In vitro GT glycosylation assays
For GT assays with TDP glucose; GT enzyme (5–20 µM), TDP-glucose (2 mM), MgCl2 (1 mM) and polyene substrates, including amphotericin B (AmB), AmB-aglycone, nystatin A1 (NysA1) and mycoheptin and dihydromycoheptin, at 100 µM concentration were incubated in a total reaction volume of 50 µl in Tris-HCl buffer (50 mM, pH 7.4) at 30 °C for 24 h. For TDP sugars other than TDP glucose with available sugar-1-phosphates, an extra step was required in which the TDP sugar was produced from the corresponding sugar-1-phosphate. In this first step, deoxythymidine triphosphate (2 mM), sugar-1-phosphate (2 mM), MgCl2 (2.2 mM) and Cps2L (50 µM) in a total volume of 50 µl in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4) were incubated for 24 h at 37 °C in a shaking thermomixer. After 24 h, the temperature was adjusted to 30 °C and GT (5–20 µM) and polyene (100 µM) were added. Assays were then incubated for a further 24 h at 30 °C. For TDP sugars with no available sugar-1-phosphate, TDP sugars were produced from the corresponding sugar. In this modified first step, deoxythymidine triphosphate (2 mM), ATP (2 mM), sugar (2 mM), MgCl2 (2.2 mM), NahK_ATCC15697 or GalkSpe4 (50 µM) and Cps2L (50 µM) in a total volume of 50 µl in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4) was incubated for 24 h at 37 °C in a shaking thermomixer. After 24 h, the temperature was adjusted to 30 °C and GT (5–20 µM) and polyene (100 µM) were added. Assays were then incubated for a further 24 h at 30 °C.
For all assays (1, 2 or 3 steps), reactions were quenched by heating to 95 °C for 5 min, followed by the addition of 1 reaction volume of methanol. The protein was then pelleted by centrifugation, and the supernatant was analysed by analytical RP-HPLC and LC–HRMS. Assays were analysed on a Shimadzu Analytical ultrahigh-performance liquid chromatography (UHPLC) apparatus with a Kinetex 5 μm XB-C18 100 × 4.6 mm (Phenomenex) column, with a flow rate of 1 ml min−1, using a solvent system of water and methanol with 0.1% formic acid and a gradient of 60–78% methanol over 12 min. Heptaenes were monitored at 405 nm and pentaenes at 350 nm. LC–HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility quadrupole time of flight (Q-TOF) LC–MS apparatus and 1290 Infinity II HPLC coupled to a 6546 LC–Q-TOF (Agilent Instruments), using a Luna Omega 5 μm 100 × 2.1 mm (Phenomenex) column, with a flow rate of 0.5 ml min−1, using a solvent system of water and methanol and a gradient of 60–95% methanol over 15 min. On LC chromatograms, tetraenes were monitored at 304 nm, pentaenes at 350 nm and heptaenes at 405 nm.
GT-catalysed reverse glycosylation of polyenes
Each of the polyenes (25–100 μM, kasufungin B, NysA3, semipurified selvamicin) were incubated with TDP (2 mM), MgCl2 (1 mM), GT (KfuSV, NysSV, SelSV, 25 μM) in 50 mM Tris-HCl buffer pH 7.5–8, 30 °C overnight, in a 50 μl reaction. The reactions were quenched with methanol (50 μl) and centrifuged (10,000 rpm, 5 min). The supernatant was analysed by HPLC and masses were confirmed by LC–MS using the same conditions as mentioned above in the in vitro GT glycosylation assays section.
MycS3 assay with dihydromycoheptin and TDP-l-digitoxose
Reverse glycosylation of NysA3 was performed to generate TDP-l-digitoxose. A 200–300 μM solution of NysA3 was incubated with TDP (2 mM), MgCl2 (1 mM) and KfuSV (50 μM) in 50 mM Tris-HCl buffer pH 7.5–8, at 30 °C overnight, in a 2-ml reaction. The reactions were quenched with an equal volume of methanol to inactivate enzyme and centrifuged (10,000 rpm, 5 min). The supernatant was concentrated under vacuum to reduce the volume to one-quarter, applied to silica filled in a small syringe (silica C18 100 Å, 30 μm, silica bed 4 ml) and washed twice with an equal volume of H2O to elute TDP-l-digitoxose. The filtrate volume was reduced to one-fifth under vacuum and used for MycS3 assays.
