Pandharkar Trupti, Zhu Xiaohua, Mathur Radhika, Jiang Jinmai, Schmittgen Thomas D, Shaha Chandrima, Werbovetz Karl A
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA.
Antimicrob Agents Chemother. 2014 Aug;58(8):4682-9. doi: 10.1128/AAC.02405-14. Epub 2014 Jun 2.
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.
芳基脒酰胺（AIA）是受二脒类抗菌剂启发而研发的，但对细胞内寄生虫显示出更强的活性。AIA DB766{2,5-双[2-(2-异丙氧基)-4-(2-吡啶基亚氨基)氨基苯基]呋喃盐酸盐}对细胞内利什曼原虫具有出色的活性，口服给药时对内脏利什曼病的小鼠和仓鼠模型有效，但其作用机制尚不清楚。在本研究中，通过持续施加DB766压力，我们培养出了对该化合物具有12倍抗性的杜氏利什曼原虫无菌无鞭毛体。这些对DB766耐药（DB766R）的寄生虫对米替福新的敏感性比野生型生物体高2倍，对甾醇14α-脱甲基酶（CYP51）抑制剂酮康唑和泊沙康唑超敏感（分别比野生型敏感2000倍和超过12000倍）。对DB766R寄生虫的蛋白质免疫印迹分析表明，虽然这些生物体中CYP51的表达略有增加，但最近在利什曼原虫中发现的与麦角甾醇代谢相关的细胞色素P450 CYP5122A1的表达在DB766R寄生虫中显著降低。体外药敏试验表明，CYP5122A1半敲除的杜氏利什曼原虫前鞭毛体对DB766的敏感性明显低于野生型，对酮康唑的敏感性高于野生型，这与在DB766R寄生虫中的观察结果一致。此外，DB766与泊沙康唑的组合在无菌和细胞内杜氏利什曼原虫无鞭毛体中均表现出协同活性。综上所述，这些研究表明CYP5122A1参与了AIA的抗利什曼原虫作用，并表明DB766与唑类药物的组合是开发协同抗利什曼原虫疗法的潜在候选药物。
