Rapamycin as an Immunomodulator in Systemic Lupus Erythematosus

Ana Gargaun

Supervisors: Maxim Berezovski Tony Durst May 3, 2012

• Systemic Lupus Erythematosus (SLE) • Rapamycin 2 • Rapamycin Biosynthesis • Rapamycin Synthesis

Systemic Lupus Erythematosus (SLE) 19 th century • Skin lesions 20 th century • Systemic autoimmune disease

Malar rash

• SLE is a multifactorial, heterogeneous group of diseases which may be fatal and can be characterized by anti-nuclear autoantibody production, resulting in organ failure Talaber G, Perl A (2011) SLE: a Metabolic Disease of T Cells? Rheumatology 1:e103 3

Epidemiologic Factors • Female to male ratio ▫ 10 to 1 between the ages of 15-45 • In Canada, the SLE prevalence is 0.05% overall ▫ Often misdiagnosed • Varies with race, ethnicity, and socioeconomic status ▫ African-Americans and Asians  More common and severe N.T. Colburn, Review of Rheumatology, © Springer-Verlag London Limited 2012 4

American College of Rheumatology Criteria for the Diagnosis of SLE • 11 criteria used to establish the disease, with 4 needed for the formal diagnosis of SLE I. Malar rash II. Discoid rash III.Photosensitivity

IV. Oral ulcers V. Arthritis VI. Serositis VII. Kidney disorder VIII.Neurologic disorder IX. Blood disorder X. Immunologic disorder XI. Abnormal antinuclear antibodies 5 http://emedicine.medscape.com/article/332244-workup

Overview of the Etiology & Pathogenesis of SLE 6 Tsokos, G.C. N Engl J Med 2011; 365:2110-21

Overview of the Etiology & Pathogenesis of SLE • Familial aggregation • Defect in apoptosis ▫ Disturbance in immune tolerance • Genes responsible for “waste disposal” ▫ C1q 7 Tsokos, G.C. N Engl J Med 2011; 365:2110-21

Overview of the Etiology & Pathogenesis of SLE • Exposure to UV light Keratinocytes Apoptosis • Viruses (e.g. Epstein-Barr virus) ▫ Cross reactivity of foreign antibody to self antigen Tsokos, G.C. N Engl J Med 2011; 365:2110-21 8

Overview of the Etiology & Pathogenesis of SLE • Major factor • Antigen-driven B Cell proliferation • B Cell hyperactivity ▫ Heightened responses  Both self and foreign antigens • Abnormal tolerance ▫ Normally precursors to autoantigens are tolerated ▫ SLE retains precursors that produce high affinity autoantibodies Tsokos, G.C. N Engl J Med 2011; 365:2110-21 9

Overview of the Etiology & Pathogenesis of SLE • Estrogen ▫ Prepubertal and postmenopausal women have similar incidence to men ▫ Men with SLE have higher concentration of estrogenic metabolites ▫ Pregnancy may aggravate symptoms of SLE ▫ Hormone replacement therapy 10 Tsokos, G.C. N Engl J Med 2011; 365:2110-21

Overview of the Etiology & Pathogenesis of SLE • DNA methylation 11 Tsokos, G.C. N Engl J Med 2011; 365:2110-21 • Hypomethylation is present in SLE • Drug induced lupus ▫ E.g. hydralazine ▫ Reversible

Autoimmunity in SLE Autoimmunity: A condition in which the body's immune system produces antibodies in response to its own tissues or blood components instead of foreign particles or microorganisms.

Impaired tolerance T-cell activation Defective immune complex clearance Hyperactive B-cell N.T. Colburn, Review of Rheumatology, 2012 © Springer-Verlag London Limited Gale Encyclopedia of Medicine. Copyright 2008 The Gale Group, Inc. 2008 12

Interaction between a T Cell and an Antigen Presenting Cell (APC) 13 Rahman, A et al. N Engl J Med 2008; 358: 929-39.

Antigen Presenting Cell: B-Cell, Dendritic Cell, or Macrophage

T-cell and B-cell Co-Stimulation 14 Rahman, A et al. N Engl J Med 2008; 358: 929-39.

Pathogenic Autoantibodies in SLE

Antigen Specificity

dsDNA Nucleosomes Ro (RNP) La (RBP) Sm (snRNP) NMDA receptor Phospholipids

Prevalence (%)

70-80 60-90 30-40 15-20 10-30 33-50 20-30

Main Clinical Effects

Kidney disease, skin disease Kidney disease , skin disease Skin disease, kidney disease, fetal heart problems Fetal heart problems Kidney disease Brain disease Thrombosis, pregnancy loss 15 Rahman, A et al. N Engl J Med 2008; 358: 929-39.