For assays with MycS3, a solution of dihydromycoheptin (50 µM) in MycS3 reaction buffer (1 mM MgCl2, 50 mM Tris-HCl, pH 7.5) was incubated with the TDP-l-digitoxose containing concentrated filtrate (25 μl) and MycS3 (25 μM) at 30 °C overnight in 50-μl reactions. The reactions were quenched with methanol (50 μl) and centrifuged (10,000 rpm, 5 min) before the supernatant was analysed by HPLC and LC–MS. In separate experiments, dried S. netropsis (DSM40846) methanolic extract containing crude dihydromycoheptin, was resuspended in MycS3 reaction buffer (1 mM MgCl2, 50 mM Tris-HCl, pH 7.5) was incubated with the TDP-l-digitoxose containing concentrated filtrate (25 μl) and MycS3 (25 μM) at 30 °C overnight in 50-μl reactions. The reactions were quenched with methanol (50 μl) and centrifuged (10,000 rpm, 5 min) before the supernatant was analysed by LC–HRMS. HPLC analysis was performed on a Shimadzu Analytical UHPLC with a Kinetex 5 μm XB-C18 100 × 4.6 mm (Phenomenex) column, with a flow rate of 1 ml min−1, using a solvent system of water and methanol (+0.1% formic acid) and a gradient of 60–78% methanol over 12 min, with UV monitored at 350 nm. LC–HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility Q-TOF LC–MS and 1290 Infinity II HPLC coupled to a 6546 LC/Q-TOF (Agilent Instruments), using a Luna Omega 5 μm 100–2.1 mm (Phenomenex) column, with a flow rate of 0.5 ml min−1, using a solvent system of water and methanol and a gradient of 60–95% methanol over 15 min.
In vitro PcsA reaction conditions
Enzyme activity assays were carried out in a reaction containing 100 µM polyene (pimaricin, rimocidin, CE-108, NysA1, NysA3, mycoheptin, dihydromycoheptin, kasufungin B or candicidin) and l-glutamine, l-glutamic acid γ-hydroxamate or l-glutamic acid γ-hydrazide (2 mM), ATP (4 mM), MgCl2 (10 mM) and 25 µl of eluted protein in Tris buffer (125 mM Tris and 25 mM NaCl, pH 7) in a total volume of 50 µl. In addition, l-glutamine analogues (l-glutamic acid γ-methylamide, l-glutamic acid γ-ethylamide (l-theanine), l-glutamic acid γ-methyl ester and l-glutamic acid γ-ethyl ester) were also tested but no activity was observed. The reactions were quenched with methanol (50 µl) after overnight incubation at 30 °C, and centrifuged (10,000 rpm for 5 min). Assays were analysed on a Shimadzu Analytical UHPLC with a Kinetex 5 μm XB-C18 100 × 4.6 mm (Phenomenex) column, using 1 ml min−1 flow rate with a solvent system of water and methanol each containing 0.1% formic acid, using a gradient of 60–78% methanol over 12 min. LC–HRMS was performed on an Agilent 1290 Infinity II coupled to a 6560 Ion Mobility Q-TOF LC–MS instrument, using a Luna Omega 5 μm 100 × 2.1 mm (Phenomenex) column, using a 0.5 ml min−1 flow rate with a solvent system of water and methanol each containing 0.1% formic acid, and a gradient of 60–80% methanol over 15 min.
Expression of S. albulus nysSV-subcluster in S. albus
The sugar subcluster from S. albulus (DSM40492), Sa-nysSV-sub, was amplified from the genomic DNA as two fragments; fragment 1 amplifying nysSVI, nysSII, nysSV, partial nysSVII and fragment 2 amplifying partial nysSVII, nysSIII and nysSIV using primer pairs (Supplementary Table 3). The plasmid pSET152-ermE* was linearized with NdeI and EcoRI, and the two fragments were assembled using HiFi assembly to form the construct pSET-ermE*-Sa-nysSV-sub. The construct was introduced into S. albus (J1074, a gift from M. Bibb at the John Innes Centre) by E. coli (ET12567) mediated conjugal transfer using standard conjugation protocols52, resulting in the S. albus::Sa-nysSV-sub strain. The empty plasmid pSET-ermE* was also introduced by conjugation resulting in S. albus::pSET-ermE* strain to be used as the negative control.
