Rapamycin as an Immunomodulator in Systemic Lupus Erythematosus

Report
Rapamycin as an Immunomodulator
in Systemic Lupus Erythematosus
Ana Gargaun
Supervisors: Maxim Berezovski
Tony Durst
May 3, 2012
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• Systemic Lupus Erythematosus
(SLE)
• Rapamycin Biosynthesis
• Rapamycin
• Rapamycin Synthesis
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Systemic Lupus Erythematosus (SLE)
19th century
• Skin lesions
20th 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
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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
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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
http://emedicine.medscape.com/article/332244-workup
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Overview of the Etiology & Pathogenesis of SLE
Tsokos, G.C. N Engl J Med 2011; 365:2110-21
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Overview of the Etiology & Pathogenesis of SLE
• Familial aggregation
• Defect in apoptosis
▫ Disturbance in immune
tolerance
• Genes responsible for “waste
disposal”
▫ C1q
Tsokos, G.C. N Engl J Med 2011; 365:2110-21
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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
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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
Tsokos, G.C. N Engl J Med 2011; 365:2110-21
▫ Normally precursors to
autoantigens are tolerated
▫ SLE retains precursors that
produce high affinity
autoantibodies
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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
Tsokos, G.C. N Engl J Med 2011; 365:2110-21
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Overview of the Etiology & Pathogenesis of SLE
• DNA methylation
• Hypomethylation is present in
SLE
• Drug induced lupus
▫
▫
Tsokos, G.C. N Engl J Med 2011; 365:2110-21
E.g. hydralazine
Reversible
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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
Hyperactive
B-cell
T-cell
activation
Defective
immune
complex
clearance
N.T. Colburn, Review of Rheumatology, 2012 © Springer-Verlag London Limited
Gale Encyclopedia of Medicine. Copyright 2008 The Gale Group, Inc. 2008
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Interaction between a T Cell and an AntigenPresenting Cell (APC)
Antigen Presenting Cell:
B-Cell, Dendritic Cell, or
Macrophage
Rahman, A et al. N Engl J Med 2008; 358: 929-39.
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T-cell and B-cell Co-Stimulation
Rahman, A et al. N Engl J Med 2008; 358: 929-39.
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Pathogenic Autoantibodies in SLE
Antigen Specificity
Prevalence
(%)
Main Clinical Effects
dsDNA
70-80
Kidney disease, skin disease
Nucleosomes
60-90
Kidney disease , skin disease
Ro (RNP)
30-40
Skin disease, kidney disease, fetal
heart problems
La (RBP)
15-20
Fetal heart problems
Sm (snRNP)
10-30
Kidney disease
NMDA receptor
33-50
Brain disease
Phospholipids
20-30
Thrombosis, pregnancy loss
Rahman, A et al. N Engl J Med 2008; 358: 929-39.
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Treatments
• Anti-malarials
• E.g. Hydroxychloroquine
• NSAIDs
• E.g. Ibuprofen
• Immunosuppressive agents
• E.g. Methotrexate
• Glucocorticoids
• E.g. Prednisone
• Cell signaling modulators
• E.g. Rapamycin
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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
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Rapamycin
Found in a soil sample from Streptomyces
hygroscopicus from Easter Island, also known as Rapa
Nui, in 1975
• Gram-positive
• Aerobic
• Filamentous
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Rapamycin (RapamuneTM)
• 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
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Mechanism of Action of Rapamycin
• FKBP is an immunophilin
• mTOR (mammalian Target of Rapamycin)
• is a serine threonine kinase
•
mTORC1 and mTORC2
Park, S. R., et al. Journal of Antibiotics. 2010. 63, 434-441
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Mechanism of Action of Rapamycin
Kd
FKBPRapamycin
0.2nM
FKBPRapamycinmTOR
2.5nM
FKBP
binding
Kd
RapamycinmTOR
260.8M
FKBPRapamycinmTOR
120.8nM
mTOR
binding
Banaszynski, L.A., et al. J. Am. Chem. Soc. 2005, 127, 4715-4721
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Sourcing Rapamycin, RapamuneTM
• 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
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Biosynthesis of Rapamycin by S. hygroscopicus
PKS
RapA
RapB
Raps1
Raps2
RapC
Raps3
NRPS
Rap
P
PIE
15 modules: 70 catalytic sites
Schwecke et al. Proc. Natl. Acad. Sci. 1995 USA Vol. 92 pp. 7839-7843
Post PKS and NRPS
oxygenases, MT, and other
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Biosynthesis of Rapamycin: PKS
13 modules, 57 steps using the enzymes: KS, AT, ACP
ER, KR, DH , using malonate or methyl malonate
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Biosynthesis: Cyclization and
Modifications
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Rapamycin: Synthesis
• 31 membered ring
• Many stereocenters
• Labile bonds
• Total Synthesis:
▫ K.C. Nicolaou
▫ S. J. Danishefsky
▫ A. B. Smith
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Retrosynthetic Analysis of
Rapamycin by K.C. Nicolaou
+
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Forward Synthesis of Rapamycin
by K.C. Nicolaou
95% yield
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Forward Synthesis of Rapamycin
by K.C. Nicolaou
70% yield
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Forward Synthesis of Rapamycin
by K.C. Nicolaou
28% yield
• Longest linear sequence from 5 sub targets is 16 steps
• Total number of steps 102
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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
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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
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Acknowledgements
Dr. Maxim Berezovski
Dr. Tony Durst
The Berezovski Lab
The Durst Lab
NSERC CREATE
Questions?

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