Transcript Slide 1

Sequence-independent Control of Peptide
Conformation in Liposomal Vaccines for
Targeting Protein Misfolding Diseases
D. Hickman, M. Deber, D. Ndao, A. Silva, D. Nand, M.
Pihlgren, V. Giriens, R. Madani, A. St-Pierre, H. Karastaneva,
L. Steger, D. Willbold, D. Riesner, C. Nicolau, M. Baldus, A.
Pfeifer, A. Muhs
Ben Kremkow
November 9th, 2011
CHEM 645 – Group 1
Motivation
Protein misfolding diseases
• Neurodegenerative:
• Population affected (US)
– Parkinsons’
– 1.5 million
– Alzheimers’
– 4 million
– Creutzfeldt-Jakob
– ~200
– Huntington
– 8,000
– Amyotrophic lateral sclerosis
– 15,000
• Non-neurodegenerative:
– Inherited cataracts
–?
– Type II diabetes mellitus
– 23.2 million
www.ninds.nih.gov
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Motivation
• Yearly cost (US $)
– Alzheimers’ – 100+ billion
– Diabetes – 156 billion
• US population
– 1900:
• Life expectancy = 47 years
• 3 million Americans
– 2000:
• Life expectancy = 77 years
• 35 million Americans
Worldclimatereport.com
Thrall, 2005.
www.ninds.nih.gov
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Motivation
Nationmaster.com
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Introduction – Protein Misfolding Defenses
• Cellular defenses:
– Chaperones
– Polyubiquitin
attachment
– Proteasome
targeting
– Aggresome
Bronstein, 2004.
Ross, et al. 2004.
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Introduction – Therapeutic Defenses
• Enhance cellular defenses:
– Geldanamycin – Modulate/enhance chaperone levels
• Reduce abnormal protein level in cell
– RNA interference – Delivery is an issue
• Small molecules
– Target protein misfolding pathway – Congo red
– Inhibit aggregation
• ID specific pathogenic mechanisms
– Proteolytic cleavage – small molecule inhibitors
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Introduction – Therapeutic Defenses
• Issues
– Inhibition of 1 step
may cause toxic
accumulation
– Unknown protein
misfolding pathway
– Diseases seem to
have common
pathways
Ross, et al. 2004.
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Objective
• Goals:
– Increased understanding of the β-sheet conformation
– Determine what variables affect liposomal protein
conformation
– Establishing a structure-conformation relationship for
liposomal Palm1-15 (ACI-24)
– Evaluate the constructs for the generation of
antibodies
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Methods
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•
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CD Spectroscopy
Thioflavin T Fluorescence
Magic Angle Spinning-NMR Spectroscopy
Size Exclusion Chromatography
• Biological Methods
– Vaccine Preparation
– Conformational Antibody Specificity
– Tissue Preparation
– Immunohistochemistry
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Common Methods
• Thioflavin T (ThT)
– Benzothiazole dye, exhibits red shift upon binding to
aggregated β-sheet peptides
– Measures β-sheet aggregation
• CD Spectroscopy
– Measures peptide secondary structure
– Minima at 220 nm is characteristic of β-sheet
conformation
Khurana, et. al, 2005.
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Magic Angle Spinning (MAS)-NMR
Spectroscopy
• Sample is spun at a magic angle, θm
– cos2 θm = 1/3
– Resolution is increased as the broad lines become
narrower
• Triple resonance (1H, 13C, 15N) MAS probe
• 16.4 T static magnetic field
– Palm1-15 amino acids Ala-2, Ser-8, and Gly-9 were
labeled with 13C and 15N
– Incorporated into DMPC, DMPG, DMTAP, cholesterol,
and MPLA liposomes
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Liposome Lipids and Molecules
• Lipids
– DMPC
– DMPG
– DMTAP
• MPLA
• Cholesterol
Avantilipids.com
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ACI-24
• Liposomal vaccine
– Tetrapalmitoylated β-amyloid 1-15 peptide
– Elicits immune response to restore cognitive
impairment of amyloid precursor protein pathway
• Link between peptide immunogen conformation and in
vivo efficacy
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Peptides Created
Avantilipids.com
Hickman, et. al, 2011.
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Conformational Analysis
• ThT Fluorescence
– 485 nm
– No liposome
interference
• Kd = 2.4 μm
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Hickman, et. al, 2011.
CD Spectra
• Lipidated Palm1-15
– Solid line
• Acetylated native
Acetyl 1-15
– Dotted line
• Liposomal Palm1-15
adopts a B-sheet
secondary structure
• Acetyl1-15 is
unstructured
• B-sheet aggregates
similar to B-amyloid
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Hickman, et. al, 2011.
Metal Ion Effects
• B-sheet aggregates are
dissociated by Cu(II)
– Similar to AB(1-42)
• Metal chelator DPTA
– Similar to before
metal ion addition
• Other metals have
reduced or no effect
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Hickman, et. al, 2011.
Sequence Order and Length
Palm15-1 (___)
scPalm15(------)
Palm1-9(xxxx)
Palm1-5(…….)
• Palm15-1, 1-9, and
scPalm1-15
– β-sheet
conformation
• Palm1-5
– Mixed β-sheet and
random coil
• Peptide sequence has
minor influence on βsheet aggregation
– Not unique to Aβ115 sequence
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Hickman, et. al, 2011.
Acetyl CD Spec
• Lack of minima around 220 nm
– Lacks β-sheets
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Hickman, et. al, 2011.
Effect of Peptide Charge
Palm1-15(D7K) (___)
Palm1-15(E3A,D7K) (----)
Palm1-15(E3K,D7K) (xxx)
Palm1-15(E3K,D7K,E11K) (…)
• β-sheet have no
dependence upon
peptide net charge
– Range: 5.2-10.0
– Minima at 220 nm
• ThT findings support
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Hickman, et. al, 2011.
Liposome Surface Charge
Anionic – dark square
Cationic – white triangle
Empty – dark circle
• Similar apparent Kd values for
liposome formulations from the
same peptide
– Charge does not weaken ThT
binding
• ThT signal due to differences in
peptide structure and aggregation
Anionic – blue
Cationic - red
• β-strand favored in anionic, rather
than cationic liposomes
– Red decrease in CB-Ala
– Red decrease in CA-Ser
• Increase in mobility
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Hickman, et. al, 2011.
Palmitoyl Chain Effects
• Vary number and position of lipid anchors
– C-terminal tetrapalmitoylated peptide forms more β-sheets
• Apparent Kd values similar, so not due to ThT binding affinity
(Supp)
– Number of lipid chains is an indicative variable
Palm(4C) (___)
Palm1-15(2C) (xxx)
Palm1-15(1N1C) (----)
Palm1-15(1C) (…….)
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Hickman, et. al, 2011.
Lipid Chain Length Effects
• Lipid chain length is an indicative variable
– Longer chains improve β-sheet conformation and
extent of aggregation
Hickman, et. al, 2011.
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Antibody Binding
• Recognition of oligomer, not
monomer
– Oligomer fraction – red
– Monomer fraction – blue
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Hickman, et. al, 2011.
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Hickman, et. al, 2011.
Summary
• Peptide N- or C-terminal lipidation is common to embed
peptides into liposome bilayers
• Palm1-15
– Adopts a β-sheet conformation, not random coil
– Similar ThT fluorescence to Aβ
• Responsible variables
– Net surface potential
– Lipidation pattern
– Lipid anchor chain length
• Induced IgG antibodies to recognize β-sheet multimers
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Thanks for your attention
Questions?
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Supplemental Figures/Info
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