1 / 28

Essentials of Glycobiology Lecture 34 May 28, 2002 Jeff Esko

Essentials of Glycobiology Lecture 34 May 28, 2002 Jeff Esko. Glycosylation Inhibitors. Overview. Indirect inhibitors and metabolic poisons Tunicamycin - Inhibition of dolichol-PP-GlcNAc assembly Plant Alkaloids - Natural inhibitors of glycosidases

yanni
Download Presentation

Essentials of Glycobiology Lecture 34 May 28, 2002 Jeff Esko

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Essentials of Glycobiology Lecture 34May 28, 2002Jeff Esko Glycosylation Inhibitors

  2. Overview • Indirect inhibitors and metabolic poisons • Tunicamycin - Inhibition of dolichol-PP-GlcNAc assembly • Plant Alkaloids - Natural inhibitors of glycosidases • Substrate analogs - Directed synthesis of inhibitors • Glycoside primers - Mimicking what already works • Inhibitors of glycolipids and GPI anchors • Neuraminidase inhibitors - Rational design from X-ray crystallography

  3. Introduction • Need to perturb structure to understand function • Inhibitors can lead to therapeutics

  4. Indirect Inhibitors and Metabolic Poisons In vitro inhibitors - nucleotide sugar analogs, acceptor analogs Sugar analogs - deoxyglucose, fluorinated sugars, mannosamine analogs Compounds that interfere with transport - Brefeldin A, ionophores, ammonium chloride Compounds that block intermediary metabolism - 6-diazo-5-oxo-L-norleucine (DON), chlorate

  5. O H N O H H O O N O R C H N H O O C H 2 O O H H N H A c H H O H O H O O H H O O H Tunicamycin • Blocks the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichyl-P (GPT) • Resistant mutants overproduce GPT • Km for UDP-GlcNAc is ~3 x 10-6 M, whereas the Ki value for tunicamycin is ~5 x 10-8M

  6. 2 a ± 3 3 a a 3 3 a a 2 2 2 2 2 2 2 2 2 a a a a a a a a a 2 2 2 3 6 3 6 3 6 3 6 3 6 a a a a a a a a a 2 a a a a b 3 6 3 6 3 6 3 6 3 6 3 6 a a a a a a a a a a a a 4 4 4 4 4 4 b b b b b b G l c N A c T I 4 4 4 4 - g l u c o s i d a s e I - g l u c o s i d a s e I I - m a n n o s i d a s e I - m a n n o s i d a s e I I 4 4 a a a a b b b b b b A s n A s n A s n A s n A s n A s n Plant Alkaloids - Natural Inhibitors of Glycosidases Australine Swainsonine Kifunensin Deoxynojirimycin Castanospermine Deoxymannojirimycin

  7. H O HO O H Plant Alkaloids CH2OH O H HO HO O H H O H N H N OH O H N H O N O H H C H O H O H 2 Australine Castanospermine Deoxymannojirimycin Swainsonine a-Glucosidase I a-Glucosidase I and II a-Mannosidase I a-Mannosidase II • Alkaloids contain polyhydroxylated ring systems that mimic the orientation of hydroxyl groups in the natural substrates • Protonation of the ring nitrogen may mimic the positive charge developed on the ring oxygen during the hydrolytic reaction

  8. Substrate Analogs - Directed Synthesis of Inhibitors • Modify the acceptor or nearby hydroxyl group in a substrate • Exhibit Ki values in the range of the Km for the parent substrate Hindgaul and coworkers

  9. Glycosidase Inhibitors • Many inhibitors of lysoomal and bacterial hydrolases are available • Generally they mimic a proposed transition state since C1 takes on a trigonal orientation PUGNAC Galactonolactone

  10. Synthetic Substrate Analogs

  11. Prepare compounds that resemble biosynthetic intermediates Conjugate to a hydrophobic aglycone to enhance uptake and activity Alkylation or acylation also works H H O H O O H O H O H H H H H O O H O O O H O O O H H Glycoside Primers - Substrate Mimicry Sarkar et al (1995) Proc. Natl. Acad. Sci. USA 92: 3323

  12. Proteoglycan 6 S 6 S O O 2 S N S N S 6 S 6 S Hydrophobic O Xylose N S N S 2 S Aglycone 6S 6S O NS NS 2S 6S 6S O Xyloside NS NS 2S Xyloside Primers Block Proteoglycan Glycosylation Cell Fritz & Esko (2001) Methods Mol Biol. 171:309

  13. OAc OAc OAc O O O O AcO AcO NHAc OAc Gal GlcNAc Naphthalene methanol Types of Primers

  14. Properties of Glycoside Primers • Priming usually exceeds normal levels of synthesis on endogenous conjugate • The enhanced rate of synthesis of oligosaccharides on primers may alter the assembly of other classes of glycoconjugates than those generated on the primer • Inhibition is rarely complete