For NysA1 assays with the lysate, both S. albus::Sa-nysSV-sub and S. albus::pSET-ermE* strains were grown in tryptone soy broth (TSB) for 3 days. The resulting seed cultures (2% v/v) were used to inoculate 10 ml of SG2 growth medium (glucose 20 g l−1, yeast extract 5 g l−1 and soytone 10 g l−1, pH 7.2). After 48 h, 1 ml of each culture was centrifuged at 12,000 rpm for 10 min. The cell pellet was resuspended in lysis buffer at four times the pellet weight (50 mM Tris Cl, 150 mM NaCl, 10% glycerol, 2 mM MgCl2, pH 7.5), followed by addition of 4–6 mg ml−1 of lysozyme. The resulting cell suspension was incubated at 30 °C for 30 min, vortexed and centrifuged at 12,000 rpm for 20 min. A 100-µl portion of the supernatant was incubated with 100 µM of NysA1 at 30 °C overnight. The reactions were quenched with equal volumes of methanol and centrifuged (10,000 rpm for 10 min). Assays were analysed on a Shimadzu Analytical UHPLC with a Kinetex 5-μm XB-C18 100 × 4.6 mm (Phenomenex) column, using 1 ml min−1 flow rate with a solvent system of water and methanol each containing 0.1% formic acid, using a gradient of 60–78% methanol over 12 min. On LC, the conversion of NysA1 to NysA3 was monitored at 304 nm. LC–HRMS was performed on an Agilent 6546 LC/Q-TOF and 1290 Infinity II HPLC instrument, using a Luna Omega 5 μm 100 × 2.1 mm (Phenomenex) column, using a 0.5 ml min−1 flow rate with a solvent system of water and methanol each containing 0.1% formic acid, and a gradient of 60–80% methanol over 15 min.
Expression of kfuSV- and Sa-nysSV-subclusters in S. nodosus
The sugar subcluster from S. kasugaensis (DSM40819), kfuSV-sub, was amplified from S. kasugaensis genomic DNA as three fragments; fragment 1 amplifying kfuSVI, kfuSII, fragment 2 amplifying kfuSIII, kfuSIV, kfuSV and fragment 3 amplifying kfuSVII using three primer pairs (Supplementary Table 3). The plasmid pSET152-ermE* was linearized with NdeI and EcoRI, and the three fragments were assembled using HiFi assembly to form the construct pSET-ermE*-kfuSV-sub. Similarly, the sugar subcluster from S. albulus (DSM40492), Sa-nysSV-sub was amplified from the genomic DNA as two fragments to form the construct pSET-ermE*-SanysSV-sub using HiFi, as described above. The two constructs pSET-ermE*-kfuSV-sub and pSET-ermE*-SanysSV-sub were introduced separately into S. nodosus (DSM 40109) by standard conjugation protocols, resulting in the S. nodosus::kfuSV-sub and S. nodosus::Sa-nysSV-sub strains, respectively52. The empty plasmid pSET-ermE* was also introduced into S. nodosus by conjugation resulting in the S. nodosus::pSET-ermE* strain to be used as the negative control. For polyene production, both strains were grown in TSB for 3 days. 2% v/v of the seed culture was inoculated into 50 ml of FSM (fructose 20 g l−1, dextrin 60 g l−1, soya flour 30 g l−1, CaCO3 10 g l−1) growth medium. Fermentation cultures were supplemented with amberlite XAD16N (50 g l−1) resin and were incubated for 7 days with shaking at 30 °C. Mycelia and amberlite XAD16N resin were pelleted by centrifugation and polyenes were extracted with methanol (1× culture volume). A second round of methanol extraction could be performed to improve polyene recovery.
