Monday, August 17, 2015
The Sterol-Binding Fungicidal Mechanism of Action of Amphotericin B
Amphotericin B (AmB) is a chemically complex polyene-macrolide natural product that has been used continuously since the 1960s as a last line of defense against systemic fungal infections. Unfortunately, AmB is plagued by an extrememly narrow therapeutic index. Indeed, because of the often dose-limiting toxicity of the antifungal drug, mortality rates for systemic fungal infections still hover near 50%.
Martin Burke’s laboratory at the University of Illinois at Urbana-Champaign has taken a keen interest in the fungicidal mode of action of AmB, which has managed to evade significant microbial resistance for over 50 years. A better understanding of the precise nature of AmB’s ‘resistance-refractory’ mechanism might lead to new antimicrobial agents with reduced toxicity. The prevailing model used to interpret biophysical studies of AmB’s role within living systems is the ‘artificial ion channel model.’ According to this theory, AmB exists in the form of small ion channel aggregates that are inserted into lipid bilayers with membrane sterols oriented in between vertically-stacked molecules of AmB, arranged in a circular pore or cavity (see Figure below, top left). The artificial channels are thought to permeabilize and ultimately kill cells. The ion channel model dictates that the strategic pathway to improving therapeutic index is by achieving selective formation of ion channels in yeast over human cells.
Burke has recently advanced an alternate mechanistic scenario to explain AmB's potent fungicidal properties. In the 'sterol sponge model' (Figure above, top right), AmB forms large extramembranous aggregates that extract the essential sterol ergosterol from phospholipid bilayers. The coincidental extraction of membrane cholesterol (unselectively with ergosterol) might then be primarily responsible the observed toxicity of AmB to human cells. This insight, if proven correct, could guide the development of novel AmB derivatives with an improved therapeutic index. With regard to evasion of resistance, AmB may simultaneously disrupt all of the cellular processes that depend on membrane ergosterol. Simultaneous mutation of all requisite fungal proteins in order to alleviate the ergosterol dependence of the pathogenic organism is then improbable and, hence, no significant resistance emerges.
In 2014, Burke’s group presented evidence from biophysical, cell-based experiments that uniformly supports the new sterol sponge model. As such, an improvement in the relative binding affinity of AmB aggregates for ergosterol over cholesterol was expected to translate to antimicrobial agents with lower toxicity towards human cells. The Illinois team approached this challenge by initially examining a recent crystal structure of an AmB derivative that reveals an intramolecular salt bridge between the C16 carboxylate and the C3’ ammonium moiety of AmB’s mycosamine subunit (See AmB molecular structure, depicted above). They hypothesized that a complex network of intramolecular noncovalent interactions, including the aforementioned salt bridge, orients the mycosamine in a conformation that is capable of forming an H-bond to the 3b-hydroxyl of both ergosterol and cholesterol (diagrammed below). The mycosamine C2’-hydroxyl was implicated in this network of interactions by previous studies which demonstrated that deletion of the C2’-hydroxyl group (in semisynthetic C2’deOAmB) leads to differential binding of ergosterol and cholesterol. A high-resolution structure of the AmB aggregate (the functional ‘sponge’), with and without bound sterol, would enable rational design of new synthetic molecules. However, in its absence, a practical and highly modular semisynthetic route to derivatize the ‘eastern’ mycosamine-bearing substructural framework of AmB was sought. Newly generated analogs would then be evaluated for sterol binding selectivity and correlation to selective toxicity to yeast.
A remarkably concise strategy for AmB derivatization was developed by Burke and co-workers. The route (shown below) involves only three reaction ‘pots’ and requires one chromatographic separation to produce pure material on gram-scale. The exceptionally mild oxazolidinone ring-opening by amine nucleophiles is noteworthy. The facile nature of this transformation, which introduces a new point of diversity for SAR studies, likely reflects inherent ring-strain residing within the trans-fused 6/5 heterocyclic ring system of the bracketed intermediate. The tetrahydropyran methyl ketal of the penultimate species is converted into its corresponding hemiacetal during the course of HPLC purification, which uses aqueous formic acid in the mobile phase. An ultracentrifugation-based membrane isolation assay indicated that the new C16-urea derivatives exhibit binding selectivity for ergosterol over cholesterol. Moreover, the sterol binding selectivity was generally associated with an improvement in therapeutic index, as indicated by a comparison of the minimum inhibitory concentration (MIC) against S. cerevisiae with the minimum hemolytic concentration (MHC) determined against human red blood cells. This project is a great example of a practical and well-executed organic chemistry strategy, applied to a long-standing pharmacological problem with profound human clinical implications. The emerging AmB mechanistic picture is an example of a rarely encountered pharmacological phenomenon – that is, noncovalent binding between a small molecule drug and an endogenous small molecule. The seemingly ‘resistance-refractory’ nature of this type of mechanism renders it a highly desirable platform for pharmacological intervention aimed at anti-infective drug development with low tox and evasion of resistance. The work also illuminates to the ubiquity and essential nature of sterols such as cholesterol and ergosterol to living systems within the cells of both humans as well as pathogenic organisms.