1. Effect of Galantamine on Amylin (1-37) Aggregation: Investigations using Atomic Force Microscopy and
Spectrofluorimetry
Bradley Rose and Latha Ramakrishnan, Ph. D.,
Department of Chemistry and Physics; St. Cloud State University; St. Cloud, MN
Acknowledgements: The authors would like to thank the Office of Sponsored Program at St. Cloud State University. The authors would also like to acknowledge the CMIA
(COSE,SCSU) and Russell L. Lidberg for the AFM studies, Dr. Sivaprakasam for the FT-IR studies, and the Department of Chemistry and Physics.
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Intensity
Wavelength (nm)
Emission Spectra Showing Amylin Aggregation
ThT Blank Amylin
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Control Amylin 100 µM Gal 1 mM Gal 100 µM Mem 1 mM Mem 100 µM Ril 1 mM Ril
Intensity
Effect of Drugs on Amylin Aggregation: ThT
Fluorescence Emission Intensity at 485 nm
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Intensity
Time (Hours)
Time-Dependent Study of Amylin Incubated with
Galantamine
ThT Control Amylin + 0 Gal Amylin + 1 µM Gal Amylin + 100 µM Gal Amylin + 1 mM Gal
Figure 1: Emission spectra of two samples incubated at 37˚ C for 120 hours. The red spectrum labeled
“Amylin” contained 10 µM amylin with 3µM thioflavin T in buffer and the black spectrum labeled
“ThT Blank” contained 3 µM thioflavin T in buffer.
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Absorbance
Wavenumbers (cm-1)
FTIR Absorbance Spectra Showing Amylin
Aggregation
Post-incubation Pre-incubation
Figure 2: FTIR spectra of two samples incubated at 37˚ C for 120 hours. A spectrum was
obtained before and after incubation for a sample containing 1.28 mM amylin in buffer.
Figure 3: Atomic force microscope images of two samples incubated at 37˚ C for 48 hours.
The sample on the left contained 10 µM amylin in buffer and the sample on the right
contained 10 µM amylin in buffer with 100 µM galantamine.
Figure 4: Fluorescence data collected for 3 drugs, galantamine (Gal), memantine (Mem) and riluzole (Ril). Each
drug was incubated at 37˚ C for 120 hours with 10 µM amylin.
Figure 5: Fluorescence data collected for 10 µM amylin incubated with galantamine at 37 C for 144 hours.
AFM Images of Amylin without Gal and
With 100 M Gal
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Conclusions
• Amylin samples tested did indeed aggregate forming a primarily β-sheet
structure, which was confirmed through ThT fluorescence emission studies
and characteristic FT-IR amide I stretching frequency (1623 cm-1)
• Of the three small molecular drugs tested to prevent aggregation of amylin,
AFM studies indicated that Gal was effective in preventing Amylin
aggregation. Results from ThT fluorescence emission studies indicate that
Gal may prevent aggregation, while Mem did not inhibit aggregation, and
the results with Ril were inconclusive. The significant increase in emission
intensity with 1 mM Ril may be due to interaction between ThT and Ril; two
structurally similar small molecular compounds.
References
1. Janson, et al. (1999) The mechanism of islet amyloid polypeptide toxicity is
membrane disruption by intermediate-sized toxic amyloid particles. Diabetes, 48.
2. LeVine, et al. (1993) Thioflavine T forms uniquely fluorescent complexes with
amyloid structures of synthetic Alzheimer’s disease β-amyloid peptides and insulin in
solution. Proc. Int. Symp. Amyloidosis, 7th.
Introduction
• Amylin, a 37 amino acid peptide co-secreted with insulin can assume
an insoluble β-sheet conformation
• Amyloid aggregations composed primarily of amylin form on β-cells
• Amylin plaques have been found in 90% of type II diabetic patients
• Amylin plaques lead to apoptosis and necrosis in β-cells within 24-48
hours
• Various drugs including short peptide inhibitors and small molecular
drugs have been tested to prevent plaque formation
• Galantamine is effective as an amyloid-β aggregation inhibitor, a
peptide which has a similar mechanism in Alzheimer’s disease
• Amyloid-β and amylin have β-sheet structures that are indistinguishable
• Thioflavin T fluorescence increases upon binding to amyloid fibrils;
this is an established method for diagnosing fibril formation
• AFM, having nanometer resolution, can provide morphological support
for fluorescence data
Objectives
• Confirm that amylin is aggregating
• Measure effectiveness of galantamine, memantine, and
riluzole as inhibitors of amylin aggregation. These three
small molecular drugs have been shown to inhibit
aggregation of amyloid Aβ implicated in Alzheimer’s
Disease.
• Apply several instruments and techniques to monitor
amylin aggregation in the absence or in the presence of
various drugs
Methods
All samples were incubated for various times at 37˚ C. A first
fluorescence study was performed on 10 µM amylin samples with
galantamine and 3 µM thioflavin T incubated for 144 hours having
fluorescence obtained at 24, 48 120 and 144 hours. An additional
fluorescence experiment was performed with galantamine, memantine and
riluzole where samples were incubated for 120 hours, adding thioflavin T to
a concentration of 3 µM after incubation, then collecting fluorescence data.
Fluorescence emission intensity indicated inhibition of amylin aggregation.
FTIR studies were performed by obtaining the spectra of a 1.28 mM
amylin sample prior to incubation for 120 hours and again afterward. The
before and after spectra were compared to confirm the presence of β-sheet
structures.
AFM samples taken from the first fluorescence experiment after 48 and
120 hours were imaged. One sample having only amylin and the other
having amylin and 100 µM galantamine were compared. Morphological
differences were used to support inhibition of amylin aggregation by
galantamine.