S. albulus and S. noursei transcriptome analysis (RT–qPCR)
The expression levels of nysSII, nysSIII and nysSVII were examined using a two-step RT–qPCR system. Starter cultures of S. noursei (ATCC 11455) and S. albulus (DSM40492) were grown at 28 °C for 48 h in TSB media before 2% (v/v) of starter culture was used to inoculate SPG fermentation media. After 48 h of incubation at 28 °C, 0.5 ml of culture was harvested, washed with phosphate-buffered saline (PBS) and total RNA was extracted using the Monarch Spin RNA Isolation Kit (NEB) as per the manufacturers’ instruction, with an on-column DNase I treatment to remove any contaminating DNA. The quantity of RNA was determined using Nanodrop (ThermoScientific) and the integrity of the purified samples was determined using a 4150 TapeStationAnalyser (Agilent). Then 1 μg of purified RNA was immediately used as a template for complementary DNA (cDNA) synthesis using the SuperScript IV VILO Master Mix (ThermoFisher) following the manufacturers’ instructions. cDNA was flash frozen and stored at −80 °C until use. Primers were designed using the PrimerQuest Tool (IDT) to amplify 80–150 base-pair fragments of the genes of interest and are listed in Supplementary Table 3. qPCR was performed using PowerTrack SYBR Green Master Mix (ThermoFisher) and a StratageneMx3000P system. Amplification conditions used were 95 °C for 2 min and then 40 cycles of 95 °C for 15 s, 60 °C for 60 s, followed by a single default dissociation cycle. Next, 16S ribosomal RNA was used as an internal control. The absence of contamination was verified with no-template and no-reverse-transcription controls. Three biological replicates were performed for each gene. For statistical analysis, multiple (three) unpaired t-tests were performed, specifying an α value of 0.05.
Production of polyenes from S. albulus and S. noursei
S. albulus (DSM40492) or S. noursei (ATCC 11455) spores were used to inoculate GYM media and the seed cultures were cultivated for 3 days at 30 °C. For fermentation, the seed cultures were used to inoculate FSM media (2% v/v inoculum, 3 × 50 ml, triplicates). Fermentation cultures were supplemented with amberlite XAD16N resin (50 g l−1) and were incubated for 7 days with shaking at 30 °C. Mycelia and amberlite XAD16N resin were pelleted by centrifugation. Polyenes were extracted with methanol (1 culture volume) overnight at 4 °C, protected from light. The resulting polyene extracts were diluted fivefold and 10 µl was injected for analysis by analytical HPLC. The relative titres of NysA1 and NysA3 were calculated by comparison with NysA1 standard calibration curve.
Production of polyenes from S. netropsis
S. netropsis (DSM40846) spores were used to inoculate TSB media and the seed cultures were cultivated for two days at 30 °C. For fermentation, the seed cultures were used to inoculate 200 ml of FSM (fructose 20 g l−1, dextrin 60 g l−1, soya flour 30 g l−1, CaCO3 10 g l−1) fermentation cultures in 1 l flasks (2% v/v inoculum). Fermentation cultures were supplemented with amberlite XAD16N (50 g l−1) resin to aid natural product recovery and were incubated for 7 days with shaking at 30 °C. Mycelia and amberlite XAD16N resin were pelleted by centrifugation. Polyenes were extracted with methanol (1× 1 culture volume). LC–HRMS was performed as described above.
Production of polyenes from S. kasugaensis
S. kasugaensis (DSM40819) spores were used to inoculate TSB and the seed cultures were cultivated for 2 days at 30 °C. For fermentation, the seed cultures were used to inoculate 200 ml of FSM (fructose 20 g l−1, dextrin 60 g l−1, soya flour 30 g l−1, CaCO3 10 g l−1) fermentation cultures in 1-l flasks (2% v/v inoculum). Fermentation cultures were supplemented with amberlite XAD16N (50 g l−1) resin and were incubated for 7 days with shaking at 30 °C. Mycelia and amberlite XAD16N resin were pelleted by centrifugation. Polyenes were extracted with methanol (2× 1 pellet volume).
