Transcript No Slide Title

Glycans in the Biotechnology and
Pharmaceutical Industries
Lecture 42
Carolyn R. Bertozzi
UC Berkeley
Lecture Outline
1. Examples of approved carbohydrate-based drugs
2. Development of drugs to treat influenza
3. Development of selectin inhibitors for inflammation
4. Imino-sugars as drug candidates for storage diseases
Examples of approved carbohydrate drugs:
Substance
Indication
Company
Acarbose
AMVISC
Hyalgan
Lovenox
Miglitol
ORTHOVISC
Relenza
Tamiflu
SOLARASE
Topamax
Voglibose
Diabetes
Opthalamic surgery
Osteoarthritis
Cardiovascular disease
Diabetes
Osteoarthritis
Influenza
Influenza
Actinic keratosis
Epilepsy
Diabetes
Bayer AG
Anika Therapeutics
FIDIA/Sanofi
Aventis
Bayer AG
Anika Therapeutics
Glaxo Smithkline
Roche
Hyal Pharmaceuticals
J&J
Takedo/Abbott
Examples of carbohydrate-based drugs
OSO3–
O
HO
HO
–O3SHN
–O2C
O
O
HO
–O3SO
O
–O3SHN
OH
O
O
HO
HO
O
–O3SHN
OCH3
O
CO2–
OSO3–
O
OSO3–
Heparin pentasaccharide – anti-coagulant
OH
HO
HO
H2 N
H3C
OH
HN
HO
NH2+
O
OH
OH
O
HO
H2N
O
NH
HO
NH
O
OH
O
HO
OH
OH
O
OHC
OH
Acarbose – diabetes
O
OH
OH
H3C
HO
O
NHCH3
O
OH
OH
HO
AcHN
HN
H2 N
Relenza – anti-flu drug
NH2
+
OH
OH
CH3
Streptomycin – antibiotic
CO2–
O
NH2+
Examples of glycosylated natural products
O
O
OH
O
OH
HO
H3C
OH
CH3O
O
CH3
OH
OH
CH3
H3C
O
OH O
O
H3C
O
OH
O
HO
O
NH3
+
O
Doxorubicin – anti-cancer drug
HO
HO
H3C
O
O
HO
H3C
O
O
HO
CH3
OH
CH3
Erythromycin A – antibiotic
HO CH
3
H3C
N(CH3)2
OH
CH3
CH3
O
H3C
O
OH
O
Digoxin – cardiovascular
O
O
Examples of approved glycoprotein drugs:
Substance
Company
Erythropoietin
Tissue plasminogen activator
Interleukin-2
Cerezyme
Monoclonal antibodies
Amgen, J&J
Genentech
Chiron
Genzyme
Many
From: Hudson, P. J.; Souriau, C. Nature Medicine 2003, 9, 129-134
Lecture Outline
1. Examples of approved carbohydrate-based drugs
2. Development of drugs to treat influenza
3. Development of selectin inhibitors for inflammation
4. Imino-sugars as drug candidates for storage diseases
Cell surface oligosaccharides are
determinants of cell recognition
bacterium
virus
toxin
hormone
glycoprotein
cell
Microbial pathogens bind to cell surface sugars
as a first step during infection
Influenza virus - “Flu”
HIV - AIDS
Helicobacter pylori - Ulcers
Escherichia coli - Meningitis
Pseudomonas aeruginosa - Pneumonia
Trypanosomes - African sleeping sickness
Plasmodium falciparum - Malaria
The influenza virus has two membrane-associated
proteins, hemagglutinin and neuraminidase
Neuraminidase
cleaves sialic acid
(enzyme)
Hemagglutinin
binds sialic acid
(receptor)
HO
HO
CO2–
OH
O
AcHN
O
HO
Sialic acid
(SA)
Host cell
Life cycle of the influenza virus
SA
Hemagglutinin
binds sialic acid SA
to initiate infection
Neuraminidase
cleaves sialic acid
and liberates new
virus
SA
Endocytosis
New viruses
assemble at
membrane
SA
Host cell
Membrane
fusion and
release of
viral particle
SA
Replication
SA
Sialic acid analogs block influenza virus infection
influenza
virus
Sialic acid
OH
HO
AcHN
OH
CO2–
OH
O
HO
AcHN
CH2CHCH2
CO2–
OH
HO
AcHN
O
O
O
HO
HO
OH
CO2–
OH
OH
CH2CHCH2
HO
CO2–
OH
HO
AcHN
O
CH2CHCH2
Cell
HO
“C-Glycoside” of
sialic acid
OH
CO2–
OH
HO
AcHN
O
HO
CH2CHCH2
Enzymes catalyze reactions by preferential binding of the
transition state vs the ground state
Ea (uncat)
Ea (cat)
Transition state analogs are potent enzyme inhibitors
Proposed mechanism of the neuraminidase catalyzed
glycoside hydrolysis reaction
OH
CO2
OH
HO
AcHN
O
OH
–
HO
AcHN
OR
OH +
O
HO
HO
CO2–
OR
AH
OH
OH
OH
HO
