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Glycans in the Biotechnology and Pharmaceutical Industries
Lecture 42
Carolyn R. BertozziUC Berkeley
Lecture Outline
1. Examples of approved carbohydrate-based drugs2. Development of drugs to treat influenza3. Development of selectin inhibitors for inflammation4. Imino-sugars as drug candidates for storage diseases
Examples of approved carbohydrate drugs:
Substance Indication CompanyAcarbose Diabetes Bayer AGAMVISC Opthalamic surgery Anika TherapeuticsHyalgan Osteoarthritis FIDIA/SanofiLovenox Cardiovascular disease AventisMiglitol Diabetes Bayer AGORTHOVISC Osteoarthritis Anika TherapeuticsRelenza Influenza Glaxo SmithklineTamiflu Influenza RocheSOLARASE Actinic keratosis Hyal PharmaceuticalsTopamax Epilepsy J & JVoglibose Diabetes Takedo/Abbott
CO2–
O
O
O
O
O
OSO3–
HOHO
–O3SHN O–O2C
HOOH
O–O3SO
–O3SHN
OSO3–
OHO O
HO
OSO3–
–O3SHNOCH3
O
OHNH3C
HOOH
O O
HOOH
O
OH
O
HOOH
OH
OH
HOHO
OH
OH
CO2–
HNNH2
+
H2N
OH
AcHN
OHHO
Heparin pentasaccharide – anti-coagulant
NHHO
O
NHOH
OH
H2NNH2
+
H2N
NH2+
O
OHO
OHC
H3C
O
NHCH3
CH3
OHOH
Acarbose – diabetes
Streptomycin – antibiotic
Relenza – anti-flu drug
Examples of carbohydrate-based drugs
O
OCH3O
OH
OH O
OH
O OH
OH3C
OHNH3
+
O
H3C
OHHO
CH3
OHCH3
CH3H3CO
OO
O
OCH3
OH
OHCH3
OHO
N(CH3)2
CH3
Doxorubicin – anti-cancer drug Erythromycin A – antibiotic
CH3
OH
H3C
O
HO
OH3C
HOO
OO
Digoxin – cardiovascular
OH3C
HOO
OH3C
HOHO
Examples of glycosylated natural products
Examples of approved glycoprotein drugs:
Substance CompanyErythropoietin Amgen, J&JTissue plasminogen activator GenentechInterleukin-2 ChironCerezyme GenzymeMonoclonal antibodies Many
From: Hudson, P. J.; Souriau, C. Nature Medicine 2003, 9, 129-134
Lecture Outline
1. Examples of approved carbohydrate-based drugs2. Development of drugs to treat influenza3. Development of selectin inhibitors for inflammation4. Imino-sugars as drug candidates for storage diseases
Cell surface oligosaccharides aredeterminants of cell recognition
bacterium
cellvirustoxin
hormone
glycoprotein
Microbial pathogens bind to cell surface sugarsas a first step during infection
Influenza virus - “Flu”HIV - AIDS
Helicobacter pylori - UlcersEscherichia coli - MeningitisPseudomonas aeruginosa - Pneumonia
Trypanosomes - African sleeping sicknessPlasmodium falciparum - Malaria
The influenza virus has two membrane-associatedproteins, hemagglutinin and neuraminidase
Hemagglutininbinds sialic acid(receptor)
Neuraminidase cleaves sialic acid(enzyme)
O
CO2–
HO OHHO
AcHNHO
O
Host cellSialic acid
(SA)
SASA
Hemagglutininbinds sialic acidto initiate infection
Host cellEndocytosis
Membranefusion andrelease ofviral particle Replication
SA
SA
New virusesassemble atmembrane
SA
SA
Neuraminidasecleaves sialic acidand liberates newvirus
Life cycle of the influenza virus
Sialic acid analogs block influenza virus infection
Cell
Sialic acid
influenzavirus
“C-Glycoside” ofsialic acid
O
CO2–
HOOH
OH
AcHNHO
O
O
CO2–
HOOH
OH
AcHNHO
CH2CHCH2
O
CO2–
HOOH
OH
AcHNHO
CH2CHCH2
O
CO2–
HOOH
OH
AcHNHO
CH2CHCH2
O
CO2–
HOOH
OH
AcHNHO
CH2CHCH2
Ea (cat)
Ea (uncat)
Enzymes catalyze reactions by preferential binding of the transition state vs the ground state
Transition state analogs are potent enzyme inhibitors
O
CO2–
HOOH
OH
AcHNHO
ORO CO2
–
HOOH
OH
AcHNHO OR
δ +
AH
O CO2–
HOOH
OH
AcHNHO
H2OO CO2
–HOOH
OH
AcHNHO
OH