Purification of polyenes and NMR structural characterization
Extracts from S. nodosus::kfuSV-sub, S. nodosus::Sa-nysSV-sub, S. albulus, S. netropsis (DSM40846) and S. kasugaensis were dried in vacuo to complete or near-complete dryness. Water (50 ml) was added to the dry material, the resulting suspension was incubated at 4 °C for 3 h and then centrifuged to produce pellets containing polyenes. All polyene pellets were then dissolved in dimethylsulfoxide (DMSO) for purification by semi-preparative RP-HPLC (Shimadzu Prominence HPLC with a Phenomenex Gemini C18 column 250 × 10 mm, 5-μm particle size). Before HPLC kasufungin B was partially purified using a Mega BE-C18, 10 g, 60-ml bond elute column (Agilent Technologies). The bond elute was washed with up to 70% methanol to remove most impurities, followed by 100% methanol to elute the pentaene compounds. The eluant was concentrated in vacuo and further purified with semi-preparative HPLC. Mycoheptin, dihydromycoheptin and mandimycin were purified from S. netropsis (DSM40846) extract using a 5 ml min−1 flow rate with a solvent system of water and methanol at an isocratic flow of 65% methanol (MeOH) for 25 min, followed by 95% MeOH for 12.5 min. Kasufungin B from S. kasugaensis was purified using 5 ml min−1 flow rate with a solvent system of water and methanol using a 65% isocratic flow of methanol over 11 min followed by a gradient of 66–84% methanol over 12.5 min. AmB-l-digitoxose from S. nodosus::kfuSV-sub was purified using 5 ml min−1 flow rate with a solvent system of water and methanol with a gradient of 70–88% methanol over 30 min. NysA3 from S. albulus was purified using methanol gradients (40–65% for the first 5 min and 65–85% over the next 18 min). Fractions containing the appropriate polyenes were dried in vacuo and dissolved in DMSO-D6 (NysA3 and AmB-l-digitoxose) or CD3OD (kasufungin B) for NMR analysis. One-dimensional (1H, 13C) and two-dimensional NMR (correlation spectroscopy, heteronuclear single quantum coherence, heteronuclear multiple-bond correlation spectroscopy and rotating-frame nuclear Overhauser effect spectroscopy) experiments were performed at 500-MHz frequency to confirm the structures of mycoheptin, dihydromycoheptin and mandimycin, and 800 MHz for NysA3, kasufungin B and AmB-l-digitoxose.
Antifungal activity tests (IC50 determination)
Antifungal susceptibility testing was performed according to the European Committee for Antimicrobial Susceptibility Testing reference microdilution method (version 9.3.2)53 and (v.7.4)54. Yeasts (C. albicans ATCC 90028, C. albicans ATCC 200955, C. glabrata NCPF3309, C. auris H.17.157 and Cryptococcus neoformans F10025) were cultured on Sabouraud’s dextrose agar (SDA) plates at 30 °C for 3 days. Inocula were prepared by suspending five distinct colonies in PBS-0.1% Tween and adjusted to 5 × 105 cells per ml in sterile distilled water. Filamentous fungi (Aspergillus fumigatus A1160, Aspergillus fumigatus cyp51ATR34/L98H, Rhizopus delemar 99-880, Mucor circinelloides 1006Phl, Fusarium oxysporum 9935 and Fusarium solani 9596) were cultured in SDA T25 vented flasks at 37 °C for 3 days, except Fusarium species that were cultured at 28 °C for 5 days. Conidia were collected with PBS-0.1% Tween and filtered through miracloth. Inocula were adjusted to 5 × 105 cells per ml in sterile distilled water. Tests were performed in flat-bottom 96-well plates (CytoOne, StarLab) containing 100 μl of 2× Roswell Park Memorial Institute-1640 medium of a twofold dilution series of antifungal agents and a drug-free control well. Compound stock solutions were prepared in DMSO and concentrations tested were 12.5–0.012 μg ml−1 for the AmB series and 50–0.048 μg ml−1 for the Pim, NysA1 and NysA3 series. Each well was inoculated with 100 μl of the respective strain at a final concentration of 5 × 104 conidia. In addition, Milli-Q water (100 μl) was added to a row of wells in each plate as a sterility control. All plates were incubated at 37 °C for 48 h and optical density at 600 nm (OD600) was measured using a BioTek Synergy 2 SL microplate reader. From the optical density measurement, the IC50 of each drug was determined by fitting inhibitory dose–response curves using variable slope model on GraphPad Prism, version 10.2.3 (347).
