Liposomal formulation of a new antifungal hybrid compound provides protection against Candida auris in the ex vivo skin colonization model

ABSTRACT The newly emerged pathogen, Candida auris, presents a serious threat to public health worldwide. This multidrug-resistant yeast often colonizes and persists on the skin of patients, can easily spread from person to person, and can cause life-threatening systemic infections. New antifungal therapies are therefore urgently needed to limit and control both superficial and systemic C. auris infections. In this study, we designed a novel antifungal agent, PQA-Az-13, that contains a combination of indazole, pyrrolidine, and arylpiperazine scaffolds substituted with a trifluoromethyl moiety. PQA-Az-13 demonstrated antifungal activity against biofilms of a set of 10 different C. auris clinical isolates, representing all four geographical clades distinguished within this species. This compound showed strong activity, with MIC values between 0.67 and 1.25 µg/mL. Cellular proteomics indicated that PQA-Az-13 partially or completely inhibited numerous enzymatic proteins in C. auris biofilms, particularly those involved in both amino acid biosynthesis and metabolism processes, as well as in general energy-producing processes. Due to its hydrophobic nature and limited aqueous solubility, PQA-Az-13 was encapsulated in cationic liposomes composed of soybean phosphatidylcholine (SPC), 1,2-dioleoyloxy-3-trimethylammonium-propane chloride (DOTAP), and N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt (DSPE-PEG 2000), and characterized by biophysical and spectral techniques. These PQA-Az-13-loaded liposomes displayed a mean size of 76.4 nm, a positive charge of +45.0 mV, a high encapsulation efficiency of 97.2%, excellent stability, and no toxicity to normal human dermal fibroblasts. PQA-Az-13 liposomes demonstrated enhanced antifungal activity levels against both C. auris in in vitro biofilms and ex vivo skin colonization models. These initial results suggest that molecules like PQA-Az-13 warrant further study and development.

The microwave reactions were conducted in a Discover LabMate apparatus (CEM Corporation, Matthews, USA).The UPLC-MS or UPLC-MS/MS analyses were done on an UPLC-MS/MS system comprising a Waters ACQUITY UPLC (Waters Corporation, Milford, USA) coupled with a Waters TQD mass spectrometer (electrospray ionization mode ESI with tandem quadrupole).Chromatographic separations were carried out using an ACQUITY UPLC BEH (bridged ethyl hybrid) C18 column: 2.1 × 100 mm with a particle size of 1.7 μm.
The column was maintained at 40 °C and eluted under gradient conditions using 95 to 0% of eluent A over 10 min, at a flow rate of 0.3 mL/min.Eluent A: 0.1% solution of formic acid in water (v/v); eluent B: 0.1% solution of formic acid in acetonitrile (v/v).A total of 10 μL of each sample was injected, and chromatograms were recorded using a Waters eλ PDA detector.The spectra were analyzed in the range of 200−700 nm with 1.2 nm resolution and at a sampling rate of 20 points/s.The UPLC/MS purity of all the test compounds and key intermediates was determined to be >95%. 1 H NMR and 13 C NMR spectra were recorded using an FT-NMR 500 MHz spectrometer (Joel Ltd., Akishima, Tokyo, Japan), and a 19 F NMR spectrum was obtained in a Varian Mercury spectrometer (282 MHz, Varian Inc., Palo Alto, USA).Chemical shifts are reported as δ values (ppm) relative to TMS δ = 0 ( 1 H) as the internal standard.The J values are expressed in hertz (Hz).Signal multiplicities are represented by the following abbreviations: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).Elemental analyses were conducted using a Vario EL III elemental analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany).Elemental analyses were found to be within ±0.4% of the theoretical values.Melting point was determined on a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Essen, Germany) using open glass capillaries and are uncorrected.Synthetic procedures (Figure 1).A mixture of 3-chloromethyl-pyrrolidine-1-carboxylic acid tert-butyl ester, 1-(3-trifluoromethyl)phenyl-piperazine, cesium carbonate, and a catalytic amount of potassium iodide in DMF (5 mL) was stirred at 120 °C for 2 h under microwave irradiation.After that time, the reaction mixture was cooled to room temperature and the mixture was extracted with ethyl acetate (20 mL).The organic layer was washed with water (20 mL) and then dried over anhydrous sodium sulfate.After evaporation of ethyl acetate, the product was purified by column chromatography over silica gel using dichloromethane/acetone (70/30, v/v) as eluent.The crude products were obtained after deprotection of the tert-butyloxycarbonyl group, conducted according to a general procedure.
Next, the reaction mixture was purified by column chromatography over silica gel using dichloromethane/methanol (9/1, v/v) as eluent.

Table S1 .
Predicted and calculated molecular properties of PQA-AZ-13.

Table S2 .
Cellular proteomics in control and PQA-Az-13 treated C. auris cells.