Transcript Amphotericin binds to insulin and Aβ 25

AMPHOTERICIN B DELAYS AMYLOID FIBRILLIZATION
Scott C Hartsel, Theodore R. Weiland, Rachel Nauss and Caitlin Cheney
Deparment of Chemistry, University of Wisconsin-Eau Claire, Eau Claire, U.S.A.
Conclusions and Significance:
Results:
ABSTRACT
Amphotericin binds to insulin and Aβ
25-35 amyloid Fibrils
195-Pos Board # B69
The antifungal agent Amphotericin B (AmB) is one of a handful of agents
shown to slow the course of animal prion disease. We propose it is possible
that AmB may act to physically prevent conversion of the largely a-helical
prion protein (PrP) to the pathological b-sheet aggregate protease resistant
isoform (PrPres) in prion disease. Congo Red and other small molecules
have been reported to directly inhibit amyloidogenesis in both prion and
Alzheimer peptide model systems by specific binding. This binding is
thought to either directly block fibril propagation or “overstabilize” the
pathogenic isoform so that it is not flexible enough to induce other proteins
to misfold. To assess whether AmB is capable of preventing
amyloidogenesis as does Congo Red, we have used the insulin fibril and Ab
25-35 amyloid model fibril systems. We find that AmB does bind strongly to
both insulin (Kd= 1.1 µM) and Ab 25-35 amyloid (Kd= 6.4 µM )fibrils, but not
to native insulin. Binding is characterized by a red-shifted AmB spectrum
indicative of a more hydrophobic environment. In kinetic fibril formation
studies, AmB was able to significantly kinetically delay the formation of Ab
25-35 fibrils at pH 7.4 but not insulin fibrils at pH 2. We have further
investigated the effect of AmB on putative channel formation by Ab 25-35.
Figure 1
From a titration of a fixed amount of AmB with increasing concentrations
of sonicated fibrils an extinction coefficient of 95,700 at 418 nm was
estimated for the AmB*fibril complex (Figures 2). The curves are fitted to a
modified hyperbola with a linear component due to the increasing
contribution of the free AmB absorbance tail
Figure 2
It has long been known that the azo dye Congo Red (CR) binds strongly and
specifically to amyloid plaques, changing to a bright red-orange color in the
process (below). In fact Congo Red binding is diagnostic for amyloidosis. It is
thought to bind at the interface of antiparallel b sheets. Molecules like Congo
Red have been shown to slow or arrest the process of amyloid fibril formation
and thus have been looked at as possible therapeutic agents for Alzheimer’s
disease and prion scrapie diseases.
O
Amyloid Plaque
AB fibrils
NH2
 Thus, we think that there are at least three possible mechanisms by
which AmB and analogues can effect the outcome of amyloidoses as
outlined below.
Physical Blockage of Fibril
Growth*
OH
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Insulin Fibrils
Kinetics of fibril growth for a 2mg/mL bovine insulin at pH 2.0 as measured
by CR binding in 25 µM CR buffer. Under these conditions AmB neither
prevented nor promoted fibrillization.
Kinetics of fibril growth for a 2mg/mL Ab 25-35 in PBS at pH 7.4 as
measured by CR binding in 25 µM CR buffer. In both cases AmB
significantly delayed the onset of unseeded fibrillization, however points
taken after 12 hours indicate the ultimate extent of fibrillization was
unaffected.
 HOWEVER, AmB’s mode of action is complex and AmB has been
shown to activate macrophages in the spleen and to stimulate the release
of cytokines from monocytes, possibly enhancing the general host
response versus amyloid deposits (similar to LPS) . Possibly the specific
amyloid binding is only coincidental.
 The most speculative and intriguing possibility is that AmB, since it has
an amyloid-like profile as suggested by CR binding, may provoke some
kind of anti-amyloid immune response in animal models. Alzheimer’s
vaccines have recently proven to be plausible. The fact that AmB is more
effective if given two weeks before an experimental prion infection is
supportive of the second and third possibilities.
 Difference spectra of the interaction of 25µM Congo Red to varying
amounts of AmB. 25 µM CR without AmB was the baseline (dashed line)
which was subtracted from all the other spectra to produce the difference.
The isosebestic points at 440 and 535 nm suggest a simple two-species
transition. The inset of a plot of the CR absorbance loss at 473 nm fitted to
a hyperbolic function suggests a saturable interaction between CR and
AmB.
Introduction
NH2
 Amphotericin B binds in a concentration dependent manner to Insulin
fibrils (Figure 1) as indicated by the spectroscopic shift of the monomer Amax
from 409 to 417 nm with a conversion of the aqueous aggregate at 340 nm
to protein-bound monomer (Figure 1). AmB did not bind to equivalent
amounts of native insulin as indicated by no increase in A417.
 We show that AmB’s anti-prion activity could be attributable to specific
binding to amyloid fibrils, possibly “capping” the fibril and stopping
propagation like some N-methylated peptides. This is seen with the
inhibition of early onset fibril formation as seen in Figure 5.
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Figure 4
O S O
OH
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NH 2
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OH
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HO
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CO2H
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Congo Red
OH
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NH 2
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Stimulation of Monocytes to Release
Cytokines
OH
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Amphotericin B
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NH 2
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CO2H
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OH
Figure 3
HYPOTHESIS
A popular membrane-active antifungal agent,
Amphotericin B (AmB) and its derivatives have been
shown to be among the very few agents which can
slow the course of prion disease in animal models.
We hypothesize that AmB may exert its effect by
selectively binding to b-amyloid structures and
preventing or slowing fibril formation and propagation.
We have tested for this proposed binding using the
low-pH insulin amyloid fibril model system and the AB
25-35.
Figure 5
Specific Immune response like
Alzheimer’s Vaccine?
OH
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NH 2
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OH
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CO2H
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CO2H
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*b-amyloid fibril model picture from Sunde M, Serpell LC, Bartlam M, Fraser PE, Pepys MB, Blake CCF
J Mol Biol (1997)273:729-739