Horse blood haemolysis
Measurement of polyene haemolytic activity on horse blood was performed with slight modifications to the method reported in ref. 28. Polyene dilutions ranging from 0.04 µM to 350 µM were prepared from 4 mM stock in DMSO. From each of the dilutions, 6 µl was added to 114 µl of PBS buffer containing 2.5% horse blood (ThermoFisher) and incubated at 37 °C for 1 h in 96-well plates, centrifuged (4,000 rpm for 15 min) and OD545 of the supernatant (100 µl) was measured using a plate reader. Compounds were added to randomly assigned rows in a 96-well plate. A total of 16 different final concentrations (0.04 µM, 0.08 µM, 0.3 µM, 1.25 µM, 10 µM, 20 µM, 30 µM, 40 µM, 50 µM, 60 µM, 70 µM, 80 µM, 90 µM, 100 µM, 150 µM, 200 µM) were tested for AmB, AmB-l-digitoxose, NysA3, Nys31, Nys32, Nys33 and Nys34, 15 final concentrations (0.6 µM, 1.3 µM, 2.5 µM, 10 µM, 30 µM, 50 µM, 70 µM, 100 µM, 120 µM, 140 µM, 160 µM, 200 µM, 250 µM, 300 µM, 350 µM) for NysA1, Nys11, Nys12, Nys13 and Nys14, 13 final concentrations (10 µM, 30 µM, 50 µM, 70 µM and 90 µM, and between 110 µM and 350 µM) for Pim, Pim1, Pim2, Pim3 and Pim4. Positive and negative controls were determined by adding 6 µl of DMSO into 114 µl of 2.5% horse blood in distilled water and PBS buffer, respectively, incubated at 37 °C for 1 h and OD545 was determined as described above. All modified polyenes and standards were tested in parallel with controls in triplicates. The values for 50% horse blood haemolysis (EC50) were determined by a four-parameter logistic curve using GraphPad Prism, version 10.2.3 (347).
Human cell toxicity tests
A549 (human alveolar basal epithelial), HEK293 and HEPG2 cell lines were maintained using standard culture conditions in Gibco DMEM high glucose, pyruvate medium. Cells were seeded in T75 flasks and incubated at 37 °C under a 5% CO2 atmosphere. After trypsinization, A549 (10,000 cells per well), HEK293 (20,000 cells per well) and HEPG2 (50,000 cells per well) cells were seeded into tissue-culture 96-well plates and incubated at 37 °C for 24 h, with 100-μl volume in each well. Polyene (AmB and Nys34) stock solutions were prepared in DMSO and the final concentrations for HEK293 were set at 400 μg ml−1, 200 μg ml−1, 100 μg ml−1, 50 μg ml−1, 25 μg ml−1, 12.5 μg ml−1, 6.25 μg ml−1 and 3.125 μg ml−1, the final concentrations for A549 were set at 800 μg ml−1, 400 μg ml−1, 200 μg ml−1, 100 μg ml−1, 50 μg ml−1, 25 μg ml−1, 12.5 μg ml−1 and 6.25 μg ml−1 and the final concentrations for HEPG2 were set at 400 μg ml−1, 200 μg ml−1, 100 μg ml−1, 50 μg ml−1, 25 μg ml−1, 12.5 μg ml−1, 6.25 μg ml−1 and 3.125 μg ml−1. The compounds were added to randomly assigned rows on tissue-culture 96-well plates keeping final DMSO concentration at 2%. The wells containing cells and media at 2% DMSO were used as viability controls whereas wells containing media at 2% DMSO and no cells were used as negative controls. The compound-treated plate was incubated at 37 °C under a 5% CO2 for 18 h. A colorimetric MTS reduction assay was performed by the addition of MTS reagent (CellTiter 96 AQueous One Solution Reagent (Promega) to quantify viable cells) to the polyene-treated 96-well plates. Absorbance at 490 nm was measured using a microtitre plate reader before and 4 h after incubation with MTS. To measure cell viability, absorbance at 490 nm before MTS treatment was subtracted from the reading taken after addition of MTS. The 2% DMSO-treated cells were considered as 100% viability. Cell viability is shown relative to DMSO-only control (100% viability). All assays were done in triplicates. The dose–response curves were plotted using Prism and IC50 values were determined by a four-parameter logistic curve using GraphPad Prism. All cell lines were tested negative for mycoplasma and were used as received from Sigma without further authentication.