Proposeδ mechanism of the neuraminiδase catalyzeδglycosiδe hyδrolysis reaction
O CO2–
HOOH
OH
AcHNHO OR
δ +
AH
Features:• Planarity at C-2• Builδup of + charge
C-2
Design of transition state analog neuraminidase inhibitors
O CO2–
HOOH
OH
AcHNHO
Planarity (sp2)2,3-Anhydro sialic acid
Ki = 10-6 MAlso inhibits human
neuraminidase
‡
CO2–
OAcHN
+H3N
Tamiflu(Hoffman La Roche)
Ki = 10-10 M
O CO2–
HOOH
OH
AcHNHN
H2NNH2
+
Relenza(Glaxo Smithkline)
Ki = 10-10 M
O CO2–
HOOH
OH
AcHNHO
Structure-based design of more potent and selective neuraminidase inhibitors
O–OBinding pocket ofFlu neuraminidase based on X-ray structure
Lecture Outline
1. Examples of approved carbohydrate-based drugs2. Development of drugs to treat influenza3. Development of selectin inhibitors for inflammation4. Imino-sugars as drug candidates for storage diseases
Blood vessel
Activated endothelium
Tissue
Leukocyte
A hallmark of inflammation is the recruitment of leukocytes from the bloodstream into surrounding tissues
The initial attachment of leukocytes to endothelial cellsat sites of inflammation is mediated by the selectins
Leukocyte
Endothelial cell
L-selectin
E-selectinP-selectin
Inflammatory diseases involving the selectins:
1. Rheumatoid arthritis2. Asthma3. Transplant rejection4. Psoriasis5. Inflammatory bowel disease6. Ischemia/reperfusion injury7. Diabetes8. Multiple sclerosis9. Many more…..
Inhibitors of selectin-mediated cell adhesion would bebroad spectrum anti-inflammatory agents
O
CO2–HO
HO
OH
AcHNHO
OOH OH
O OOH
OHO
OAcHN
OR
OH3C
HOOH
OH
SiaGal
Fuc
GlcNAc
Sialyl Lewis x binds all three selectins (Kd = 1–2 mM)
ORO
OHO
ONHAc
OOHOH
OOH
O
HO OHHO
AcHNHO
CO2–
O
OHOH
OHH3C
ORO
OHOHO
NHAcO
OHOH
OOH
O
HO OHHO
AcHNHO
CO2–
ORO
OHOHO
NHAcO
OHOH
HOOH
An example of an enzymatic oligosaccharide synthesisused for the production of a clinical candidate
N-Acetyllactosamine(from chemical synthesis)
CMP-Sialic acid
a2,3-Sialyltransferase
GDP-Fucosea1,3-Fucosyltransferase
Sialyl Lewis x: An anti-inflammatory agent δevelopeδ by Cytel
Limitations of sLex as a therapeutic agent
• Lack of potency
• Poor pharmacokinetics
• Difficult and expensive synthesis
Possible solutions
• Glycomimetics
• Oligomerization
• Glycoprotein constructs
OO
NHAc
OOR
OH
OOH OH
OOH
OH3C
HOOH
OH
O
CO2–
HO
AcHN OHHO
OH
NHNH2
+
H2N
O
NH
H
O
–
O
OH
Ca2+
Proposed model for sialyl lewis x bound to E-selectin and important functional groups for selectin binding
Glu 80
Tyr 94
Arg 97
Asn 105
OO
NHAc
OOR
OH
OOH OH
OOH
OH3C
HOOH
OH
O
CO2–
HO
AcHN OHHO
OH
OO
NHAc
OOR
OH
OOH OH
OOH
OH3C
HOOH
OH
–O2C
OOO
OH OH
OOH
OH3C
HOOH
OH
–O2C
OH3C
HOOH
OH
NH
ONH
HOHO
O–O2C
Similar E-selectin binding affinityas sialyl lewis x
Two-fold lower E-selectinbinding affinity than
sialyl Lewis xE-selectin binding affinitysimilar to sialyl Lewis x
Evolution of a sialyl Lewis x mimetic with comparable E-selectin binding affinity (Wong and coworkers)
Sialyl Lewis x
OH
O
HOOH
OH
NH
O
HOOH3C
HOOH
OH
N
ONH
HOHO
O–O2C
HO–O2C
ONH
Two-fold more potent than sialyl Lewis xfor E-selectin binding
O
HOOH
OHOH
O
CH3
H3C
O–O2C
Three-fold more potent than sialyl lewis xfor E-selectin binding
Fucose-based sialyl Lewis x mimetics
O
NH(CH2)13CH3
13-fold more potent than sialyl Lewis Xfor E-selectin binding
Mannose-based sialyl Lewis x mimetics
O
HOOH
OHOH
OOPOOH
OH
OO
O
104-fold more potent than sialyl Lewis xfor P-selectin binding
(in clinical development at Texas Biotech.)