AcHN
OH
O
HO
CO2–
H 2O
OH
HO
AcHN
HO
O
CO2–
Design of transition state analog neuraminidase inhibitors
‡
OH
HO
AcHN
OH +
O
CO2–
C-2
HO
Features:
• Planarity at C-2
• Buildup of + charge
OR
AH
Planarity (sp2)
OH
2,3-Anhydro sialic acid
OH
-6
O
Ki = 10 M
HO
Also inhibits human
AcHN
neuraminidase
HO
CO2–
Structure-based design of more potent and selective
neuraminidase inhibitors
OH
HO
AcHN
OH
OH
HO
AcHN
CO2–
HO
–O
OH
O
Binding pocket of
Flu neuraminidase
based on X-ray
structure
O
CO2–
O
O
HN
AcHN
NH2+
CO2–
+
H3N
H2N
Relenza
(Glaxo Smithkline)
Ki = 10-10 M
Tamiflu
(Hoffman La Roche)
Ki = 10-10 M
Lecture Outline
1. Examples of approved carbohydrate-based drugs
2. Development of drugs to treat influenza
3. Development of selectin inhibitors for inflammation
4. Imino-sugars as drug candidates for storage diseases
A hallmark of inflammation is the recruitment of leukocytes
from the bloodstream into surrounding tissues
Tissue
Leukocyte
Blood vessel
Activated endothelium
The initial attachment of leukocytes to endothelial cells
at sites of inflammation is mediated by the selectins
Leukocyte
L-selectin
E-selectin
P-selectin
Endothelial cell
Inflammatory diseases involving the selectins:
1. Rheumatoid arthritis
2. Asthma
3. Transplant rejection
4. Psoriasis
5. Inflammatory bowel disease
6. Ischemia/reperfusion injury
7. Diabetes
8. Multiple sclerosis
9. Many more…..
Inhibitors of selectin-mediated cell adhesion would be
broad spectrum anti-inflammatory agents
Sialyl Lewis x binds all three selectins (Kd = 1–2 mM)
Gal
GlcNAc
Sia
HO
CO2–
OH
HO
AcHN
O
OH OH
HO
O
O
OH
HO
O
O
O
OR
AcHN
O
H3C
Fuc
OH
HO
OH
An example of an enzymatic oligosaccharide synthesis
used for the production of a clinical candidate
OH
OH
O
HO
OH
HO
NHAc
HO
O
CMP-Sialic acid
OR
O
OH
2,3-Sialyltr ans fe ras e
HO
AcHN
CO2–
OH
O
OH
OH
O
O
NHAc
HO
O
OH
HO
N-Ace tyllactos am ine
(fr om che m ical s ynthe s is )
GDP-Fucos e
1,3-Fucos yltrans fe ras e
OH
OH
HO
HO
AcHN
CO2–
OH
O
HO
H3C
OH
OH
O
O
O
NHAc
O
O
OH
OH
O
OH
OR
Sialyl Lewis x: An anti-inflammatory agent developed by Cytel
O
OH
OR
Limitations of sLex as a therapeutic agent
• Lack of potency
• Poor pharmacokinetics
• Difficult and expensive synthesis
Possible solutions
• Glycomimetics
• Oligomerization
• Glycoprotein constructs
Proposed model for sialyl lewis x bound to E-selectin
and important functional groups for selectin binding
Glu 80
–
Tyr 94
O
O
Ca 2+
O
HO
O
Arg 97
H3 C
H
NH
NH2 +
H2 N
OH
CO2 –
O
O
OH
HO
O
OH
AcHN
OH
HO
N
H
OH
O
OH
Asn 105
H
OH
NHAc
O
O
OR
O
OH
Evolution of a sialyl Lewis x mimetic with comparable
E-selectin binding affinity ( Wong and coworkers)
HO
HO
OH
H3C
CO2–
OH
O
OH OH
O
O
OH
OH
HO
AcHN
–
O
O2C
OH
O
OH OH
O
OR
O
O
H3C
NHAc
O
OH
NHAc
O
O
OR
O
OH
OH
O
OH
Sialyl Le w is x
Sim ilar E-s e le ctin binding affinity
as s ialyl le w is x
OH
HO
HO
H3C
OH
HO
O
OH
H3C
HO
HO
O2C
N
H
NH
–
O2C
O
OH OH
O
O
–
OH
O
OH
O
O
OH
O
E-s e le ctin binding affinity
s im ilar to s ialyl Le w is x
Tw o-fold low er E-s e le ctin
binding affinity than
s ialyl Le w is x
Fucose-based sialyl Lewis x mimetics
HO
H3C
HO
HO
–
NH
O2C
O
N
H
OH
O
OH
O
HO
OH
HO
HO
OH
O
OH
–
O2C
N
H
O
Two-fold more potent than sialyl Lewis x
for E-selectin binding
O
N
NH(CH2)13CH3
O
13-fold more potent than sialyl Lewis X
for E-selectin binding
Mannose-based sialyl Lewis x mimetics
–
CH3
O2C
HO
HO
OH
O
H3C
O
OH
OH
O
Three-fold more potent than sialyl lewis x
for E-selectin binding
(in clinical development at Texas Biotech.)
