Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Fungicidal amphotericin B sponges are assemblies of staggered asymmetric homodimers encasing large void volumes.

Amphotericin B (AmB) is a powerful but toxic fungicide that operates via enigmatic small molecule-small molecule interactions. This mechanism has challenged the frontiers of structural biology for half a century. We recently showed AmB primarily forms extramembranous aggregates that kill yeast by extracting ergosterol from membranes. Here, we report key structural features of these antifungal 'sponges' illuminated by high-resolution magic-angle spinning solid-state NMR, in concert with simulated annealing and molecular dynamics computations. The minimal unit of assembly is an asymmetric head-to-tail homodimer: one molecule adopts an all-trans C1-C13 motif, the other a C6-C7-gauche conformation. These homodimers are staggered in a clathrate-like lattice with large void volumes similar to the size of sterols. These results illuminate the atomistic interactions that underlie fungicidal assemblies of AmB and suggest this natural product may form biologically active clathrates that host sterol guests.

A modification of γ-encoded RN symmetry pulses for increasing the scaling factor and more accurate measurements of the strong heteronuclear dipolar couplings.

Symmetry based γ-encoded RNnν elements are broadly used in magic-angle spinning solid-state NMR experiments to achieve selective recoupling of the heteronuclear dipolar interactions. The recoupled dipolar couplings in such experiments are scaled by a factor, Ksc, which theoretically depends on the chosen symmetry numbers N, n, and ν. However, the maximum theoretical value of Ksc for γ-encoded RNnν pulses is limited to ~0.25, resulting in long RNnν experiment times. Also, the dependence of Ksc on the experimental parameters can result in systematic errors in the experimental determination of the dipolar couplings, especially at low and moderate MAS rates. In this manuscript, we investigate the use of MODifiEd RNnν symmetry (MODERNnν(ϕM)) pulses that increase the dipolar scaling factor by at least 1.45 fold compared to γ-encoded RNnν. The second advantage of MODERNnν(ϕM) pulses with respect to traditional RNnν pulses is the reduced influence of experimental parameters on Ksc, which allows for more accurate measurement of short-range distances. The robustness of MODERNnν(ϕM) is compared with γ-encoded R1423 symmetry pulses. The enhanced performance is demonstrated on two uniformly-13C-enriched samples, N-acetyl valine and the microcrystalline protein GB1, at a 31.111 kHz MAS rate.