Limitations of sLex as a therapeutic agent
• Lack of potency
• Poor pharmacokinetics
• Difficult and expensive synthesis
Possible solutions
• Glycomimetics
• Oligomerization
• Glycoprotein constructs
Multivalent inhibitors are much more potent for multivalent receptor-ligand interactions
Cell
Cell
Cell
Cell
+Multivalent inhibitor
Aqueous Interior
Lipid Bilayer
+
1. Self assembly2. UV-initiated polymerization
O
O–
OO
OH
ON
OH
OOH OH
OOH
OH3C
HOOH
OH
–O2CS
HN
O
OO
OH
ON
OH
OOH OH
OOH
OH3C
HOOH
OH
–O2CS N
H
O
–O
O
OO
OH
ON
OH
OOH OH
OOH
OH3C
HO OHOH
–O2C
S NH
O
–O
O
Ac
Ac
Ac
Functionalized polymerized liposomes are nanomolar selectin inhibitorsNagy and coworkers
Multivalent "glycopolymers" as potent selectin inhibitors
OOSO3
–OH
–O3SOOH
OHN
OO
OSO3–OH
–O3SOOH
OHN
O
n
500-fold more potent than sialyl lewis xin blocking P-selectin binding to HL-60 cells
Kiessling and coworkers
Limitations of sLex as a therapeutic agent
• Lack of potency
• Poor pharmacokinetics
• Difficult and expensive synthesis
Possible solutions
• Glycomimetics
• Oligomerization
• Glycoprotein constructs
L-selectinGlyCAM-1CD34MAdCAM-1
P-selectin P-selectin glycoprotein ligand-1(PSGL-1)
E-selectin E-selectin ligand-1 (ESL-1), perhaps
Approaches to selectin inhibitors inspired by the discovery of their biological ligands
L-selectin ligands
OSO3–
OSO3–
OSO3–
–O3SO
–O3SO
–O3SOOSO3
––O3SO OSO3
–
OSO3––O3SO
–O3SO
OSO3–
OSO3–OSO3
––O3SOOSO3
–
OSO3–
OSO3–
OSO3–
–O3SO
–O3SO
–O3SOOSO3
––O3SO OSO3
––O3SO
–O3SO
OSO3–
–O3SOOSO3
–
OSO3–
OSO3–
OSO3–
–O3SO
–O3SO
–O3SO–O3SO
OSO3–
OSO3–
–O3SO
OSO3–
–O3SO
OSO3–
OSO3–
–O3SO
–O3SO
OSO3–
SS
GlyCAM-1 CD34 MAdCAM-1 PSGL-1
Endothelial-derivedsulfomucins
Leukocyte-derivedtyrosine-sulfated mucin
P-selectin ligand
Structure of the biological selectin ligands
Determinants of PSGL-1 required for P-selectin binding
–O3SO
OSO3–
OSO3–
–O3SO
–O3SO
OSO3–
SS
PSGL-1
A potent P-selectin inhibitor based on PSGL-1:Recombinant PSGL-Ig (TS-1)
Features:• Good potency (nM Kd)• Good PK (serum 1/2-life = 2-3 wks)• Also inhibits L-selectin
–O3SO–O3SO
–O3SO OSO3–
OSO3–
OSO3–
Recombinant PSGL-IgGenetics Institute/Wyeth
Human IgG1 Fc(inactivated)
47 N-terminalresidues fromPSGL-1
Leukocyte
L-Selectin{
Endothelial cell
Leukocyte adhesion to endothelial cells is mediated by L-selectin binding to sulfated sialyl Lewis x
6-Sulfo sialyl Lewis x O
CO2–OH OH
AcHNHO
OHO OH
O OOH
OOSO3
–
ONHAc
OH3C
HOOH
OH
O
O
HO
AcHNO
HO OH
O OOH
O
HO
OCO2–OH OH
AcHNHO
OHO OH
OOH
OH3C
HOOH
OH
HO O OOSO3
–
OAcHN
O
CO2–OH
HO
OH
AcHNHO
O
HO OH
O OOH
OOSO3
–
ONHAc
OH3C
HOOH
OH
O
O
O
HO
AcHNSer/Thr
OOH OH
O O
OH
Alternative approaches to blocking selectin-mediated cell adhesion: Inhibition of key biosynthetic enzymes
Fucosyltransferaseinhibitors
Sulfotransferaseinhibitors
O
CO2–OH
HO
OH
AcHNHO
OOH OH
OOH
OOSO3
–
OO
NHAc
L-Selectin ligandOH3C
HOHO
OH
Lecture Outline
1. Examples of approved carbohydrate-based drugs2. Development of drugs to treat influenza3. Development of selectin inhibitors for inflammation4. Imino-sugars as drug candidates for storage diseases
Defects in glycolipid degradation lead to lysosomal storage disorders
From: Dwek, R. A., et al. Nature Rev. Drug Disc. 2001, 1, 65-75.
Imino-sugars can block biosynthesis of glycolipid precursors
Numerous companies have been foundedon glycobiology platforms
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