O
O
O
OH
O P O
O
OH
OH
OH
O
OH
104-fold more potent than sialyl Lewis x
for P-selectin binding
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
Multivalent inhibitor
+
Cell
Cell
Functionalized polymerized liposomes are nanomolar selectin inhibitors
Nagy and coworkers
HO
–O
2C
OH
H3C
O OH
OH
OH OH
O
O
O
O
O
OH
OH
H
N
S
N
O
Ac
+
O
O–
HO
–O C
2
–O C
2
1. Self assembly
2. UV-initiated polymerization
OH
H3C
O OH
OH OH
O
O
O
O
OH
HO OH
O
OH
S
N
O
OH
H3C
O OH
OH
OH OH
O
O
O
O
O
OH
OH
Ac
N
HO
–O
O
S
N
Ac
N
H
Aqueous
Interior
O
–O
Lipid Bilayer
Multivalent "glycopolymers" as potent selectin inhibitors
Kiessling and coworkers
n
OH
OSO3–
O
–
HN
O
O3SO
OH
OH
O
OSO3–
O
O
–
HN
O
O3SO
OH
500-fold more potent than sialyl lewis x
in blocking P-selectin binding to HL-60 cells
Limitations of sLex as a therapeutic agent
• Lack of potency
• Poor pharmacokinetics
• Difficult and expensive synthesis
Possible solutions
• Glycomimetics
• Oligomerization
• Glycoprotein constructs
Approaches to selectin inhibitors inspired by the
discovery of their biological ligands
L-selectin
GlyCAM-1
CD34
MAdCAM-1
P-selectin
P-selectin glycoprotein ligand-1
(PSGL-1)
E-selectin
E-selectin ligand-1 (ESL-1), perhaps
Structure of the biological selectin ligands
P-selectin ligand
L-selectin ligands
–O
OSO3–
OSO3–
3SO
–O SO
3
–O SO
3
–O
3SO
–O
3SO
OSO3–
OSO3–
OSO3–
OSO3–
OSO3–
–O SO
3
–O
3SO
GlyCAM-1
–O
OSO3–
–O
3SO
–O
3SO
3SO
–O SO
3
OSO3–
OSO3–
OSO3–
OSO3–
3SO
–O SO
3
–O SO
3
–O
OSO3–
–O SO
3
–O SO
3
–O SO
3
OSO3–
OSO3–
OSO3–
OSO3–
–
–
OSOOSO
3
3
–
OSO3
CD34
Endothelial-derived
sulfomucins
–O
3SO
–O SO
3
–
SO
–OO3
3SO
–O
3SO
OSO3–
OSO3–
OSO3––
OSO3
OSO3–
OSO3–
S
S
MAdCAM-1
PSGL-1
Leukocyte-derived
tyrosine-sulfated mucin
Determinants of PSGL-1 required for
P-selectin binding
A potent P-selectin inhibitor based on PSGL-1:
Recombinant PSGL-Ig (TS-1)
OSO3–
–O SO
3
–O SO
3
–O SO
3
OSO3–
OSO3–
–O
3SO
–O SO
3
–O SO
3
OSO3–
OSO3–
OSO3–
47 N-terminal
residues from
PSGL-1
Human IgG1 Fc
(inactivated)
S
S
Recombinant PSGL-Ig
Genetics Institute/Wyeth
PSGL-1
Features:
• Good potency (nM Kd)
• Good PK (serum 1/2-life = 2-3 wks)
• Also inhibits L-selectin
Leukocyte adhesion to endothelial cells is mediated by
L-selectin binding to sulfated sialyl Lewis x
HO
6-Sulfo
sialyl Lewis x
OH
OH
O
H3C
HO OH
OH
OH CO2 –
O
O
HO
O
O
O
AcHN
OH
HO
HO
OSO3 – HO
OH
OH OH CO –
2
O
O
O
HO
O
O
O
O
AcHN
OH
AcHN
HO
H3 C
O
OH
OH
HO
Leukocyte
NHAc
O
O
OSO3–
HO
OH
O
OH
O
O
AcHN
O
{
Endothelial cell
L-Selectin
Alternative approaches to blocking selectin-mediated
cell adhesion: Inhibition of key biosynthetic enzymes
HO
OH
O
H3C
OH OH
HO
AcHN
HO
CO2
O
HO
AcHN
O
CO2–
OH OH
O
OSO3–
OH
H3C
O
HO
O
O
NHAc
OH
Sulfotransferase
inhibitors
HO
OH OH
O
O
O
O
HO
O
O
OSO3–
O
HO
NHAc
O
OH
HO
OH OH
OH
–
O
O
Fucosyltransferase
inhibitors
OH
OH
O
O
AcHN
L-Selectin ligand
O
Ser/Thr
Lecture Outline
1. Examples of approved carbohydrate-based drugs
2. Development of drugs to treat influenza
3. Development of selectin inhibitors for inflammation
4. 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 founded
on glycobiology platforms
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