Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal

Abstract

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 bilayers^2-6. 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 annually^7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids⁹.

Extended Data Fig. 1 ∣. Cholesterol binding primarily drives renal toxicity of AmB, not ion channel formation.

a, Synthesis of C35MeOAmB, a non-ion-channel forming antifungal probe starting from AmB. b, C35MeOAmB binds cholesterol during UV-Vis binding assay. c, C35MeOAmB does not permeabilize human red blood cells. Pre-complexation with cholesterol mitigates renal toxicity of both d, AmB and e, C35MeOAmB against human primary renal cells. Cholesterol (53 mg/g) in β-Me cyclodextrin (MβCD) obtained from Sigma Aldrich (C495; lot no SLCJ3255). Results are means ± SD (n = 3 biological replicates/time point). In d, all pairwise comparisons with corresponding (1:0) at each concentration were performed using two-way ANOVA with Tukey’s multiple comparison test; *P = 0.028, **P = 0.0086, **P = 0.0019, ***P = 0.0004, ***P = 0.0003, ****P < 0.0001. In e, all pairwise comparisons with corresponding (1:0) at each concentration were performed using two-way ANOVA with Tukey’s multiple comparison test; ***P = 0.0004, ****P < 0.0001.

Extended Data Fig. 2 ∣. AmB in one state binds to multiple sterols in AmB-sterol complexes.

a, UV-vis spectra of AmB (black) and AmB upon titration with increasing molar ratios of Erg (0.5, yellow, 1, green, 2, cyan, and 3, blue). The red shift and “finger”-like pattern at higher sterol ratios is consistent with the AmB polyenes separating to accommodate sterols. b, AmB:sterol stoichiometric ratios (average of three replicates) after three rounds of complex washing as described in Methods and a picture of AmB-sterol complexes undergoing sterol washing in chloroform. c, ¹³C-¹³C 2D 50 ms DARR spectrum of ¹³C-AmB:Chol showing AmB in one state. d, ¹³C-¹³C 2D SPC5 spectrum with one bond correlations in green and two-bond correlations in blue for a ¹³C-AmB:¹³C-Erg sample with Erg crosspeaks highlighted. The largest chemical shift difference observed for each Erg carbon is summarized graphically (SI Table 7). UV-Vis sterol binding interaction between AmB and sterols from ergosterol biosynthetic pathway e, lanosterol, f, zymosterol, and g, episterol.

Extended Data Fig. 3 ∣. Erg interacts with AmB through contacts between the sterol rings and conserved GPM motif.

a, A diagram summarizing AmB-Erg interactions shown in c, d, and e, highlighting ¹³C-¹³C and ¹⁹F-¹³C interactions between the Erg A and B rings and the conserved C11-C20 motif and mycosamine. b,¹³C detected ¹H-¹H polarization transfers from water to the ¹³C-AmB C13/C1’ signal for homogenized ¹³C-AmB (black line) and ¹³C-AmB:Erg complexes (blue lines). Each data point comes from one peak (n = 1). Error bars represent uncertainties from the signal-to-noise ratio of each spectrum. c, Dephasing curves (red stars for experimental data and black lines for simulated curves) from ¹³C{¹⁹F} frequency selective REDOR experiments performed on a ¹³C-AmB:C6F-Erg sample and corresponding distances calculated from the dipolar couplings. Data presented as mean +/− SD of n = 11, 5, and 10 technical replicates, respectively. Error bars indicate uncertainty from the spectrum signal-to-noise ratio. d, AmB chemical shift perturbations relative to Erg bound ¹³C-AmB for the apo AmB states, previously reported in Nat. Chem. Bio. 28, 972 (2021), and ¹³C-AmB bound to ¹³C-Erg, C6F-Erg, and Chol (SI Table 6). e, Overlay of ¹³C-¹³C 2D PAR spectra collected at 8.4 ms (blue), 12.6 ms (magenta), and 16.8 ms (cyan) mixing times obtained from a ¹³C-AmB:¹³C-Erg sample highlighting interactions between the two primary Erg ring states and AmB.

