In infected mammalian cells, Leishmania parasites reside within specialized compartments called parasitophorous vacuoles (LPVs). We have previously shown that Retro-2, a member of a novel class of small retrograde pathway inhibitors caused reduced LPV sizes and lower parasite numbers during experimental L. mexicana sp. infections. The purpose of this study was to determine if structural analogs of Retro-2cycl reported to have superior potency in the inhibition of retrograde pathway-dependent phenomena (i.e., polyomavirus cellular infection by polyomavrius and Shiga toxin trafficking in cells) are also more effective than the parent compound at controlling Leishmania infections. In addition to their effects on LPV development, we show that two optimized analogs of Retro-2cycl, DHQZ 36 and DHQZ 36.1 limit Leishmania amazonensis infection in macrophages at EC50 of 13.63+/-2.58μM and10.57+/-2.66μM, respectively, which is significantly lower than 40.15μM the EC50 of Retro-2cycl. In addition, these analogs caused a reversal in Leishmania induced suppression of IL-6 release by infected cells after LPS activation. Moreover, we show that in contrast to Retro-2cycl that is Leishmania static, the analogs can kill Leishmania parasites in axenic cultures, which is a desirable attribute for any drug to treat Leishmania infections. Together, these studies validate and extend the published structure-activity relationship analyses of Retro-2cycl.
Clinical manifestations of leishmaniasis range from self-healing, cutaneous lesions to fatal infections of the viscera. With no preventative Leishmania vaccine available, the frontline option against leishmaniasis is chemotherapy. Unfortunately, currently available anti-Leishmania drugs face several obstacles, including toxicity that limits dosing and emergent drug resistant strains in endemic regions. It is, therefore, imperative that more effective drug formulations with decreased toxicity profiles are developed. Previous studies had shown that 2-(((5-Methyl-2-thienyl)methylene)amino)-N-phenylbenzamide (also called Retro-2) has efficacy against Leishmania infections. Structure–activity relationship (SAR) analogs of Retro-2, using the dihydroquinazolinone (DHQZ) base structure, were subsequently described that are more efficacious than Retro-2. However, considering the hydrophobic nature of these compounds that limits their solubility and uptake, the current studies were initiated to determine whether the solubility of Retro-2 and its SAR analogs could be enhanced through encapsulation in amphiphilic polymer nanoparticles. We evaluated encapsulation of these compounds in the amphiphilic, thermoresponsive oligo(ethylene glycol) methacrylate-co-pentafluorostyrene (PFG30) copolymer that forms nanoparticle aggregates upon heating past temperatures of 30°C. The hydrophobic tracer, coumarin 6, was used to evaluate uptake of a hydrophobic molecule into PFG30 aggregates. Mass spectrometry analysis showed considerably greater delivery of encapsulated DHQZ analogs into infected cells and more rapid shrinkage of L. amazonensis communal vacuoles. Moreover, encapsulation in PFG30 augmented the efficacy of Retro-2 and its SAR analogs to clear both L. amazonensis and L. donovani infections. These studies demonstrate that encapsulation of compounds in PFG30 is a viable approach to dramatically increase bioavailability and efficacy of anti-Leishmania compounds.
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