Amodiaquine

All toxins were purchased from List Biological Laboratories. FP59 was a kind gift from S. Leppla (National Institute of Allergy and Infectious Diseases/National Institutes of Health, Bethesda, MD). An FDA-approved drug library comprising of 1,581 drugs was purchased from Johns Hopkins, titled, Johns Hopkins Clinical Compound Library (JHCCL) version 1.0. The drugs arrived as 10 mM stock solutions in sealed microtiter plates and were made using DMSO or water as solvents. Drugs were arrayed in 96-well plates and screened at a stock concentration of 10 mM. The library was stored at −20 °C until use. Prior to use, the library of drugs was thawed at 25 °C. Compounds that were determined to be compounds of interest, were isolated and reproduced from 10 mM solutions. Amodiaquine, Chloroquine, Cinchonine, Primaquine, and Quinidine were purchased from Sigma-Aldrich (St. Louis, MO, USA). All drugs were prepared at 10 mM using DMSO as a solvent. Desethyl-Amodiaquine and desethyl-Chloroquine were purchased from Toronto Research Chemicals Inc. Anti–N-terminal MEK-2, anti-tubulin, and anti-PA antibodies were purchased from Santa Cruz Biotechnology. Purified human cathepsin B protein used for NMR experiments was purchased from ACROBiosystems.

Seongsanamide A (>98%, Figure 1) was isolated from the fermentation broth extracts of Bacillus sp. KCTC 12796BP by as previously reported [7 (link)]. Acetaminophen, amodiaquine, bupropion, chlorzoxazone, dextromethorphan, trimipramine, uridine diphosphoglucuronic acid (UDPGA), nicotinamide adenine dinucleotide phosphate (NADP⁺), glucose-6-phosphate (G6P), and G6P dehydrogenase (G6PDH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Coumarin, midazolam, omeprazole, and tolbutamide were purchased from Toronto Research Chemicals (Toronto, ON, Canada). Pooled HLMs (XTreme 200) were supplied by XenoTech (Lenexa, KS, USA). We purchased rP450 isoforms (rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C19, rCYP2D6, rCYP2E1, rCYP2J2, rCYP3A4, and rCYP3A5) from Corning life sciences (Woburn, MA, USA). All solvents used in the analyses were LC-MS grade (Fisher Scientific Co., Pittsburgh, PA, USA).

Diabetes was induced in eight-week-old male C57/BL6J mice (Center for Research Animals, Seoul, Korea) by the intraperitoneal injection of STZ (Sigma Chemicals, Missouri) at a dose of 50 mg/kg for 5 consecutive days. In the intervention study, the following four groups of mice (n = 5 in each group) were used for three separate experiments: (1) a normal control group, (2) a diabetic control group, (3) a diabetes + Chloroquine (50 mg/kg) group, and (4) a diabetes + amodiaquine (20 mg/kg) group. Chloroquine (Sigma-Aldrich, St. Louis, MO, USA) and amodiaquine (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in saline and administered to the mice at the indicated doses via intraperitoneal injections at 48-h intervals for 14 weeks beginning 2 weeks after STZ administration. All the mice were sacrificed 16 weeks after STZ administration, and their kidney tissues were collected for analysis. During the experiments, bodyweights and serum glucose concentrations were measured weekly.

All high-throughput compound screening was performed at the Centre for Microbial Chemical Biology (McMaster University). The chemical library we screened contained 3840 diverse small molecules assembled from Sigma-Aldrich and MicroSource. Screening stocks (5 mM) were stored at −20 °C in DMSO. The following compounds were ordered from Sigma-Aldrich: 3′-azido-3′-deoxythymidine (AZT), 5-fluorouracil (5-FU), 5-flurocytosine (5-FC), ciprofloxacin, colistin, amodiaquine, cantharidin, indatraline. Berbamine and cetrimonium bromide were sourced from Cedarlane. Compounds were routinely dissolved in DMSO at a concentration of 10 mg mL⁻¹ and stored at −20 °C.

Drug hits that inhibited at least 66% of LF FRET reaction were also tested for their ability to inhibit FRET reactions of human furin (New England Biolabs), calpain 1 (BioVision), cathepsin B (EMD Millipore), and caspases-1 and -3 (BioVision). The fluorescently labelled substrate peptide for furin FRET assay was purchased from Peptides International. The positive control furin Inhibitor I was purchased from EMD Millipore. The calpain Inhibitor 1, ALLN, was purchased from BioVision and used as a control. Amodiaquine (Sigma-Aldrich) was used as a control inhibitor of cathepsin B. The universal caspase inhibitor Z-VAD-FMK (BioVision) was used as a control. Rates of reactions were quantified by the Microsoft Excel LINEST function.

YL-IPA08 and AC-5216 internal standard (IS) (Figure 1) were supplied by chemical synthesis laboratory of our Institute (Beijing, China) with purity greater than 99%. Midazolam (MDZ), 1′-OH-MDZ, phenacetin, acetaminophen, diclofenac, S-mephenytoin, 4-OH-diclofenac, 4-OH-mephenytoin, bupropion, OH-bupropion, amodiaquine, N-desethylamodiaquine, dextromethorphan, dextrorphan, atenolol, propranolol, and digoxin were all purchased from Sigma-Aldrich (St. Louis, MO). Human liver microsomes (pool of 50, mixed gender), male rat liver microsomes (pool of 495), human intestinal microsomes (pool of 15, mixed gender) and male rat intestinal microsomes (pool of 100) were purchased from XENOTECH (Lenexa, KS). NADPH was purchased from Roche Life Science (Basel-Stadt, Switzerland). Other reagents were of HPLC grade or better.