Drug tolerability and pharmacokinetic studies in mice
CD1 mice were purchased from the Jackson laboratory and transferred to the University of Manchester animal unit for use under licence no. PP0175051. The mice were quarantined and acclimatized to the facility for a minimum of 1 week before any regulated procedures took place. Mouse husbandry was performed in individually ventilated cages on a 12-hour light and dark cycle with temperature regulated to 21 °C (±2 °C) and 40–50% humidity. Mice were kept in cages containing nesting material and wood chips. Access to water and food was provided ad libitum for the entirety of the study. While on protocol, mice were checked at the start and end of the working day and a third check was introduced when mice started showing signs of infection or 10% body weight loss.
Maximum tolerated dose finding was performed in rising concentrations starting at 1 mg kg−1, 2 mg kg−1, 5 mg kg−1 and 10 mg kg−1 delivered by i.p. injection with 1 mouse receiving each dose. Mice were observed continuously over the course of 1 h to monitor for immediate adverse effects and then hourly for a total of 4 h. Mice were anaesthetized with isoflurane (Isofane) before terminal cardiac puncture blood collection followed by cervical dislocation.
Pharmacokinetic (PK) analysis of the Nys34 compounds was performed with 20 CD1 male and female mice (10 males and 10 females of roughly 20 g). Blood microsamples were taken twice per mouse from the tail vein in a heparinized capillary tube and a third terminal cardiac puncture sample was taken from each mouse to limit the number of mice used for this experiment in line with the 3R’s principles. Microsamples from mice were taken at 0.5 h, 1 h, 2 h, 4 h and 8 h postdosing with the cardiac punctures performed at the 8 h and 24 h postdosing time points following the scheme outlined in ref. 55.
The organ homogenates were analysed for Nys34 concentration by means of the addition of 200% v/w MeOH, followed by sonication at room temperature for 30 min and vigorous vortexing. Extracts were then clarified by means of centrifugation, and the extraction repeated for a total of three times. Combined liver extracts were directly injected for analysis on LC–MS. Combined extracts of lung, spleen and kidney samples were dried under a stream of nitrogen and resuspended in a given amount of MeOH for analysis by means of LC–MS. Lung and spleen samples were resuspended in 50 µl of MeOH, and kidney samples in 80 µl. Whole blood samples were analysed from 5-µl aliquots, which were frozen and lyophilized until dry. Dry samples were resuspended in 23 µl of MeOH, sonicated at room temperature for 30 min and vortexed vigorously. Samples were then clarified by centrifugation and injected for analysis on LC–MS. LC–HRMS for biological sample analysis was performed on a 1290 Infinity II HPLC coupled to a 6546 LC/Q-TOF Agilent instrument, using a Luna Omega 5 μm 100 × 2.1 mm (Phenomenex) column, with a flow rate of 0.5 ml min−1, using a solvent system of water and methanol and a gradient of 60–95% methanol with 0.1% formic acid over 8 min.
Population pharmacokinetics modelling was performed in R using nlmixr2. A one-compartment model with first-order absorption and first-order elimination was fitted to the whole blood concentration–time data. The model was parameterized using the absorption rate constant (ka), clearance (CL) and volume of distribution (V). Inter-individual variability was included on CL and V using exponential random effects. Residual unexplained variability was modelled using a proportional error structure. Model parameters were estimated using the stochastic approximation expectation–maximization algorithm implemented in nlmixr2. Model adequacy was assessed using standard goodness-of-fit plots and residual diagnostics.
To support regimen selection, toxicity risk was evaluated using peak exposure (Cmax) and total exposure (area under the curve). A regimen corresponding to 10 mg kg−1 administered every 8 h for 4 doses (q8h ×4) was defined as a toxic reference regimen. Using the population pharmacokinetics model derived from nlmixr2, Monte Carlo simulations were performed for this regimen to generate a distribution of 0–32 h peak concentrations. The median simulated Cmax for the toxic reference regimen was used as the primary toxicity threshold for subsequent screening analyses.