Extended Data Fig. 4 ∣. SSNMR AmB-Erg structure and water MD simulations.

a, An overlay of the AmB structures from the 10 lowest energy lattices with the average and standard deviation of the dihedral angle at the mycosamine attachment b, Overlay of AmB and one Erg from the minimal subunit for the 6 structures, taken from the 10 lowest energy lattices of the calculation, in which the 3β-hydroxyl oxygen is within 4 Å of the C2’ hydroxyl oxygen. Water interactions from MD simulations for Erg-bound AmB, c, and Erg-bound C2’epiAmB, d, sponges. left, overlays of observed water molecules within the sponges highlighting regions of high water propensity. Right, single models taken from the overlays on the left showing representative positions of persistent waters and distances (in Å) consistent with hydrogen bonding interactions. See Extended Data Table 1 for SSNMR structure statistics.

Extended Data Fig. 5 ∣. AmB ¹³C-¹³C Correlations in the AmB-Erg Complex.

a, A diagram summarizing AmB-AmB ¹³C-¹³C correlations observed in 500 ms CORD and 12.6 ms PAR spectra between AmB carbons with intramolecular distances >8.5 Å. Blue ovals represent sterol molecules. b, ¹³C-¹³C 2D 500 ms CORD and 12.6 ms PAR spectra obtained from ¹³C-AmB:C6F-Erg and ¹³C-AmB:¹³C-Erg samples, respectively, highlighting intermolecular AmB-AmB interactions.

Extended Data Fig. 6 ∣. Mechanistic probing of C2’epiAmB’s decreased potency.

a, Synthesis of C2’epiAmB starting from AmB. b, Evaluations of C2’epiAmB efficacy in disseminated candidiasis mice model infected with C. albicans SN250, 24 h post single IV dose (n = 3 mice/group). Both compounds were administered as 1:2 deoxycholate in D5W to improve aqueous solubility. All dose units are mg/kg. Pairwise comparisons with 24 h placebo group were performed using (one-way ANOVA with Tukey’s multiple comparison test; ***P = 0.0003; ****P < 0.0001. c, Structure and numbering system of skip labeled ¹³C-erg. Sterol sequestration mechanism of antifungal action is conserved in C2’epiAmB as probed in d, PRE experiments, showing a decrease in the PRE effects of resolved Erg resonances in the presence of C2’epiAmB, indicating a decrease in sterol proximity to doxyl-labeled lipids (samples 40:1 POPC/ ¹³C-Erg ± 5 mol% 16-DOXYL-PC; Data presented as mean +/− SD of 3 measurements. Error bars indicate uncertainty from the spectrum signal-to-noise ratio.) and decreased efficacy upon pre-complexation with ergosterol for both e, AmB (n = 3 biological replicates/conc.) and f, C2’epiAmB (n = 3 biological replicates/conc.). Both molecules have similar stability profiles in g, C. albicans SN250 (n = 4 biological replicates/conc.) and h, A. fumigatus 1163 cultures (for 8 μM AmB n = 3, for all other n = 4 biological replicates). Both molecules (3 μM) have similar ion channel forming ability and have similar time-delay between compound addition and efflux in i, wild-type C. albicans SN250 and j, instant ion-channel formation in protoplast. Improvement of C2’epiAmB efficacy upon cotreatment with erg biosynthetic inhibitors Ketoconazole and Posaconazole against k, l, A. fumigatus 1163 and m, n, A. fumigatus 91 reveals that the extraction rate-driven kinetic model of efficacy is conserved against moulds. o, Unlike C2’epiAmB, AmBMU, though better tolerated than AmB, retains cholesterol binding and p, causes kidney damage in mice (n = 4 mice/group). Pairwise statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparison test; *P = 0.0422; ***P = 0.0008, ***P = 0.0005, ***P = 0.001, ****P < 0.0001. Results are means ± SD.

Extended Data Fig. 7 ∣. Combining C2’epimerization and C16 amidation results in a renal sparing potent antifungal.

