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Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal

Nature volume 623, pages 1079–1085 (2023)Cite this article

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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 bilayers2,3,4,5,6. 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.

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Fig. 1: AmB sterol sponge clathrates similarly encapsulate Erg and Chol.
Fig. 2: Controlled destabilization of AmB–sterol clathrates results in Erg selectivity.
Fig. 3: Kinetics of Erg extraction enabling tuning of antifungal efficacy.
Fig. 4: In vivo tolerability and efficacy of AM-2-19_DP2K.

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Data availability

Data that support the findings of this study are available within the paper and its Supplementary Information. SSNMR source data are provided in BMRbig entry IDs bmrbig84 and BMRB 21102. Molecular dynamics simulation trajectories are provided for Extended Data Fig. 4c,d at https://usegalaxy.org/u/taras.pogorelov/h/amb-simulations-2023. Further requests can be directed to the corresponding authors. Source data are provided with this paper.

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Acknowledgements

We thank L. Zhu for assistance with the NMR experiment set up; J. G. Weers, A. Blake, S. Ekaputri and T. Tyrikos-Ergas for helpful discussions; Y. Gu for help with the mucormycosis animal studies; the SCS NMR Lab at University of Illinois Urbana-Champaign for technical support; and the Beckman Institute at University of Illinois Urbana-Champaign for the fellowship of A.M. This work was supported by US NIH grants 5R01-AI135812-04 and R35-GM118185 to M.D.B., R01-GM112845 and R01-GM123455 to C.M.R. and R01-AI063503 to A.S.I.. Studies carried out at the National Magnetic Resonance Facility at Madison were supported by NIH grant P4-GM136463. The Bruker 500-MHz NMR spectrometer was obtained with the financial support of the Roy J. Carver Charitable Trust, Muscatine, Iowa, USA. The work was also supported by Sfunga Therapeutics.

Author information

Author notes

  1. These authors contributed equally: Arun Maji, Corinne P. Soutar

Authors and Affiliations

  1. Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Arun Maji, Taras V. Pogorelov & Martin D. Burke

  2. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Arun Maji, Corinne P. Soutar, Jiabao Zhang, Agnieszka Lewandowska, Brice E. Uno, Su Yan, Yogesh Shelke, Ganesh Murhade, Evgeny Nimerovsky, Justin D. Lange, Jonnathan P. Marin-Toledo, Jordan T. Holler, Anuj Khandelwal, Anna M. SantaMaria, Taras V. Pogorelov & Martin D. Burke

  3. Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL, USA

    Arun Maji, Corinne P. Soutar, Jiabao Zhang, Agnieszka Lewandowska, Su Yan, Yogesh Shelke, Ganesh Murhade, Justin D. Lange, Yinghuan Lyu & Martin D. Burke

  4. Molecule Maker Lab, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Arun Maji & Collin G. Borcik

  5. Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA

    Corinne P. Soutar, Agnieszka Lewandowska & Chad M. Rienstra

  6. Department for NMR-Based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany

    Evgeny Nimerovsky

  7. National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI, USA

    Collin G. Borcik & Chad M. Rienstra

  8. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Andres S. Arango & Taras V. Pogorelov

  9. Department of Pathology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, USA

    Yinghuan Lyu

  10. Department of Veterinary Clinical Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Keith L. Bailey, Patrick J. Roady & Timothy M. Fan

  11. National Institute of Child Health and Human Development, Bethesda, MD, USA

    Anna M. SantaMaria

  12. Department of Medicine, Section of Infectious Disease, University of Wisconsin-Madison, Madison, WI, USA

    Hiram Sanchez & David R. Andes

  13. Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Praveen R. Juvvadi & William J. Steinbach

