Chloroquine diphosphate

The Approved Oncology Drugs Set VIII screening panel was obtained from the NCI-Chemotherapeutic Agents Repository, through Fisher Bioservices. Marizomib was obtained from Adipogen. N-acetyl cysteine (NaC) and Q-VD-OPh were obtained from Sigma–Aldrich. Carfilzomib was obtained from Selleckchem. Chloroquine diphosphate was obtained from Sigma–Aldrich.

Stock solution of 4-hydroxytamoxifen (4-HT) (Sigma) was dissolved in ethanol and stored at -20°C. Stock solutions of chloroquine diphosphate (CQ), desipramine (DSP), chlorpromazine (CPZ) and promethazine (PMZ) (all from Sigma), were prepared fresh before each experiment, by dissolving in phosphate buffered saline (PBS) and filter sterilization. Stock solutions of L-leucyl-L-leucyl methyl ester (Sigma) was prepared freshly before each experiment, by dissolving in DMSO. Stock solution of Z-Phe-Ala fluoromethyl ketone (Z-Phe-Ala; Sigma) was prepared in DMSO. Vehicle controls consisted of equivalent amounts, all < 1%, of DMSO (LeuLeu-OMe), PBS (CQ, CPZ, PMZ) and ethanol (4-HT). Monensin (Sigma) was prepared at a final concentration of 100 nM in ethanol. The concentration of Monensin was optimized to be the lowest concentration that inhibited lysotracker accumulation in lysosomes, yet did not obviously affect cell growth after 6 hours (data not shown).

Opti-MEM without phenol red (Gibco) was purchased from ThermoFisher Scientific (San Jose, CA, USA). DMEM low glucose, with phenol red, fetal bovine serum (FBS), penicillin/streptomycin, l-glutamine, phosphate buffer saline (PBS) and trypsin/versene were all purchased from Biochrom (Berlin, Germany). Thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and isopropanol were purchased from Merck KGaA (Calbiochem^®, Darmstadt, Germany). N-hydroxybenzotriazole (HOBt) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium (HBTU) were purchased from Anaspec (San Jose, CA USA). N,N-diisopropylethylamine (DIPEA) and chloroquine diphosphate were purchased from Sigma-Aldrich Ltd. (Poole, UK). MitoTracker^® Green (Cell Signaling Techology Inc., Beverly, MA, USA) FM was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Doxorubicin hydrochloride and hyperbranched poly(ethyleneimine) (PEI) of 1300 molecular weight (Lupasol^® G20, water-free, 98%) were kindly donated by Regulon SA (Athens, Greece) and BASF (Ludwigshafen, Germany), respectively.

Mammalian expression vector pcDNA3.1 containing α-syn and its mutants were transfected into wild type and Ate1 knockout mouse embryonic fibroblasts^{10 (link)} or 293 T cells at 70–90% confluence using Lipofectamine®2000 Transfection Reagent (Life Technologies). After 8 hrs in culture, cells were either collected directly into SDS sample buffer for Western blotting, or split into two equal dishes, grown overnight, and treated with 20 µM MG132, 100 nM of bafilomycin A1 (Sigma B1793), or 100 nM of chloroquine diphosphate (Sigma C6628), or DMSO (control).

Phorbol 12-myristate 13-acetate (PMA, Sigma P1585) was used in the concentration of 1–10 μM. Twenty millimolar of NEM (Sigma E3876) and 100 μg/ml of cycloheximide was used (Sigma C7698). MG132 is Z-Leu-Leu-Leu-al, used in the concentration of 10 μM (Sigma C2211). Chloroquine diphosphate was used in the concentration of 10 μM (Sigma C6628). Protease inhibitor used was EDTA-free Halt protease inhibitor cocktail (Thermo Scientific 87785). 2-D08, a SUMOylation inhibitor III (Calbiochem 505156, EMD-Millipore) was used at the concentration of 6 μM. Transporter inhibitors in MPP⁺ uptake were included: GBR 12909 dihydrochloride (EMD-Millipore 505732), Mazindol (Sigma M2017), Desipramine hydrochloride (Sigma D3900), and Citalopram (Sigma C7861).

Artesunate, mefloquine hydrochloride, amodiaquine dihydrochloride dihydrate, chloroquine diphosphate, primaquine phosphate, quinine hemisulfate, atovaquone, methylene blue, pyronaridine tetraphosphate, doxycycline hyclate, clindamycin and praziquantel were purchased from Sigma-Aldrich. Ferroquine was obtained from Sanofi-Synthelabo, proguanil and cycloguanil from Jacobus Pharmaceutical Company, tigecycline from Wyeth, and mirincamycin hydrochloride enantiomers from Maldevco [45 (link)]. All compounds were dissolved in sterile DMSO except for quinine for which methanol was used and pure M199 medium (without additives) was used to dissolve proguanil, cycloguanil, clindamycin, and pyronaridine. The stock concentration was 50 mM for Artesunate, amodiaquine, chloroquine, atovaquone, quinine, and primaquine, and 100 mM for praziquantel, proguanil, cycloguanil, methylene blue, pyronaridine, clindamycin, doxycycline, and mirincamycin enantiomers, respectively. Mefloquine was dissolved to 24 mM and Ferroquine to 12.5 mM. All stocks were freshly prepared for the study and stored at -20°C. Maximum concentration of the solvent (DMSO, methanol) in the in vitro assays did not exceed 0.8% and did not interfere with parasite viability.

The initial AA (500 mg) of 97 % purity used in the preliminary studies was a kind donation from Prof Van Heerden (University of KwaZulu Natal). Further quantities of AA (97 % purity) were purchased together with Giemsa stain, dimethyl sulfoxide (DMSO), chloroquine diphosphate (CHQ) from Sigma-Aldrich (St. Louis, MI, USA). All other chemicals and reagents were of analytical grade.