a. in vitro efficacy of potent AmB amides. Head-to head comparison of in vitro toxicity of AmB, C2’epiAmB, AM-243-2 and AM-2-19 against b, H9C2 cells (rat cardiomyocyte; n = 3 biological replicates/conc.) c, Hep-G2 cells (human liver cell; n = 3 biological replicates/conc.) d, K562 (human red blood cells progenitors; n = 3 biological replicates/conc.) e, SHSY-5Y (human neural blastoma; n = 3 biological replicates/conc.) and f, RPTEC (primary renal proximal tubule epithelial cells; n = 3 biological replicates/conc.). C2’epiAmB and AM-2-19 both g, do not lyse human red blood cells (n = 3 biological replicate/conc.), and h, retain an AmB-like drug-drug interaction profile and i, do not elevate kidney injury biomarkers after 24 h of single IV 45 mg/kg dose (n = 4 mice/group). Pairwise statistical analyses were performed using two-way ANOVA with Tukey’s multiple comparison test; **P = 0.0022, ****P < 0.0001. AM-2-19 binds j, ergosterol but not k, cholesterol in the UV-Vis binding assay and is l, highly efficacious against pathogens that were resistant to C2’epiAmB (100% inhibition reported). m, Comparison of solution stability between AM-2-19 and C2’deOAmB in PBS buffer at pH 6. Pairwise two-way ANOVA with Tukey’s multiple comparison tests was performed at each time point; ***P = 0.0005, ****P < 0.0001. AM-2-19 does not bind n, lanosterol, o, zymosterol, and shows little to no binding with p, episterol. q, Head-to-head in vitro efficacy comparison between AM-2-19, C2’deOAmB, Natamycin and Nystatin. Data in j and k are representative of at least 3 independent experiments.

Extended Data Fig. 8 ∣. AM-2-19_DP2K is highly efficacious and resistance evasive.

a, Representative examples of several drug-resistant strains that are susceptible to AM-2-19_DP2K treatment, unlike its comparators. The study was conducted at UT Health, San Antonio, and 100% inhibition was reported. b, AM-2-19_DP2K is highly efficacious against hundreds of clinical fungal isolates tested at different locations (number of isolates in parenthesis; 100% inhibition reported). For AM-2-19_DP2K and comparators, dosing concentrations (μM and μg/ml) are reported based on the active pharmaceutical ingredient (API). The MIC values of DSG-PEG 2000 (DP2K), tested at UIUC, were >16 μM against all isolates. AmB or AmBisome^® is used as a comparator because, unlike AM-2-19, AmB does not form a homogenous solution with DSG-PEG 2000. c, in vivo efficacy of AM-2-19_DP2K and comparator AmBisome^® was evaluated in male CD-1 non-neutropenic disseminated candidiasis model infected with C. albicans SC5314 after 7 days of daily IV dosing (n = 6 mice/group). Pairwise statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparison test; ***P = 0.0008, ****p < 0.0001. Dose dependent mitigation of fungal burden on day 4 in neutropenic mucormycosis model, infected with R. delemar (first dose 48 h post infection; q24h; n = 10 mice/group) was measured by qPCR for d, lung (one-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001) and e, brain tissues (one-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001). f, Gross pathology, and histological examination of lung tissues harvested from mice infected with R. delemar, and treated with placebo, AM-2-19_DP2K or AmBisome^®. Notice the diffused filamentation in the lung from placebo or 0.3 mg/kg of AM-2-19_DP2K treated mice. Fewer/no hyphae are evident in lungs of mice treated with AM-2-19_DP2K 1.5, 7.5 and 30 mg/kg or AmBisome^®. Arrows in upper panel refer to fungal abscesses. Scale bar 100 μm for histopathological analysis (bottom). Tissue fungal burden of g, lungs (one-way ANOVA with Tukey’s multiple comparison test; *P = 0.0147, ****P < 0.0001) and h, brain (one-way ANOVA with Tukey’s multiple comparison test; *p = 0.0243, ****P < 0.0001) of mice (n = 10 mice/group) infected with M. circinelloides, treated with either drug and euthanized on Day +4 post infection. Spontaneous mutation frequency study indicates AmBisome-like resistance-refractory property of AM-2-19_DP2K against i, C. tropicalis ATCC 90874, j, C. albicans ATCC90028 k, C. glabrata ATCC 90030 and l, C. krusei ATCC 6258 after 20 passage and MIC recorded after every three passages. All dosing units are mg/kg. In mice, fungal burdens below the limit of detection (LOD) were given a nominal value CFU/g = 1. Results are means ± SD. In mice, fungal burdens below the limit of detection (LOD) were given a nominal value of CFU/g = 1.