Treatments • Anti-malarials • E.g. Hydroxychloroquine • NSAIDs • E.g. Ibuprofen • Glucocorticoids • E.g. Prednisone 16 • Immunosuppressive agents • E.g. Methotrexate • Cell signaling modulators • E.g. Rapamycin

Survival rate • 1950s ▫ 5 year survival rate was 50% • Today ▫ 15 year survival rate of 80% ▫ Patients diagnosed at 20 years of age, have a 1 in 6 chance of dying by 35 years of age from lupus or infection Tsokos, G.C. N Engl J Med 2011; 365:2110-21 17

Rapamycin Found in a soil sample from Streptomyces hygroscopicus from Easter Island, also known as Rapa Nui, in 1975 18 • • • Gram-positive Aerobic Filamentous

Rapamycin (Rapamune TM ) • Currently in clinical trials for SLE • • Andras Perl, MD/PhD David Fernandez, MD/PhD Candidate Michigan Upstate University Immunosuppressant used to prevent rejection during organ transplant ▫ Approved by the FDA in 1999 and is marketed as Rapamune by Pfizer (formerly Wyeth) Potent antifungal and anticancer agent 19

Mechanism of Action of Rapamycin • • FKBP is an immunophilin mTOR (mammalian Target of Rapamycin) • is a serine threonine kinase • mTORC1 and mTORC2 20 Park, S. R., et al. Journal of Antibiotics. 2010. 63, 434-441

21 Mechanism of Action of Rapamycin FKBP Rapamycin FKBP Rapamycin mTOR

Kd

0.2nM

2.5nM

mTOR binding Rapamycin mTOR FKBP Rapamycin mTOR

Kd

26  0.8

 M 12  0.8nM

Banaszynski, L.A., et al. J. Am. Chem. Soc. 2005, 127, 4715-4721 FKBP binding

Sourcing Rapamycin, Rapamune TM • How is it made on a commercial scale?

▫ Fermentation of S. hygroscopicus ▫ $388 million in revenue for Pfizer in 2010 • Oral solution (5mg) and tablets (0.5mg, 1mg, 2mg) • How much does it cost?

▫ ~$1,200/90 2mg tablets ▫ Dosage for organ transplants: 2mg/day 22

Biosynthesis of Rapamycin by S. hygroscopicus PKS NRPS Post PKS and NRPS 23 Raps1 Raps2 Raps3 15 modules: 70 catalytic sites PIE oxygenases, MT, and other Schwecke et al. Proc. Natl. Acad. Sci. 1995 USA Vol. 92 pp. 7839-7843

Biosynthesis of Rapamycin: PKS 24 13 modules, 57 steps using the enzymes: KS, AT, ACP ER, KR, DH , using malonate or methyl malonate

Biosynthesis: Cyclization and Modifications 25

Rapamycin: Synthesis • • • 31 membered ring Many stereocenters Labile bonds • Total Synthesis: ▫ K.C. Nicolaou ▫ S. J. Danishefsky ▫ A. B. Smith 26

Retrosynthetic Analysis of Rapamycin by K.C. Nicolaou 27

+

Forward Synthesis of Rapamycin by K.C. Nicolaou 28 95% yield

Forward Synthesis of Rapamycin by K.C. Nicolaou 29 70% yield

Forward Synthesis of Rapamycin by K.C. Nicolaou 30 28% yield • • Longest linear sequence from 5 sub targets is 16 steps Total number of steps 102

Conclusion • “Pathophysiology of SLE is enigmatic, potentially deadly, and it is yet unsolved” • Pathogenic autoantibodies are the primary cause of tissue damage in patients with lupus • Many different elements of the system are potential targets for therapeutic drugs in patients with SLE 31

Conclusion continued • • • Rapamycin is in clinical trials for patients with severe cases of SLE Its biosynthesis involves elaborate enzyme complexes • Polyketide synthase and Non-Ribosomal Peptide Synthetase Its total synthesis is an impressive challenge for any chemist 32

Acknowledgements 33

Dr. Maxim Berezovski Dr. Tony Durst

The Berezovski Lab The Durst Lab

NSERC CREATE

Questions?