AMPHOTERICIN B DELAYS AMYLOID FIBRILLIZATION

1. 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. ABSTRACT 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. Physical Blockage of Fibril Growth* Amyloid Plaque Stimulation of Monocytes to Release Cytokines Specific Immune response like Alzheimer’s Vaccine? • Conclusions and Significance: • 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. • 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. • 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. • Results: • 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. • 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 • 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. • 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. Amphotericin binds to insulin and Aβ 25-35 amyloid Fibrils Figure 1 Introduction 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. Insulin Fibrils AB fibrils Figure 4 Congo Red Amphotericin B 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 *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