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5 protocols using proguanil

1

Anti-malarial Drug Sensitivity Testing

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Experiments reported in this study were performed using P. falciparum parasite strains Dd2 (86 (link)) and NF54 (87 (link)). The identities of these strains were confirmed by their expected drug-sensitivity profiles and were Mycoplasma-free by PCR test. Parasite culturing was done in Roswell Park Memorial Institute medium (RPM1-1640, Thermo Fisher 23400021) supplemented with 2.5 g/L Albumax I Lipid-Rich BSA (Thermo Fisher 11020039), 15 mg/L hypoxanthine (Sigma H9636), 110 mg/L sodium pyruvate (Sigma P5280), 1.19 g/L HEPES (Sigma H4034), 2.52 g/L sodium bicarbonate (Sigma S5761), 2 g/L glucose (Sigma G7021), and 10 mg/L gentamicin (Invitrogen Life Technologies 15750060). Parasite cultures were maintained at 2% hematocrit in deidentified human erythrocytes obtained from the University of Utah Hospital blood bank, at 37°C, and at 5% O2, 5% CO2, 90% N2. For growth assays with decyl-ubiquinone (dQ, Caymen Chemicals 55486005) and/or proguanil (Sigma 637321), the growth medium was supplemented with 15 µM dQ (DMSO) and/or 1 µM proguanil (DMSO), final concentration. In all cases, the final DMSO concentration was limited to ≤0.3% w/v.
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2

Antimalarial Compound Screening Protocol

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L-Dihydroorotic acid (DHO), CoQD, DCIP, antimycin A, atovaquone (ATV), mefloquine (MQ), artemisinin, dihydroartemisinin (DHA), and proguanil were purchased from Sigma-Aldrich (St. Louis, MO). Genz-669178, was kindly provided by Genzyme, a Sanofi Company (Waltham, MA). IDI-6273 was purchased from ChemDiv. DSM1 and DSM74 were prepared following the literature procedure28 (link) and were recrystallized from ethanol. 1H NMR spectra matched that reported, and HPLC analysis indicated >95% purity.
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3

Synthesis and Characterization of Antimalarial Compounds

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Chemicals, solvents and commercially available compounds [Proguanil (30), 4-chlorophenylbiguanide (31), pyrimetamine (32) and trimethoprim (33)] were purchased from Sigma-Aldrich (Milan, Italy). Mps: Büchi apparatus, uncorrected. 1H NMR and 13C NMR spectra were recorded on a Varian Gemini-200 instrument at 200 and 50 MHz, respectively; DMSO-d6; δ in ppm rel. to Me4Si as internal standard. J in Hz. Elemental analyses were performed on a Carlo Erba EA-1110 CHNS instrument in the Microanalysis Laboratory of the Department of Pharmacy of Genoa University.
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4

Proguanil Bioavailability Study

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The proguanil used in the study was 98.2% pure and obtained from Sigma Aldrich (Saint Louis, MO, USA). It was dissolved in DMSO where it was stable for an extended period of time. The bioavailability of proguanil when taken orally is typically 100% according to the literature [29 (link)]; however, some studies have reported lower values [30 ].
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5

Biguanide Compound Screening Protocol

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Phenformin, phenyl biguanide (PubChem CID: 5932) (Sigma-Aldrich Ltd.), isopropyl biguanide (PubChem CID: 9570185), chloroPhenformin (PubChem CID: 67587204) (AKOS GmbH), and benzyl biguanide (PubChem CID: 9570091) (Angene International Ltd.) were added from aqueous stock solutions, and proguanil (Sigma-Aldrich Ltd.) and bis-isopropyl biguanide (PubChem CID: 23437065) (European Directorate for the Quality of Medicines and Healthcare) from stock solutions in DMSO.
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