title: Studies on the antileishmanial mechanism of action of the arylimidamide DB766: azole interactions and role of CYP5122A1.
abstract: Arylimidamides (AIAs) are inspired by diamidine antimicrobials but show superior activity against intracellular parasites. The AIA DB766 {2,5-bis[2-(2-i-propoxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride} displays outstanding potency against intracellular Leishmania parasites and is effective in murine and hamster models of visceral leishmaniasis when given orally, but its mechanism of action is unknown. In this study, through the use of continuous DB766 pressure, we raised Leishmania donovani axenic amastigotes that displayed 12-fold resistance to this compound. These DB766-resistant (DB766R) parasites were 2-fold more sensitive to miltefosine than wild-type organisms and were hypersensitive to the sterol 14α-demethylase (CYP51) inhibitors ketoconazole and posaconazole (2,000-fold more sensitive and over 12,000-fold more sensitive than the wild type, respectively). Western blot analysis of DB766R parasites indicated that while expression of CYP51 is slightly increased in these organisms, expression of CYP5122A1, a recently identified cytochrome P450 associated with ergosterol metabolism in Leishmania, is dramatically reduced in DB766R parasites. In vitro susceptibility assays demonstrated that CYP5122A1 half-knockout L. donovani promastigotes were significantly less susceptible to DB766 and more susceptible to ketoconazole than their wild-type counterparts, consistent with observations in DB766R parasites. Further, DB766-posaconazole combinations displayed synergistic activity in both axenic and intracellular L. donovani amastigotes. Taken together, these studies implicate CYP5122A1 in the antileishmanial action of the AIAs and suggest that DB766-azole combinations are potential candidates for the development of synergistic antileishmanial therapy.
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA tpandhar@nd.edu werbovetz.1@osu.edu.
authors.forename: Trupti
authors.initials: T
authors.lastname: Pandharkar
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Cell Death and Differentiation Research Laboratory, National Institute of Immunology, New Delhi, India.
authors.forename: Radhika
authors.initials: R
authors.lastname: Mathur
authors.affiliation: Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Jinmai
authors.initials: J
authors.lastname: Jiang
authors.affiliation: Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Schmittgen
authors.affiliation: Cell Death and Differentiation Research Laboratory, National Institute of Immunology, New Delhi, India.
authors.forename: Chandrima
authors.initials: C
authors.lastname: Shaha
authors.affiliation: Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA tpandhar@nd.edu werbovetz.1@osu.edu.
authors.forename: Karl A
authors.initials: KA
authors.lastname: Werbovetz
mesh_terms: D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D061245:Axenic Culture; D015153:Blotting, Western; D006224:Cricetinae; D004338:Drug Combinations; D004351:Drug Resistance; D004357:Drug Synergism; D005663:Furans / Q000494:pharmacology*; D005786:Gene Expression Regulation; D007654:Ketoconazole / Q000494:pharmacology*; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008018:Life Cycle Stages / Q000187:drug effects* / Q000235:genetics; D051379:Mice; D010767:Phosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D058886:Sterol 14-Demethylase / Q000235:genetics* / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology*
chemical_list: C552046:2,5-bis(2-(2-propoxy)-4-(2-pyridylimino)aminophenyl)furan; D000578:Amidines; D000981:Antiprotozoal Agents; D004338:Drug Combinations; D005663:Furans; D015800:Protozoan Proteins; D014230:Triazoles; D010767:Phosphorylcholine; C039128:miltefosine; C101425:posaconazole; D058886:Sterol 14-Demethylase; D007654:Ketoconazole