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 KremkowNovember 9th, 2011CHEM 645 – Group 1

Motivation

Protein misfolding diseases• Neurodegenerative:

– Parkinsons’– Alzheimers’– Creutzfeldt-Jakob– Huntington– Amyotrophic lateral sclerosis

• Non-neurodegenerative:– Inherited cataracts– Type II diabetes mellitus

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• Population affected (US)– 1.5 million– 4 million– ~200– 8,000– 15,000

– ?– 23.2 millionwww.ninds.nih.gov

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

3

Worldclimatereport.comThrall, 2005.www.ninds.nih.gov

Motivation

4Nationmaster.com

Introduction – Protein Misfolding Defenses

• Cellular defenses:

– Chaperones

– Polyubiquitin attachment

– Proteasome targeting

– Aggresome

5Bronstein, 2004.Ross, et al. 2004.

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 6

Introduction – Therapeutic Defenses

• Issues

– Inhibition of 1 step may cause toxic accumulation

– Unknown protein misfolding pathway

– Diseases seem to have common pathways

7Ross, et al. 2004.

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

• 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

10Khurana, et. al, 2005.

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

12Avantilipids.com

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

14Avantilipids.comHickman, et. al, 2011.

Conformational Analysis

• ThT Fluorescence

– 485 nm

– No liposome interference

• Kd = 2.4 μm

15Hickman, 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

16Hickman, 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

17Hickman, et. al, 2011.

Sequence Order and Length

• Palm15-1, 1-9, and scPalm1-15– β-sheet

conformation• Palm1-5

– Mixed β-sheet and random coil

• Peptide sequence has minor influence on β-sheet aggregation– Not unique to Aβ1-

15 sequence

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Palm15-1 (___)scPalm15(------)Palm1-9(xxxx)Palm1-5(…….)

Hickman, et. al, 2011.

Acetyl CD Spec

• Lack of minima around 220 nm – Lacks β-sheets

19Hickman, et. al, 2011.

Effect of Peptide Charge

• 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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Palm1-15(D7K) (___)Palm1-15(E3A,D7K) (----)Palm1-15(E3K,D7K) (xxx)Palm1-15(E3K,D7K,E11K)

(…)

Hickman, et. al, 2011.

Liposome Surface Charge

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• 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

• β-strand favored in anionic, rather than cationic liposomes

– Red decrease in CB-Ala

– Red decrease in CA-Ser

• Increase in mobility

Anionic – blueCationic - red

Anionic – dark squareCationic – white

triangleEmpty – dark circle

Hickman, et. al, 2011.

Palmitoyl Chain Effects

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• 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)

(…….)

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 23Hickman, et. al, 2011.

Antibody Binding

• Recognition of oligomer, not monomer– Oligomer fraction – red– Monomer fraction – blue

24Hickman, et. al, 2011.

25Hickman, 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

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Questions?

Supplemental Figures/Info

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