Infection experiments in mice
For efficacy experiments requiring infections with Aspergillus fumigatus conidia; mice between 4 and 5 weeks of age were immunocompromised 24 h before infection by subcutaneous injection of Kenalog (Triamcinolone Acetonide, Bristol Myers Squibb) at 40 mg kg−1, on the introduction of immunosuppression the water was supplemented with 2 g l−1 antibiotic (Neomycin, Sigma) and changed daily. Mice were infected intranasally with freshly prepared 5 × 105 spores of CEA10 in 40 µl of sterile saline. Ten CD1 (five male and five female) mice were randomly allocated to each treatment arm of the efficacy experiments. Mice were dosed i.p. with 5 mg kg−1 Nys34 or the drug carrier vehicle 10% DMSO in saline q8h ×4 i.p. and, AmB at 1 mg kg−1 every 24 h 12 h after infection. Mice were weighed and health checked at each timepoint and culled 32 h after the first dosing.
To determine A. fumigatus lung fungal burden, mice lungs were harvested immediately after euthanasia, weighed and transferred to a sterile 2-ml microcentrifuge tube containing 1 ml of sterile PBS supplemented with 0.1% Tween-20. Lungs were then homogenized using a sterile tissue homogenizer until no visible fragments remained. Tenfold serial dilutions of each lung homogenate were prepared in sterile PBS containing 0.1% Tween-20 and 100 µl from each dilution were plated onto SDA plates supplemented with chloramphenicol (50 µg ml−1). All samples were plated in technical duplicates and plates were incubated at 30 °C for 72 h. After incubation, A. fumigatus CFUs were enumerated and the mean colony count from replicate plates was used to calculate total fungal burden. To allow comparison across mice with different lung sizes, fungal burden was normalized to tissue mass (CFU per gram of lung tissue). Researchers were blinded to sample identity for CFU counts.
UV–Vis studies to determine polyene-sterol binding
Stock solutions of ergosterol (100 mM) were prepared in chloroform and diluted in DMSO to a final concentration of 1 mM. Polyene stocks (AmB, NysA1, Nys34) were prepared in DMSO as 1 mM solutions. Polyene-sterol complexes were prepared in triplicate in a 96-well microplate by combining polyene and sterol stock solutions at defined molar ratios (1:0 to 1:5) as detailed in Supplementary Table 6. Sterol complexes were incubated at room temperature for 30 min before UV–visible absorption spectra were recorded using a microplate spectrophotometer. NysA1 derivatives were scanned from 280 nm to 340 nm and AmB derivatives from 380 nm to 440 nm, with a wavelength interval of 1 nm6.
Effect of sterol precomplexation on polyene antifungal activity
Ergosterol was recrystallized from ethanol and prepared as a stock solution in CHCl3. Relevant amounts of ergosterol were dispensed before the solvent was removed under nitrogen gas and fully dried under vacuum overnight. A DMSO solution of polyene was added to solid Erg at a 5:1 molar ratio of Erg:polyene. The resulting suspension was gently vortexed and heated at 80 °C for 1 hour before leaving to cool to room temperature to allow complex formation.
S. cerevisiae was grown on SDA solid media for 3 days at 30 °C. To prepare the inoculum, five distinct colonies (greater than 1 mm) were picked and resuspended in PBS with 0.1% Tween-20 and diluted to an OD600 of 0.5. This was further diluted tenfold to give a final inoculum of 5 × 105 cells per ml. For tests with A. fumigatus A1160, it was grown on SDA solid medium at 37 °C, spores were harvested in PBS + Tween-20 (0.1%) and inocula were adjusted to 5 × 105 spores per ml in sterile distilled water. Compounds were prepared as 2.7 mM (AmB) or 5.4 mM (NysA1 and Nys34) stock solutions in DMSO with or without precomplexation with ergosterol. Stocks were serially diluted twofold with DMSO before diluting 100-fold in 2× YPD media (S. cerevisae) or Roswell Park Memorial Institute media (A. fumigatus). Then 100 µl of each media–polyene solution was added to a CytoOne 96-well plate containing 100 µl of yeast or fungal inoculum in triplicate. Plates were incubated at 30 °C for 24 h before measuring OD600. Control wells with 0.5% DMSO were tested to confirm viability and a water only inoculum was included as a sterility control54.
Reporting summary
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