  15. Properties of Glycoside Primers • Primers can reveal alternate glycosylation pathways • Primers represent starting points for making membrane permeable analogs that might have inhibitory activity without acting as a primer • Glycosides can be absorbed through the gut • Many glycosides occur naturally, especially in plants

  16. Glucosides Alkaloids Arabinosides Flavonoids Xylosides Carotenoids Ribosides Steroids Rhamnosides and others and others Natural Inhibitors of Glycosylation

  17. Psittacanthus Alchornea Maieta cucullaris triplinervia guianensis 1 2 3 4 1 2 3 4 1 2 3 4 1 mg/ml 0.33 0.11 .037 .012 .004 0 Natural Glycoside Primers Plant extracts were prepared by graded solvent extraction (1-4) and assayed for GAG priming activity Taylor et al. (1998) J. Biol. Chem. 273:22260

  18. O O 5 " O 4 " 5 " O 4 " H O 1 " 2 " O H H O H O 1 " 2 " O H O 4 3 " 4 M e O H 3 " M e O H C = O 5 7 " 3 5 3 2 6 1 " ' O 2 6 O 2 " ' O 6 " ' 1 O 7 7 1 3 " ' 7 ' 1 ' 5 " ' O 7 ' 1 ' O O O H H O 4 " ' O 2 ' 6 ' 2 ' 6 ' O H 5 ' 3 ' 5 ' 3 ' O M e H O M e 4 ' H 4 ' O H O H Natural GAG primers EAGX) 2 3,3'-Di-O-methyl ellagic acid 4-O-(3"-galloyl)-b-D-xylopyranoside 3,3'-Di-O-methyl ellagic acid- 4-O-b-D-xylopyranoside

  19. Mannosamine derivatives (ManNR) can be used by this pathway Glycolysis Gal Glc Fuc Man GlcNAc ATP ATP ATP Pi Gal-1-P Man-6-P Glc-6-P Fru-6-P Glycogen Fuc-1-P -NH UDP-Glc Glutamine UTP 3 GlcN-6-P Man-l-P Glc-1-P UDP-Gal UDP-Glc NAD AcCoA GTP GTP Dol-P + NAD ATP GDP-Man GlcNAc-6-P Dol-P-Glc UDP-GlcA NADP -CO Dol-P 2 GlcNAc-l-P GDP-Fuc UTP UDP-Xyl Dol-P-Man UTP ATP UDP- GlcNAc GalNAc UDP-GalNAc GalNAc-1-P ManNAc ATP CMP-Neu5Gc ManNAc-6-P PEP NADP CTP CMP-Neu5Ac Neu5Ac-9-P Neu5Ac

  20. Mannosamine Analogs Mahal et al. (1997) Science 276:1125

  21. Metabolism of ManAzide results in display of cell surface azides. Reaction with exogenously delivered phosphine via the Staudinger ligation creates an amide-linked cell surface conjugate. Saxon & Bertozzi (2001) Annu. Rev. Cell Dev. Biol. 17:1

  22. -O-Cer -O-Cer b b a3 b4 b -O-R O Inhibitors of Glycolipid Assembly Xylosides have a mild effect on glycolipid formation, due to the assembly of a GM3 like compound Glucosylceramide acts like a primer; an analog containing an exocyclic epoxide inhibits glycolipid formation (IC50 ~8 µM)

  23. N-butyldeoxynojirimycin inhibits Glc-Cer synthesis The best inhibitors are analogs of the long chain base (sphingosine analogs) O N O CH2OH HO H O N H DL-threo-PDMP N HO HO Inhibitors of Glycolipid Assembly

  24. O O H O Inhibitors of GPI anchors • Mannosamine inhibits GPI anchor formation: ManNH2-Man-GlcN-PI is a poor substrate for the 2mannosyltransferase • Trypanosomes selectively take up and exchange fatty acid analogs (10-(propoxy)decanoic acid) for acyl chains on glycosylphosphatidylinositol

  25. Neuraminidase Inhibitors • A neuraminidase inhibitor was deduced by assuming a carbocation arises at C1 in the transition state, which would cause C1 to adopt a trigonal (sp2) planar configuration • Compounds mimicking this geometry had inhibitory activity (µM Ki value)

  26. Rational design from X-ray crystallography

  27. O H O H H O C O O H H N O C H C 3 C H 2 N H H N 2 N H Potent Sialic Acid Analogs • A sialic acid analog containing a positively charged guanidinium group instead of O4 had a Ki of 10-11 M due to an additional salt bridge • Non-carbohydrate analogs based on of benzoic acid mimic the partially planar ring of the proposed intermediate O C 4-guanido-Neu5Ac-2-ene

  28. Therapeutics • Relenza and Tamiflu are drugs based on these sialic acid analogs • N-butylated alkaloids are under development as anti-viral and anti-tumor agents • Other pharmaceutical applications of glycosylation and glycosidase inhibitors will be discussed later

More Related