Transcript Amyloids, Prions and Congo Red

Amphotericin B
Two-edged sword or Swiss army
knife? Bridging the gap between
Alzheimer’s and Prion diseases.
What are some Amyloid
Diseases?
Alzheimer’s disease
Atrial Amyloidosis
Hereditary Renal
Amyloidosis
Secondary Systematic
Amyloidosis
Injection-Localized
Amyloidosis
What are some Prion
Diseases?
Chronic Wasting
Disease (CWD)
Scrapie
BSE- Mad Cow
Disease
Kuru
Creutzfeldt-Jakob
Disease
Amyloids vs Prions
Prions are known to
be infectious in their
spreading to different
hosts, e.g. CWD,
BSE, kuru, etc…
Amyloid diseases not thought to be
infectious agents
but….
Amyloids vs. Prions-news!
It has been recently shown that mice who were fed amyloid
fibrils (from the spleen of infected mice) orally (in H2O)
developed amyloid deposits upon stimulation.
Amyloids vs. Prions, pt. II
Similar propagation
of fibrils in vitro.
Similar symptoms
(inflammatory
neuropathies) and
adverse affects from
having the disease.
These diseases involve fibril
deposits: What is a fibril?
Normal PrP or Ab protein
may misfold into a beta
sheet structure
The beta sheets form
extended aggregate fiber
structures by “recruiting”
properly folded proteins
These fibrils are protease
resistant and insoluble
This is the most prevalent
characteristic of amyloid
and prion diseases.
Alzheimer’s
Disease Amyloid
hypothesis
Possible approaches to treating
Amyloid and Prion Diseases.
1. Physical blockage of fibril growth (by small
molecules).
2. Alter processing/clearance of protein
(enzyme inhibitors)
3. Damage Control of existing fibrils(?)
(NSAIDs -- Non-steroidal AntiInflammatory Drugs, Statins)
4.Symptomatic (anticholinergics)
5. Effecting an immune response.
 Specific-vaccine
 Non-Specific-general stimulant
How would Physical Blockage
work?
Congo Red: a molecule that
binds to and inhibits fibril
growth.
Congo Red is an azo dye that
was used by Alois Alzheimer
to characterize brains with
Alzheimer’s disease
Some other in vitro success
using N-methylated peptides,
anti-sense peptides and other
small molecules.
AD model system #1: Characterizing fibrils
Congo Red binds to fibrils
very specifically.
Absorbance at 540 nm can
be used to quantitate [fibril]
formation
The Insulin Fibril system was
used as it is a proven
amyloid model system.
Hypothesis:
AmB is one of the only agents known to slow
prion diseases in animal models
(hamster,mouse)!
Perhaps AmB could act by the 1st mechanism
by binding to existing fibrils and preventing
growth, thus preventing the propagation of the
disease.
Why AmB?
Amphotericin B is an
antifungal drug used to
treat immunocompromised people
(AIDS, cancer) who have
deep fungal infections.
Amphotericin B is also an
interesting molecule that
has a polyene side and a
polyol side, which may
hint to its binding
characteristics.
It is also a relatively
inexpensive drug that is
already on the market.
Does AmB bind to fibrils?
417 nm
Yes it does!
Although the actual
binding site(s) are
unknown, it does bind
to fibrils specifically and
not to protein in native
form.
1 mole AmB/2 mole
insulin
Kd~1.1µM
Does AmB B inhibit insulin
Fibril Formation?
No it doesn’t.
Luckily it does not
induce fibrils either
Thus, AmB neither
inhibits nor
promotes fibril
formation under
these conditions (pH
2-HCl).
AD model system #2: AD APP-fragment #2535= GSNKGAIIGLM
Ab 25-35
Minimal “Alzheimer’s unit?”
Rapidly fibrillizes(~1 hr)
Can promote protein
denaturation-”anti chaperone”
Is cytotoxic and neurotoxic
likeAb
Our titration of the Ab25-35 fibrils
with Congo Red shows ~1 “fibril
peptide” per Congo Red.
Ab 25-35 fibrils do bind AmB
But…does AmB B inhibit Ab25-35
Fibril Formation?
But…does AmB B inhibit Ab25-35
Fibril Formation?
Yes it does!! At reasonable
therapeutic concentrations,
too (7.5 and 15µM)
However, it only delays the
onset of fibrillogenesis;it
does not change final
concentration
Ab 1-40 and PrP peptides
will be the real test.
How else might Amphotericin B
work in addition to fibril inhibition?
Amphotericin B may work non-specifically. Because of its properties
and known prior use, it may be able to activate the generalized
immune system acute phase response (IL-1, IL-6) and promote
removal of amyloid or prion deposits. Fibrils may also help sequester
AmB in tissues most strongly affected.

Previous reports have shown that introducing AmB 2 weeks prior or
immediately with the systematic prion inoculum effectively slows the
course of the prion infection. Later treatment is less effective.
It might bind to and modify oligomer ion channel properties
associated with amyloid diseases.
AmB would selectively associate with cholesterol-rich membrane
“rafts” and alter processing (Ab) or conversion(PrP).
Vaccination analog, i.e. could AmB-protein adduct resemble a fibril
and lead to antibody/T-cell response?(But.. SCID mice.)
Conclusions
Amphotericin B binds specifically to two different b
fibril models suggesting a possible mechanism for its
demonstrated antiprion activity.
This is further supported by the kinetic inhibition of Ab 25-35
fibrillization by AmB and suggests a possible screening assay
for future potential drugs
Further testing on PrP and
Ab
2
confirm this hypothesis
As a sidelight, it seems worth investigating whether the
fungal amyloid-like protein hydrophobin may be important to
AmB susceptibility/resistance
THANKS !
Projects in our lab
Physical characterization of Amphotericin B drug
delivery vehicles (stability, ion channel formation)
Pharmaceutical testing of new AmB preparations
(with K. Wasan)
Cytokine expression profiles caused by AmB preps in
immune cells (with L. Turtinen)
Localization and metabolism of liposomal doxorubicin
in single cells (with E. Arriaga)
AmB potential effects on amyloid diseases