Glycans in the Biotechnology and Pharmaceutical Industries Lecture 42 Carolyn R. Bertozzi UC Berkeley

Lecture Outline Examples of approved carbohydrate-based drugs Development of drugs to treat influenza Development of selectin inhibitors for inflammation Imino-sugars as drug candidates for storage diseases

Examples of approved carbohydrate drugs: Substance Indication Company Acarbose Diabetes Bayer AG AMVISC Opthalamic surgery Anika Therapeutics Hyalgan Osteoarthritis FIDIA/Sanofi Lovenox Cardiovascular disease Aventis Miglitol Diabetes Bayer AG ORTHOVISC Osteoarthritis Anika Therapeutics Relenza Influenza Glaxo Smithkline Tamiflu Influenza Roche SOLARASE Actinic keratosis Hyal Pharmaceuticals Topamax Epilepsy J & J Voglibose Diabetes Takedo/Abbott

Examples of carbohydrate-based drugs

Examples of glycosylated natural products

Examples of approved glycoprotein drugs: Substance Company Erythropoietin Amgen, J&J Tissue plasminogen activator Genentech Interleukin-2 Chiron Cerezyme Genzyme Monoclonal antibodies Many From: Hudson, P. J.; Souriau, C. Nature Medicine 2003, 9, 129-134

Lecture Outline Examples of approved carbohydrate-based drugs Development of drugs to treat influenza Development of selectin inhibitors for inflammation Imino-sugars as drug candidates for storage diseases

Cell surface oligosaccharides are determinants of cell recognition bacterium virus cell toxin hormone glycoprotein

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) Sialic acid (SA) Host cell

Life cycle of the influenza virus Neuraminidase cleaves sialic acid and liberates new virus Hemagglutinin binds sialic acid to initiate infection SA SA New viruses assemble at membrane Endocytosis SA SA Membrane fusion and release of viral particle Replication Host cell SA SA

Cell Sialic acid analogs block influenza virus infection influenza “C-Glycoside” of sialic acid

Enzymes catalyze reactions by preferential binding of the transition state vs the ground state Ea (cat) Ea (uncat) Transition state analogs are potent enzyme inhibitors

‡ Design of transition state analog neuraminidase inhibitors Planarity (sp2) 2,3-Anhydro sialic acid Ki = 10-6 M Also inhibits human neuraminidase

Structure-based design of more potent and selective neuraminidase inhibitors Binding pocket of Flu neuraminidase based on X-ray structure Relenza (Glaxo Smithkline) Ki = 10-10 M Tamiflu (Hoffman La Roche) Ki = 10-10 M

Lecture Outline Examples of approved carbohydrate-based drugs Development of drugs to treat influenza Development of selectin inhibitors for inflammation 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 P-selectin E-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

Limitations of sLex as a therapeutic agent • Lack of potency • Poor pharmacokinetics • Difficult and expensive synthesis Possible solutions • Glycomimetics • Oligomerization • Glycoprotein constructs

(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

Nagy and coworkers

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

Approaches to selectin inhibitors inspired by the discovery of their biological ligands

Structure of the biological selectin ligands

Determinants of PSGL-1 required for P-selectin binding

A potent P-selectin inhibitor based on PSGL-1: Recombinant PSGL-Ig (TS-1) 47 N-terminal residues from PSGL-1 Human IgG1 Fc (inactivated) 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 6-Sulfo sialyl Lewis x L-Selectin { Endothelial cell

Lecture Outline Examples of approved carbohydrate-based drugs Development of drugs to treat influenza Development of selectin inhibitors for inflammation 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