Hartselab Research-Past and Present

Amphotericin B is an antifungal and potentially anti-AIDS drug used to treat nasty fungal infections frequently seen in AIDS, chemotherapy and transplant patients. 
Health professionals ruefully refer to this highly toxic yet very effective drug as "amphoterrible."  In the past, the usual model for the mechanism of action of Amphotericin has invoked a "barrel stave" organization of Amphotericin + sterol molecules forming a fungus-killing membrane pore. Our research (1,2,3) has proven conclusively that specific cation selective pores do not need sterol to form. To paraphrase Huxley, this was an example of the "Tragedy of  Science: a beautiful Hypothesis slain by an an ugly little Fact." Some researchers have protested our iconoclasm, but the results have been confirmed again and again. In addition we have shown that pores formed in the presence of the fungal sterol ergosterol are structurally and  functionally different (4). More recently, we have explored new, simple (and practical!) ways of reducing the toxicity of this drug (5,6). This heat-treatment method of reducing toxicity has recently been proposed as a practical, inexpensive way to treat serious fungal diseases in Thrird World contries (7). It's nice when something we do here in Eau Claire might actually have global benefits! For more on fungal infections and treatments (including cost analyses), see my favorite web site, Dr. Fungus!

The longest ongoing project in our lab is to tease apart which specific properties of Amphotericin B pharmaceutical preparations make them less toxic (and more effective) in hopes of finding a simpler way to increase the drug's therapeutic index. We are specifically interested in how Liposomal and other supramolecular formulations of Amphotericin B alter toxicity and channel formation properties of this drug.  We use stopped-flow fluorescence, absorbance and CD methods to probe the structure and activity of Amphotericin preparations. In addition we are beginning studies aimed at characterizing the biological response of  monocytes to stimulation by different formulae. We propose that there are essentially three factors which influence the toxicity and efficacy of AmB preparations: 1) direct membrane toxicity via ion channel formation, 2) differences in distribution and delivery to tissues due to differences in serum lipoprotein/protein binding, and 3) initiation of an inflammatory cytokine response. 

Another interetsing feature of Amphotericin B (AmB) is that it is one of the few agents shown to slow the course of prion diseases (like chronic wasting disease and mad cow disease) in animals. Prions and amyloid diseases like Alzheimer's have many features in common.   
Chiefly, both diseases involve protein misfolding events which can induce other proteins to misfold into largely beta-sheet fibrillar or oligomeric isoforms (see EM photo of fibrils). These structures seem to be the key to the disease pathogenesis by various proposed mechanisms. Congo Red is a dye that has been reported to directly inhibit amyloidogenesis in both prion and Alzheimer peptide model systems by specific binding. This binding affinity has long been used as a spectroscopic marker for amyloid protein. We propose it is possible that AmB may act similarly to physically prevent fibril formation in prion disease. To assess whether AmB is capable of binding specifically to amyloid fibrils as does Congo Red, we have used the insulin fibril and amyloid precursor protein peptide (from Alzheimers) amyloid model system.  In addition, AmB interacts specifically with Congo Red, a known fibril-binding agent. In kinetic fibril formation studies, AmB was able to significantly kinetically delay the formation of Abeta 25-35 fibrils and their final extent at pH 7.4 but not insulin fibrils at pH 2 at near therapeutic AmB levels.The polyol region of AmB suggests that it could be an effective hydrogen bond donor/acceptor and may be able to terminate and/or stabilize the beta-sheet structure of a growing amyloid fibril. We are interested in physically explaining AmB's unique actvitiy in the hopes that it could lead to therapeutic strategies for both prion and amyloid diseases(8). 

More recently, Dr.Turtinen and I have collaborated  on the molecular mechanism of AmB's ability to induce a cytokine response and how drug delivery systems can modify this (9). We have found that there are essentially two categories of AmB drug delivery preparations; those that stimulate TNF-a and IL-6 in monocytes and those that do not. We use new chemiluminescent antibody-array detection (see illustration). Why would anyone care? Well, TNF and IL-6 both stimulate HIV replication. So if you are prescribing antifungal medication to an AIDS patient with serious fungal disease, which would you choose, the one that may increase viral load or the one that does not? Another possibly practical benefit of our research! How is it that diferent liposomal forms produce different responses? It is likely some combination of AmB-induced membrane potential changes, specific ion currents, calcium fluxes or TLR receptor activation. That is a subject of ongoing research. 
 In another collaboration wiuth Dr. David Lewis at UW-EC, we are trying to develop new fluoresccent probes and fluorescent tags for Amphotericin. We are currently comparing a naphthalamide probe which can distribute into acidic organelles like lysosomes and the Golgi apparatus. It functions much the the commercial Lysotracker^tm probes from Molecular Probes. Hence we refer to this probe as InstantLyso.

Images of InstantLyso and other dyes.  An epifluorescence image of InstantLyso with live fibroblasts at 75 nM and excited with blue filter light (~460-490 nm WIB set). Notice the clearly visible Golgi apparatus which is particularly targeted in this cell line.

We have also developed fluorescent stains for cholesterol rich domains (InstantLipo) which may also effecitvel stain so-called lipid "rafts" and pathological cholesterol inclusions. This probe may be useful for diagnosing cholesterol and other lipid disorders.

 Most interesting are the totally novel mitochondrial stains developed here at UW-EC. A photo of the filamentous mtiochondria in fibroblasts is shown below. All photos were imaged in the fluorescence microscope facility at UW-Eau Claire. 

A recent very exciting area of research has come from collaboration with Alan Dispirito of Iowa State University. We are studying a peptide, Methanobactin, that strongly binds and reduces metals in the environment, especially copper (see these papers, 10 and  11 for more detail). It can also reduce Au(III) to gold nanoparticles. This molecule could have major impacts on weathering of minerals and mobilization of toxic metals in the environment.  We are beginning 1 and 2D NMR studies on the structure of this peptide-like molecule.