Studies on the Antileishmanial Mechanism of Action of the Arylimidamide DB766: Azole Interactions and Role of CYP5122A1

Abstract

c 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 14␣-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 halfknockout 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.

Antileishmanial Efficacy and Pharmacokinetics of DB766-Azole Combinations

“…In vitro synergy has been described between the squalene synthase inhibitor E5700 and both posaconazole and itraconazole against intracellular L. amazonensis (29). We earlier reported in vitro synergy between DB766 and posaconazole in an intracellular assay using ␤-lactamase-expressing L. donovani parasites (7). However, the results described here, obtained from a high-content assay using wild-type L. donovani parasites, indicates an additive or indifferent interaction between these compounds.…”

“…Analysis of the interaction between DB766 and posaconazole using fixed-ratio dilutions. A synergistic interaction was previously reported between DB766 and posaconazole against intracellular L. donovani using parasites expressing a ␤-lactamase gene (7). The interaction between DB766 and posaconazole was examined using the high-content assay described above.…”

“…As part of an investigation into the mechanism of action of DB766, L. donovani axenic amastigotes resistant to this compound were raised through DB766 pressure in culture. The resultant DB766-resistant parasites were hypersensitive to the azole antifungal drugs posaconazole and ketoconazole, decreased expression of the protein CYP5122A1 was observed in the resistant parasites, and synergy was reported between DB766 and posaconazole against L. donovani ␤-lactamase-expressing parasites in an intracellular assay (7). CYP5122A1 is a novel cytochrome P450 essential for L. donovani survival that plays an unknown role in ergosterol biosynthesis in Leishmania (8), implicating sterol metabolism in the mechanism of action of DB766.…”

The antifungal compound butenafine eliminates promastigote and amastigote forms of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis

“…In this regard, it was verified that butenafine was able to eliminate amastigote forms of L. amazonensis and L. braziliensis, species that cause the most serious forms of cutaneous leishmaniasis in Latin America, the anergic diffuse and mucocutaneous leishmaniasis. Antifungal drugs such as ketoconazole, voriconazole, fluconazole, itraconazole and other azoles already were related to possess effect against amastigote forms of L. mexicana, L. amazonensis, L. major and L. donovani [2,5,6,17,20]. Similarly, the effect of antifungal allylamines was evaluated in leishmaniasis, and different studies showed that terbinafine, naftifine, and SF 86-327 drugs were able to eliminate intracellular or axenic amastigotes [6,33].…”

Synthesis and pharmacological evaluation of mono-arylimidamides as antileishmanial agents

“…Our efforts to explore the mechanism of antileishmanial action of the bis-AIAs led to the observation that DB766 is synergistic with posaconazole against Leishmania donovani in vitro 17 ; other investigators reported in vitro antileishmanial synergy between posaconazole and the squalene synthase inhibitor E5700. 18 A combination study with 1b and posaconazole was thus performed against intracellular L. amazonensis amastigotes.…”