Amphotericin B

1
Amphotericin B

• Two-edged sword or Swiss army knife? Bridging the
  gap between Alzheimers and Prion diseases.

2
What are some Amyloid Diseases?

• Alzheimers disease
• Atrial Amyloidosis
• Hereditary Renal Amyloidosis
• Secondary Systematic Amyloidosis
• Injection-Localized Amyloidosis

3
What are some Prion Diseases?

• Chronic Wasting Disease (CWD)
• Scrapie
• BSE- Mad Cow Disease
• Kuru
• Creutzfeldt-Jakob Disease

4
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.

5
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.

6
Amyloids vs. Prions, pt. II

• Similar propagation of fibrils in vitro.
• Similar symptoms (inflammatory neuropathies) and
  adverse affects from having the disease.

7
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.

• Prion Protein-PrP

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Alzheimers Disease Amyloid hypothesis
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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 Anti- Inflammatory Drugs,
  Statins)
• 4.Symptomatic (anticholinergics)
• 5. Effecting an immune response.
• Specific-vaccine
• Non-Specific-general stimulant

10
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 Alzheimers
  disease
• Some other in vitro success using N-methylated
  peptides, anti-sense peptides and other small
  molecules.

11
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.

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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.

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Why AmB?

• Amphotericin B is an antifungal drug used to
  treat immuno-compromised 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.

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Does AmB bind to fibrils?

• 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
• Kd1.1µM

15
Does AmB B inhibit insulin Fibril Formation?

• No it doesnt.
• Luckily it does not induce fibrils either
• Thus, AmB neither inhibits nor promotes fibril
  formation under these conditions (pH 2-HCl).

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AD model system 2 AD APP-fragment 25-35
GSNKGAIIGLM
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Ab 25-35

• Minimal Alzheimers 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.

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Ab 25-35 fibrils do bind AmB
19
Butdoes AmB B inhibit Ab 25-35 Fibril Formation?
20
Butdoes 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
  fibrillogenesisit does not change final
  concentration
• Ab 1-40 and PrP peptides will be the real test.

21
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.)

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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

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THANKS !

• Ted Weiland

• Emily Bauer and Evan Kwong

• NSF for

• Rachel Nauss

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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