout piglets at 2 weeks of age. 2 But, only one allele was knocked out!! 1,3GT expression was still possible from the copy of the gene on the non- knocked out allele Solution: Breeding experiments, expected progeny: +/+, +/, and / at a 1:2:1 ratio The First -Gal Knockout Pigs were Born Xmas Day, 2002

52 Phelps et al, Science (2003) Production of -/- 1,3-galactosyltransferase-deficient pigs Our results have demonstrated that removal of 1,3Gal epitopes on pig cells did not preclude development in utero the baby pigs appeared to be OK! 1,3-Galactosyltransferase knockout pigs are available for xenotrans- plantation: But, are glycosyltransferases still relevant? Uh oh – the double null animals still expressed Gal !! (albeit at a lower level, and only on glycolipids) Rearing and Caring for a Future Xenograft Donor Pig Reduced sperm adhesion to zona pellucida Increased sensibility to sepsis Increased sensibility to autoimmune diseases Cataract formation The Gal knockout pigs needed special care due to concerns about Wrapping up the Loose Ends (and new pitfalls)

53 Strategy #2. Knocking out the -Gal epitope (ca ) Why/How did the -/- GT Knock Out Pigs still Express Gal? The pig genome was not sequenced until 2010 Maybe there were other genes in the pig genome with GT activity Wrapping up the Loose Ends (and new pitfalls)

54 Strategy #2. Knocking out the -Gal epitope Hyperacute rejection is the first hurdle that has to be overcome; a reaction to the 'foreign' organ by the body's normal immune system. Humans and primates differ from other animals in that they lack an enzyme ( 1,3 galactosyltransferase) that places a particular sugar group (Gal) on the branched sugar chains which occur on cell surfaces. Our bodies recognize its presence on grafted pig organs as a signal to attack. Revivicor's has inactivated the gene in pigs which makes the enzyme that attaches this Gal sugar group, producing the worlds first 1,3 galactosyltransferase (Gal) knock-out pigs. Organs from Gal knock-out pigs transplanted into non-human primates did not undergo HAR; thus the initial attack of HAR was overcome by the use of these GE pigs. From Revivicors website: In Any Event, The Low(er) Residual Levels of -Gal were Not a Huge Problem Hyperacute Rejection *has* Been Solved!

55 But theres Still (Much!) More Work to Do While the presence of the foreign Gal sugar is by far the major signal for initiating an attack by the immune system, there are other mediators of immune rejection at play. Revivicor has also added a human gene to the pigs to produce a protein called CD46 that moderates the action of the immune system. This gene addition strategy, combined with Gal knock-out and immune suppression drugs, demonstrated encouraging results of pig hearts in primates, with survival and function for up to 8 months. Overcoming hyperacute rejection is only the first, but essential, step in Revivicor's comprehensive approach.... If interested, you can consult the companys website: Hyperacute Rejection *has* Been Solved!

56 Back to the Overview of Todays Lecture First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Organ TransplantationMetabolic Oligosaccharide Engineering Finally

57 In the past (up to the present day, really) a widespread / working assumption has been that glycan structures are controlled at the level of glycosyltransferases. By contrast, the nucleotide sugar building blocks (e.g., CMP-Neu5Ac) have been assumed to be at saturating levels We recently demonstrated that metabolic flux *is* critical: Almaraz, R. T., Tian, Y., Bhattarcharya, R., Tan, E., Chen, S.-H., Dallas, M. R., Chen, L., Zhang, Z., Zhang, H., Konstantopoulos, K., and Yarema, K. J. (2012) Metabolic flux increases glycoprotein sialylation: implications for cell adhesion and cancer metastasis. Mol Cell Proteomics, /mcp.M Back to the Overview of Todays Lecture

58 Glycosylation pathways Exogenous (e.g., dietary) sugars Naturally-occurring cell surface oligosaccharides 1 R 1 = Werner Reutters Laboratory ManNAc R1R1 Neu5Ac R1R1 Kayser et al, Journal of Biological Chemistry, 1992 Moving from the Rate to the Type of Flux This approach now is generally known as: metabolic oligosaccharide engineering or metabolic glycoengineering

