Potassium antimonyl tartrate

Miltefosine (MIL), paromomycin (PMM) and potassium antimonyl tartrate (Sb^III) were purchased from Sigma Aldrich (Diegem, Belgium); sodium stiboglucanate (Sb^V) was obtained from Calbiochem (EMD Millipore Corporation, MA, USA); amphotericin B (AmB, Fungizone^®) was obtained from Bristol-Myers-Squib (Gilead Sciences, CA, USA). The lead compounds (Table S1) in the nitroimidazole, oxaborole and aminopyrazole chemical series were selected after a SAR analysis and provided by DNDi (Geneva, Switzerland). Stock solutions for the in vitro assays were prepared in PBS for MIL (20 mM) and Sb (5.12 mg/mL) and in demineralized water for PMM (20 mM). AmB stock solutions (5.4 mM) were freshly prepared in 5% dextrose. Stock solutions (20 mM) of the DNDi lead compounds were prepared in DMSO and stored at 4 °C until use. Stock solutions were further diluted in demineralized water, except for DNDI-6148 that was diluted in 0.25% methylcellulose in water. In the in vitro assays, the final in-test concentration of DMSO was <1%.

The stock solutions of Sb(iii) (1000 mg L⁻¹) and Sb(v) (1000 mg L⁻¹) were prepared by potassium antimonyl tartrate and potassium hexahydroxyantimonate (Sigma-Aldrich), respectively. The working solutions were prepared by series dilution of the stock solutions immediately prior to their use. The other chemicals were obtained from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. All chemicals used in this work were of analytical grade and used without further purification. All the metal stock solutions (1000 mg L⁻¹) were obtained from the National Research Center for Standard Materials (NRCSM, Beijing, China). The solution of sodium tetrahydroborate 0.7% was prepared by dissolving 7 g of NaBH₄ powder and 4 g of NaOH in 1000 mL of water. A stock solution of potassium iodide 50% (w/v) containing l-ascorbic acid 10% (w/v) was prepared by dissolving 25 g of KI and 5 g of l-ascorbic acid in a 50 mL of water. 8-Hydroxyquinoline stock solution 1% (w/v) was prepared by a dissolving 1 g of 8-hydroxyquinoline was in a 10 mL of methanol, and then diluted with water to make 100 mL. All experimental and reagent solutions were prepared with deionized water. The solutions were prepared weekly and stored in a refrigerator. In order to prevent metal contamination, all the glassware was kept overnight in a 10% (v/v) HNO₃ solution with posterior cleaning with deionized water prior to use.

Five L. infantum puro/puro mrpA^−/- Sb-resistant mutants (mrpA^−/- CL1 to mrpA^−/- CL5) were independently selected from mrpA^−/- in 25 cm² flasks containing 5 mL SDM-79 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) and 5 μg/mL hemin in the presence of increasing Sb^III concentrations. Potassium antimonyl tartrate (Sigma-Aldrich, St Louis, MO, USA) was used as the source of Sb^III. The stepwise drug selection ranged from 3 μM up to 180 μM of Sb^III. Last-level mrpA^−/- Sb-resistant mutants were grown in the absence of drug pressure for 20 passages to revert resistance (rev).