Amphotericin primarily kills yeast by simply binding ergosterol

Abstract

Amphotericin B (AmB) is a prototypical small molecule natural product that can form ion channels in living eukaryotic cells and has remained refractory to microbial resistance despite extensive clinical utilization in the treatment of life-threatening fungal infections for more than half a century. It is now widely accepted that AmB kills yeast primarily via channel-mediated membrane permeabilization. Enabled by the iterative cross-coupling-based synthesis of a functional group deficient derivative of this natural product, we have discovered that channel formation is not required for potent fungicidal activity. Alternatively, AmB primarily kills yeast by simply binding ergosterol, a lipid that is vital for many aspects of yeast cell physiology. Membrane permeabilization via channel formation represents a second complementary mechanism that further increases drug potency and the rate of yeast killing. Collectively, these findings (i) reveal that the binding of a physiologically important microbial lipid is a powerful and clinically validated antimicrobial strategy that may be inherently refractory to resistance, (ii) illuminate a more straightforward path to an improved therapeutic index for this clinically vital but also highly toxic antifungal agent, and (iii) suggest that the capacity for AmB to form protein-like ion channels might be separable from its cytocidal effects.

Fig. 1.

Chemistry and biology of polyene macrolides. (A) Molecular structures of the polyene macrolide natural products AmB, natamycin, and a series of their functional group-deficient derivatives prepared via chemical synthesis. (B) Representations of two mechanisms for the antifungal activity of AmB: ergosterol binding and membrane permeabilization. (C) Representations of the two leading models for the structure of the AmB ion channel. Both models predict that the hydroxyl group at C35 is critical for ion channel self-assembly. (D) Iterative cross-coupling, a small molecule synthesis strategy, analogous to iterative peptide coupling, in which bifunctional building blocks are sequentially linked using only one reaction recursively.

Fig. 2.

Synthesis of C35deOAmB via iterative cross-coupling. Inset shows three building blocks for the synthesis of C35deOAmB having all of the required functional groups preinstalled in the correct oxidation states and with the required stereochemical relationships. A single cross-coupling reaction was used in an iterative fashion to sequentially assemble BB₁, BB₂, and BB₃ with complete stereocontrol. Reagents and conditions are, as follows: a, pinacol, NaHCO₃, MeOH, 45 °C; b, BB₂, 10 mol % PdCl₂dppf, K₃PO₄, DMSO, 23 °C, 53% over two steps; c, BB₃, 5 mol % Pd(OAc)₂, 10 mol % XPhos, aqueous NaOH, THF, 45 °C; d, LiOH, THF : MeOH : H₂O, 35 °C; e, MNBA, DMAP, DCM, 23 °C, 56% over three steps; f, TBAF, THF, 23 °C; g, HF/pyridine, MeOH, 40 °C, 15% over two steps; h, HCl, MeCN : H₂O, 0 °C, 32%; and g, penicillin G amidase, H₂O, 37 °C, 28%. Bn, benzyl; DCM, dichloromethane; DMAP, 4-dimethylaminopyridine; dppf, 1,1′-bis(diphenylphosphino)ferrocene; MNBA, 2-methyl-6-nitrobenzoic acid; PA, phenylacyl; PMP, para-methoxyphenyl; Pyr, pyridine; TBAF, tetrabutylammonium fluoride; TBS, tert-butyldimethylsilyl; THF, tetrahydrofuran; TMSE, trimethylsilylethyl; XPhos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl.

Fig. 3.

Ergosterol binding and membrane-permeabilizing activities of polyene macrolides. (A) Total exotherm, as measured by ITC, for solutions of each polyene macrolide titrated with either sterol-free or 10% ergosterol-containing POPC LUVs. AmB, natamycin, and C35deOAmB all bind ergosterol, whereas AmdeB and natamycin aglycone do not. Values represent the mean of at least three experiments ± SD. * p ≤ 0.035; NS, not significant. (B, C) Potassium efflux from 10% ergosterol-containing POPC LUVs (B) and live S. cerevisiae cells (C) after treatment with polyene macrolides. Percent of potassium release was monitored via a potassium-sensitive electrode and expressed relative to the total potassium released upon the addition of Triton X-100 (B) or digitonin (C) at the end of the experiment. In both LUVs and live yeast cells, robust membrane permeabilization is observed with AmB, whereas AmdeB, natamycin, natamycin aglycone, and C35deOAmB are all inactive.

Fig. 4.

Antifungal activities of polyene macrolides. (A) MIC as measured by broth dilution. Relative to AmB against S. cerevisiae, C35deOAmB, which cannot permeabilize membranes but retains the capacity to bind ergosterol, is only six times less active. Also note that the MICs for C35deOAmB and natamycin are very similar. A similar series of results is observed with C. albicans. (B) Killing kinetics of S. cerevisiae (Left) and C. albicans (Right) cells exposed to various antifungal agents. In contrast to the fungistatic agent ketoconazole, both AmB and C35deOAmB are powerful fungicidal agents. All compounds were tested at four times their MICs. (C) The number of molecules of AmB and ergosterol, as determined by HPLC, per yeast cell employed in the broth dilution experiments. At the MICs for both S. cerevisiae and C. albicans there is ample AmB available for binding ergosterol and thereby killing the yeast cells. CFU, colony forming units. * p ≤ 0.0003.