Miltefosine

Amphotericin B was purchased from Sigma-Aldrich (Madrid, Spain). Miltefosine was acquired from Cayman Chemicals (Vitro SA, Madrid, Spain). Staurosporine was isolated from the Streptomyces sanyensis PBLC04 strain (collected in Ecuador) (16 (link)). Laurinterol was obtained from the Laurencia johnstonii red algae, which was collected in Baja California, Mexico (17 (link)). Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO) and maintained at −20°C until required.

The cytotoxicity of active compounds was evaluated in J774A.1 macrophage cell line. Serial dilutions of compounds 1–9 were plated and incubated with the appropriate cell concentration of macrophages. After 24 h, cell viability was determined using AlamarBlue^® method [24 (link)]. Miltefosine (Cayman Chemicals, Vitro SA, Madrid, Spain) and benznidazole (Sigma-Aldrich, Madrid, Spain) were used as reference drugs.

The loss of membrane integrity and translocation of phosphatidylserine (PS) to the outside of the cellular membrane can be determined by annexin V (AV) and propidium iodide (PI) staining. Double staining with AV-fluorescein isothiocyanate (FITC) and PI during flow cytometry analysis leads to discrimination between four populations: double negatives or alive cells (AV^-/PI^-), double positive or late necrotic/late apoptotic cells (AV⁺/PI⁺), PI-positive and AV-negative or early necrotic cells (AV^-/PI⁺), and PI-negative and AV-positive or early apoptotic cells (AV⁺/PI^-) [22 (link), 23 (link)].
Parasites were harvested after incubation for 6 h, 10 h, and 24 h in the presence of compounds 1, 2 and miltefosine (Cayman Chemical Company, MI, USA), respectively, the latter serving as positive control for the induction of apoptotic cell death. DMSO was used as solvent control. Cell staining was performed using an AV-FITC Apoptosis detection kit (Sigma-Aldrich, Saint Louis, MO, USA). Cells were harvested, washed and suspended in 500 μL of binding buffer, 5 μL of AV-FITC and 10 μl of PI. The cells were incubated for 10 min in the dark at RT. Finally, stained samples were immediately analyzed by flow cytometry using MACSQuant analyzer or a flow cytometer (Becton Dickinson, San José, CA, USA).

In this study, 46 cyanomethyl vinyl ethers, whose chemical synthesis
has already been described and published by Delgado-Hernández
et al. (2021), were used. Amphotericin B (Sigma-Aldrich, Madrid, Spain)
and miltefosine (Cayman Chemicals, Vitro SA, Madrid, Spain) were used
as positive control drugs against N. fowleri.

The Leishmania infantum (MHOM/MA/67/ITMAP-263) wild-type strain (WT) and the in vitro generated resistant mutants Sb2000.1, AmB1000.1 and MF200.5 [41 (link)–46 (link)], which are resistant to 2000 μM of Sb, 1000 nM of AmB and 200 μM MF, respectively, were grown in M199 medium at 25°C supplemented with 10% fetal bovine serum, 5 μg/mL of haemin at pH 7.0 and 2000 μM Sb (Potassium antimonyl tartrate, Sigma-Aldrich), 200 μM of MF (Miltefosine, Cayman Chem.) or 1 μM AmB (Amphotericin B solution, Sigma). Antileishmanial values in promastigotes were determined by monitoring the growth of parasites after 72 h of incubation at 25°C in the presence of increasing antimony concentrations, by measuring A600 using a Cytation 5 machine (BioTek, USA). EC₅₀ values were calculated based on dose-response curves analyzed by non-linear regression with GraphPad Prism 8.0 software (GraphPad Software, La Jolla California, USA). An average of at least three independent biological replicates was performed for each determination.

M199 media, Roswell park memorial institute (RPMI) 1640 media, penicillin-streptomycin antibiotic cocktail, and Fetal bovine serum (FBS) were purchased from GIBCO (Life Technologies). Miltefosine and MTT reagents were purchased from Cayman Chemical and Merck, respectively. Propidium iodide (PI), Annexin-V/FITC (Fluorescein isothiocyanate) apoptosis kit, H₂DCFDA dye, JC-1dye, MitoSOX red, and TUNEL assay kit were procured from Invitrogen (Thermo Fisher Scientific). Sesamol was purchased from Sigma Aldrich and dissolved in DMSO to yield a stock solution (10 mM).

Miltefosine was purchased from Cayman Chemical (Ann Arbor, MI, USA). Liquid methafilcon and methafilcon contact lens blanks were purchased from Kontur Kontact Lens (Hercules, CA, USA). Poly(lactide-co-glycolide), 85% lactide, 15% glycolide (MW 190 KDa) was purchased from Merck and Cie (Schaffhausen, Switzerland). Hexafluoroisopropanol (HFIP) was purchased from Oakwood Chemical (Estill, NC, USA). Gibco phosphate-buffered saline (PBS), Dulbecco’s Modification of Eagle’s Medium, penicillin-streptomycin, and L-glutamine were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Biopsy punches were purchased from Sklar Instruments (Westchester, PA, USA). All other reagents were purchased from Millipore-Sigma (St. Louis, MO, USA), except where otherwise noted.