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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