Fig. 1 ∣. AmB sterol sponge clathrates similarly encapsulate Erg and Chol.

a, SSNMR structure of the AmB–Erg complex, computed with Xplor-NIH simulated annealing, with expansions highlighting interactions between the C6 region of Erg and C13, and polyene and mycosamine regions of AmB observed in ¹³C{¹⁹F} REDOR experiments. b, Molecular structures of AmB, Erg and Chol. c, ¹³C¹³C 2D spectra of homogenized ¹³C[AmB] (black cross-peaks) and ¹³C[AmB]–Erg complexes prepared with AmB/Erg molar ratios of 1:0.5 (cyan peak) and 1:3 (blue peak) showing the C1–C2 cross-peak collapsing from two peaks to one peak following sterol titration. U-¹³C, uniformly ¹³C labelled. d, ¹³C¹³C 2D 50-ms dipolar assisted rotational resonance (DARR) overlay of ¹³C[AmB]–Chol (red) and ¹³C[AmB]–Erg (blue) showing AmB in the same, single state with each sterol. e,¹³C{¹⁹F} REDOR broadband difference spectrum (ΔS = S₀ – S) for U-¹³C[AmB]–C6F[Erg] at 3.84-ms dephasing showing intensity from ¹³C[AmB] signals in close proximity to the ¹⁹F label at the Erg C6 position. f,¹³C-detected ¹H¹H polarization transfers from water to the ¹³C[AmB] polyene signal for homogenized ¹³C[AmB] (black line) and the ¹³C[AmB]–Erg complex (blue lines). Each data point comes from one peak (n = 1). Error bars represent uncertainties from the signal-to-noise ratio of each spectrum. Norm., normalized. g,h, UV–Vis spectra of AmB following titration with increasing molar ratios (0, 0.5, 1, 2 and 3) of Erg (g) and Chol (h). Data in g,h are representative of at least three biological replicates; a.u., arbitrary units.

Fig. 2 ∣. Controlled destabilization of AmB–sterol clathrates results in Erg selectivity.

a, Comparison of apo-AmB and sterol-bound AmB clathrate structures highlighting changes in the mycosamine orientation, with the inset showing chemical shift perturbations relative to the apo state A. b, Expansion of the sterol-bound complex showing proximity of the sterol 3β-OH to the C2′OH and disruption of this interaction following in silico epimerization of the C2′centre. c, ITC assays comparing C2′epiAmB and AmB binding affinity to sterol-free, Erg-containing and Chol-containing large unilamellar vesicles (LUVs; n = 3). (AmB–Erg binding data reported in ref. .) Pairwise statistical analysis by two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test; *P = 0.0245, **P = 0.0019, ****P < 0.0001, **P = 0.0022, **P = 0.0089. NS, not significant. d,e, UV–Vis spectra of C2′epiAmB following titration with increasing molar ratios (0, 0.5, 1, 2 and 3) of Erg (d) and Chol (e); a.u., arbitrary units. f, C2′epiAmB does not kill human primary renal cells in vitro up to 50 μM concentration (n = 3). g, C2′epiAmB does not elevate kidney damage biomarkers in female CD-1 mice measured 24 h post single IV dosage (n = 4 per group). Pairwise statistical analyses by two-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001. h, Heatmap representation of kidney histopathological scores (A, mixed-cell infiltrate, interstitial; B, tubular basophilia, cortex, medulla; C, Tubular cellular casts, cortex; D, tubular cellular casts, medulla; E, tubular degeneration and necrosis, cortex; F, tubular degeneration and necrosis, medulla; G, tubular dilatation, cortex; H, tubular protein casts, cortex; I, tubular protein casts, medulla; J, vascular congestion, medulla) in mice 24 h post single IV dosage of AmB and C2′epiAmB (n = 4 per group). Both AmB and C2′epiAmB were formulated as 1:2 deoxycholate to improve aqueous solubility. i, Comparison of AmB and C2′epiAmB MICs. Results are means ± s.d. Data presented in d,e are all representative of at least three independent experiments.

