A Molecular Harpoon?

A Molecular Harpoon?

Kinetic fluorescent methods for measuring functional delivery of membrane active antibiotics or Dr. Scott C. Hartsel University of Wisconsin-Eau Claire Overview What is Amphotericin B? The problem with Amphotericin B/A simple solution: Hot-Zone! Applied Photophysics instrumentation to measure: Activity of hot-zone by fluorescence Stability/structure of hot-zone by CD Kinetic stability in serum by kinetic diode array What is Amphotericin B? O


OH O Binds weakly to AmB CO2 H O HO OH OH OH NH 2 Cholesterol:

Cholesterol humans Nystatin Nystatin O Me O Me OH OH Me HO Me

H O OH OH OH OH O OH Amphotericin Amphotericin B O OH NH 2

CO2 H OH Binds strongly to AmB HO Ergosterol: fungi Ergosterol Q:How can you reduce toxicity? A: Associate TOXIC AGGREGATE with liposomes. Bolards model Reducing effective chemical

potential of AmB by tying up or by macrophage consumption is key. A Simple Solution: Hot Zone If lowering chemical potential is most important, can we change Amphotericins properties without expensive and troublesome lipids? YES! Heat treating Fungizone (70oC, aqueous, for 20 minutes) creates a new self-associated form. A superior therapeutic index for Hot-Zone was shown in animal fungal disease models- Francois Gaboriau, Jacques Bolard Nickname: Hot-Zone

A Simple Solution: Hot Zone We wanted to find out How and Why? by asking: How has the structural arrangement of AmB changed? Is the new arrangement stable? Is the membrane channel forming activity different? Does heat treatment change interaction with serum components and the immune system? Hot-Zone-Analysis Applied Photophysics Stopped-Flow diode array and conventional spectrophotomet er, spectrofluorimet er, and circular

dichroism (CD) spectrometer Hot-Zone-Absorption Spectra 50 45 40 HEAT 35 30 25 20 15

10 5 0 300 320 340 360 Wavelength, nm 380 400 420

440 CD Spectra (circular dichroism) CD signal Right and left hand circularly polarized light Preferential absorption of right hand polarization Polarized light beam Optically active sample CD Spectra

Self-associated molecules may absorb light as an aggregate by exciton coupling. If the molecules are twisted relative to one another in space they will absorb right and left-handed circularly polarized light differently. This gives rise to circular dichroism by the coupled oscillator mechanism. The spectrum will have two equal and opposite bands. The shape and intensity of the CD bands are very sensitive to small changes in the geometry of the molecules. Absorption Out of phase In phase CD Spectra AmB molecules normally have no CD spectrum in the visible light region, but when selfassociated (oligomers)they have intense CD spectra. The dimer is the minimal unit of CD activity.

This property gives a very sensitive handle on AmBs supramolecular geometry and changes in that structure. - + Hot-Zone-CD Spectra Circular dichroism: sensitive to small changes in supramolecular structure HEAT - Hot-Zone-CD Spectra Circular dichroism: sensitive to small changes in supramolecular structure -

Persistence of Hot-ZoneLyophilization studies show stability 1200 1000 HFZ in Dextrose, before 800 HFZ in Dextrose, after 600 400 200 0 -200 -400

-600 -800 295 315 335 355 Wavelength 375 395 415 Experimental

Membrane Activity of Hot-Zone Extruded System 1000 Liposome Membrane Vesicles Membranes with 10% ergosterol are fungal models; with cholesterol they are mammalian models. With KCl gradient K+ permeation creates a voltage, (K+ selective for AmB). H+ equilibrates with Pyranine fluorescence responds to pH linearly from ~6.2-7.8. Fluorescence decrease means net cation (K+) selectivity. Amphotericin High K+ , Cl2 mM pyranine - SO

SO3- 3 - SO 3 O- H+ Fluorescence K+ - + + +

Low K+, Cliso-osmotic H+ Hot-Zone/Membrane Channel Activity Model mammalian membranes with cholesterol Hot-Zone has much less activity Model fungal membranes With ergosterol Hot-Zone has similar activity ! Membrane Activity in the Presence of Serum Components 0.1

0 -0.1 -0.2 -0.3 -0.4 -0.5 in pH Change -0.6 -0.7 -0.8 -0.9 0 10 20 30 40 50

60 70 80 90 100 Time (seconds) 15mg/mL HSA in External Buffer 3uM Heat-treated Fungizone 5uM Heat-treated Fungizone 10uM Heat-treated Fungizone 3uM Fungizone

5uM Fungizone 10uM Fungizone 0.3uM Valinomycin Change in pH versus Time showing fluorescence detected ion currents on model mammalian membranes comparing Fungizone and Hot-Zone in the presence of 15mg/mL human serum albumin in external buffer (315mM sucrose, 15mM K2HPO4, pH 7.20 at 37C) Hot-Zone-Kinetic Stability (HSA)* 500 Fungizone aggregates are destabilized by serum albumin-500 sec/37C 0 Hot-zone aggregates are much more stable in the

presence of serum albumin Hot-Zone-Kinetic Stability (HSA)* Extra stability of Hot-Zone probably buys enough time so that AmB aggregates can be safely removed from circulation and monomers subsequently released (like liposomes). Fungizone micelles, on the other hand are unstable. The Amphotericin becomes mobile and remains in circulation longer at toxic levels Fungizone active AmB oligomer AmB channel w/ cholesterol or no sterol "Hot-Zone"

AmB monomer Engulfing and slow release from macrophages AmB/sterol channel w/ ergosterol A Happy Ending? The Applied Photophysics system has been used to measure activity, structure and stability of a new drug delivery system for Amphotericin B Hot-Zone is a cheap, easy-to-make and stable formulation of Amphotericin B from Fungizone In model membrane and animal systems, Hot-Zone is less toxic and equally effective An altered pattern of serum distribution and increased stability may also contribute to lower toxicity

A Happy Ending? A Happy Ending?

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