59 Is Metabolic Glycoengineering Useful? Glycosylation pathways Exogenous (e.g., dietary) sugars 1 R 1 = Werner Reutters Laboratory ManNAc R1R1 Neu5Ac R1R1 Keppler et al, Glycobiology 2001

60 Soon Chemical Biologists Dominated the Field R 1 = Carolyn Bertozzis Group The ketone group Mahal et al, Science, 1997 Glycosylation pathways Exogenous (e.g., dietary) sugars Naturally-occurring cell surface oligosaccharides 1 R 1 = Werner Reutters Laboratory ManNAc R1R1 Neu5Ac R1R1 The azide and alkyne, the reaction partners for click chemistry

61 Ac 4 ManNAz Sialic acid pathway Cu(I) Copper catalyzed [3+2] cycloaddition reaction (aka click chemistry) Sia5Az Saxon & Bertozzi, Science, 2000 *That was in ,300,000 in ,200,000 in ,000,000 in 2013 Click Chemistry – 1,530,000 Google Entries! * Applied to metabolic glycoengineering

62 Ac 4 ManNAz Sialic acid pathway Cu(I) Copper catalyzed [3+2] cycloaddition reaction (aka click chemistry) Sia5Az Saxon & Bertozzi, Science, 2000 *It works best when the cells/animals can be sacrificed (i.e., when they are dead) (the copper is somewhat toxic, this problem is solved on the next slide) This Technology can be used as a Glycoproteomics Tools

63 Ac 4 ManNAz Sialic acid pathway Cu(I) Copper catalyzed [3+2] cycloaddition reaction (aka click chemistry) Sia5Az Saxon & Bertozzi, Science, 2000 Strain-promoted [3+2] cycloaddition** Sia5Az Agard et al, JACS, 2004 Cell-surface glycans shine in this microscopy image of the head of a three-day-old zebrafish embryo treated with the new technique. *The copper catalyst is toxic * **Copper-free click reactions can now be done in living cells and in vivo. ** New Bio-orthogonal Chemistries can be used In Vivo

64 Cell-surface glycans shine in this microscopy image of the head of a three-day-old zebrafish embryo treated with the new technique. In addition to cell surface sialic acid, metabolic glycoengineering now can target cell surface GalNAc and fucose (GlcNAc analogs mainly label intracellular O-GlcNAc ) Additional Pathways (beyond Sialic Acid) can be Targeted

65 For Example, Remember Fucose ? 1,3,4-O-Bu 3 ManN(R) R = >25 functional groups adds chemical FGs (Refs 3,4) This actually *should* have been fucose! (from earlier in todays lecture) Additional twists Works on secreted proteins New bioorthogonal chemistry

66 Du et al, Glycobiology, 2009 (A) The ketone is the first example of an bio-orthogonal chemical functional group installed in the glycocalyx (B) and (C) Either click functional group (azides, B or alkynes, C) can be installed in the glycocalyx (D) and (E) Photoactivated functional groups can be installed in the glycocalyx (F) Thiols can be incorporated into an unusual cellular locale, the glycocalyx* *contact me for information on our labs efforts to use sialic acid-displayed thiols for tissue engineering Expanding the Repertoire of Bioorthogonal Chemistries

67 Almaraz et al, Ann Biomed Eng, 2012 OK – Finally – about those 25 R Groups.....

68 Where does Metabolic Oligosaccharide Engineering Go Next? From Chemical Biology To The Clinic ??

69 Commercialization and translational efforts were slow to be realized: In the Bigger Picture, Progress Continues.... The Bittersweet Promise of Glycobiology Nature Biotechnology, 2001 (doi: /nbt ) The Sweet and Sour of Cancer: Glycans as Novel Therapeutic Targets Nature Reviews Cancer, 2005 (doi: /nrc1649) By 2008 we had learned a valuable first do no harm lesson In the past decade, progress has accelerated: 2003: 2006: 1,3,4-O-Bu 3 ManNAc 2008: Our Technology An novel scaffold for drug design (over 100,000 permutations)

70 Back to the Overview of Todays Lecture – All Done! First – What is Carbohydrate Engineering? Sugars are critical for $250 billion $$s worth of drugs Organ TransplantationMetabolic Oligosaccharide Engineering

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