Fig. 3 ∣. Kinetics of Erg extraction enabling tuning of antifungal efficacy.

a, Competitive kinetics model of sterol encapsulation for AmB analogues. b, C2′epiAmB extracts Erg more slowly than AmB from C. albicans SN250 at 5 μM (n = 4 for 0.167 and 0.5 h and n = 3 for 1.0 and 2.0 h time points). Pairwise comparison with AmB at each time point using two-way ANOVA with Tukey’s multiple comparison test. c, Cotreatment of C. albicans SN250 with the Erg biosynthesis inhibitor ketoconazole promotes faster killing and restores potency for C2′epiAmB. CFUs, colony-forming units. d, At 5 μM, hyper potent AmB amides extract Erg more quickly than the parent AmB (n = 3 per time point) from C. albicans SN250. Pairwise comparison with AmB at each time point using two-way ANOVA with Tukey’s multiple comparison test; **P = 0.0064, ****P < 0.0001. e, Kinetics versus efficacy for C. albicans SN250, showing T_50% (time required for 50% Erg extraction) versus observed MIC. f, Rate of cell killing at 4 μM (n = 2 per sample per time point) for C. albicans SN250. g, ITC data showing retained Chol binding for AM-243-2 and selectivity for Erg over Chol for AM-2-19 (n = 3). Pairwise comparison using two-way ANOVA with Tukey’s multiple comparison test; **P = 0.0012, **P = 0.0038, ****P < 0.0001. h, The hybrid design strategy combines the renal-sparing nature of C2′ epimerization and potency-promoting C41 amidation in AM-2-19. i, AM-2-19 extracts Erg more quickly than C2′epiAmB (5 μM; n = 3 per time point) from C. albicans SN250. Pairwise comparison with C2′epiAmB at each time point using two-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001. j, AM-2-19 kills C. albicans SN250 more quickly than C2′epiAmB (4 μM). Results are means ± s.d. For c,f,j a nominal value of 20 CFUs ml⁻¹ was assigned on the basis of the dilution factor for plates with no fungal growth.

Fig. 4 ∣. In vivo tolerability and efficacy of AM-2-19_DP2K.

a, Histopathological analysis of kidneys collected 24 h post single IV dosing from mice treated with AmBisome (n = 3 per group) or AM-2-19_DP2K (n = 4 per group) (A, mixed cell infiltrate, interstitial; B, tubular basophilia, cortex; C, tubular cellular casts, cortex; D, tubular cellular casts, medulla; E, tubular degeneration and necrosis, cortex; F, tubular degeneration and necrosis, medulla; G, tubular dilatation, cortex; H, tubular protein casts, cortex; I, tubular protein casts, medulla; J, vascular congestion, medulla). b, Representative examples of in vitro efficacy of AM-2-19_DP2K and the comparator AmBisome tested at UT Health San Antonio (number of isolates in parenthesis; 100% inhibition reported). c, In vivo efficacy of AM-2-19_DP2K and the comparator AmBisome in a neutropenic disseminated candidiasis mouse model infected with C. glabrata ATCC2001 after 5 days of daily IV dosing (n = 6 per group). LOD, limit of detection. One-way ANOVA with Tukey’s multiple comparison test; ***P = 0.0008, ****P < 0.0001, **P = 0.0036. d, Efficacy of AM-2-19_DP2K and the comparator AmBisome in a neutropenic disseminated aspergillosis mouse model infected with A. fumigatus 1163 after 4 days of daily IV dosing (n = 6 per group). One-way ANOVA with Tukey’s multiple comparison test; ****P < 0.0001. e, Neutropenic invasive pulmonary aspergillosis mouse model infected with A. fumigatus ATCC 204305 after 4 days of daily IV dosing (n = 8 per group). One-way ANOVA with Tukey’s multiple comparison test; *P = 0.0117, **P = 0.0032, ****P < 0.0001. f, Neutropenic disseminated aspergillosis mouse model infected with A. terreus 49 after 10 days of daily IV dosing (n = 10 per group). One-way ANOVA with Tukey’s multiple comparison test; ***P = 0.0008, ****P < 0.0001. g,h, Survival curves of a neutropenic invasive pulmonary mucormycosis mouse model infected with R. delemar (n = 10 per group; g) and Mucor circinelloides (n = 10 per group; h) after 4 days of daily IV dose. Pairwise comparisons with the placebo using a log-rank (Mantel–Cox) test. Dosing units are milligrams per kilogram. Results are means ± s.d. Fungal burdens below the limit of detection were given a nominal value of 1 CFU g⁻¹. Concentrations (a–h) listed reflect the mass of active ingredients.