  14. Sfunga Therapeutics, Champaign, IL, USA

    Gina Johns, Ken Bartizal & Kieren A. Marr

  15. Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS, USA

    Michael J. Hageman & Joanna Krise

  16. Division of Infectious Diseases, The Lundquist Institute, Torrance, CA, USA

    Teclegiorgis Gebremariam, Eman G. Youssef & Ashraf S. Ibrahim

  17. Arkansas Children’s Research Institute, Little Rock, AR, USA

    William J. Steinbach

  18. David Geffen School of Medicine, UCLA, Los Angeles, CA, USA

    Ashraf S. Ibrahim

  19. Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Thomas F. Patterson

  20. Department of Pathology and Laboratory Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

    Nathan P. Wiederhold

  21. Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA

    David R. Andes

  22. School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Taras V. Pogorelov

  23. National Center for Supercomputing Applications, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Taras V. Pogorelov

  24. Computational Biomolecular Magnetic Resonance Core, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA

    Charles D. Schwieters

  25. Cancer Center at Illinois, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Timothy M. Fan & Martin D. Burke

  26. Morgridge Institute for Research, University of Wisconsin-Madison, Madison, WI, USA

    Chad M. Rienstra

  27. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Martin D. Burke

  28. Carle-Illinois College of Medicine, University of Illinois at Urbana-Champaign, Champaign, IL, USA

    Martin D. Burke

  29. Molecule Maker Lab Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA

    Martin D. Burke

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  1. Arun Maji
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A.M., C.P.S., C.M.R. and M.D.B. wrote the manuscript. C.P.S., A.L., E.N., C.D.S., C.G.B. and C.M.R. conducted SSNMR experiments and/or contributed to processing data and conducting calculations for structure determination. C.P.S., A.L., J.T.H. and S.Y. prepared labelled probes and samples for SSNMR. A.S.A. and T.V.P. carried out molecular dynamics simulations. B.E.U., A.K. and J.Z. synthesized and investigated the mechanism of C2′epiAmB. A.K., J.Z. and A.M.S. worked with AmB ureas. A.M. designed, synthesized and studied AmB amides and C2′epi-amides. A.M. and M.D.B. analysed and interpreted results. A.M., G.M., S.Y. and Y.S. scaled up the synthesis of AM-2-19. Y.S., A.M. and G.M. designed, synthesized and studied C35MeOAmB. J.D.L. carried out the in vitro efficacy and tolerability assay for the compounds. A.M., C.P.S. and J.P.M.-T. synthesized C2′deOAmB. Y.L. conducted C2′epiAmB stability and azole cotreatment studies. T.F.P. and N.P.W. studied the in vitro efficacy of the molecules. A.M., J.Z., K.L.B., P.J.R. and T.M.F. conducted the in vivo tolerability study in mice. H.S., D.R.A., P.R.J., W.J.S. and A.S.I. carried out the in vivo efficacy studies in mice. A.M. and M.D.B. designed and developed the DP2K formulation. J.K. and M.J.H. contributed to the further understanding of the formulation. T.G. conducted experiments related to mucormycosis studies. E.G.Y. carried out histopathological examination. A.S.I. designed and supervised the studies related to testing AM-2-19_DP2K in mucormycosis studies and edited the manuscript. K.A.M., K.B., G.J. and M.D.B. contributed to preclinical study design, data interpretation and presentation.

Corresponding authors

Correspondence to Chad M. Rienstra or Martin D. Burke.

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Competing interests

A.M., J.Z., S.Y., C.M.R. and M.D.B. are inventors on patents PCT/US20/45566, PCT/US20/45399, PCT/US 2021/45205, PCT/US2020/45387 and/or UIUC2022-022-01, submitted by University of Illinois Urbana-Champaign. M.D.B. and K.A.M. disclose consulting income and equity in Sfunga Therapeutics.

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Nature thanks Ian Seiple and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

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.

Source Data

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.

Source Data

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.

Source Data

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.

Source Data

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.

Source Data

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.

Source Data

Extended Data Table 1 SSNMR structure calculation statistics
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Maji, A., Soutar, C.P., Zhang, J. et al. Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal. Nature 623, 1079–1085 (2023). https://doi.org/10.1038/s41586-023-06710-4

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