Evaluation of phthalazinone phosphodiesterase inhibitors with improved activity and selectivity against Trypanosoma cruzi

Abstract

Background Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. Objectives As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. Methods In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. Results Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. Conclusions The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

Efficacy of Novel Pyrazolone Phosphodiesterase Inhibitors in Experimental Mouse Models of Trypanosoma cruzi

“…These observations are suggestive of osmotic stress and may be related to increased levels of cAMP. In this context, we investigated the possibility of a PDE target for NPD-227 and observed that it induced a significant increase in the cellular cAMP concentration, confirming the targeting of at least one PDE parasite isoform, consistent with reports on other PDE inhibitor chemotypes (25,26).…”

“…The rounding of parasites induced by NPD-227 suggests an impact on the osmoregulation system, already described as an outcome of PDEC inhibition in T. cruzi (9). Indeed, we have previously shown that exposure to other classes of PDE inhibitors, specifically imidazoles (25) and phthalazinones (26), causes profound ultrastructural damage in the Golgi, flagellar pocket, and plasma membrane of T. cruzi. These observations are suggestive of osmotic stress and may be related to increased levels of cAMP.…”

“…Combined therapy has been proposed as an ideal approach, considering its potential to enhance efficacy and delay drug resistance by acting upon different cellular targets (28,29). This strategy represents an important tool to optimize the potential of PDE inhibitors within combined approaches, particularly in view of their incomplete effects on trypomastigotes, and our successful experience with some combination approaches (26,(30)(31)(32). In the in vivo efficacy proof of concept, coadministration of Bz and NPD-227 resulted in improved efficacy in acute mouse models of T. cruzi infection, leading to ϳ90% reduction in parasitemia and lower mortality rates (0 to 17%) than seen with either drug alone (Bz and NPD monotherapy: 40 to 63% and 40 to 50%, respectively).…”

“…Following 48 h of interaction, the infected cultures were rinsed, fixed with Bouin for 5 min, stained with Giemsa, and evaluated by light microscopy. The percentage of infected host cells and the number of parasites per cell were determined for the calculation of the infection index as reported (26).…”

Cloning and functional complementation of ten Schistosoma mansoni phosphodiesterases expressed in the mammalian host stages

“…The essentiality of the cAMP signaling pathways, including PKA, prompted us to extend our work on cAMP phosphodiesterases of protozoan parasites [ 28 – 32 ] to S . mansoni , anticipating that, as in human-infective protozoa, inhibition of PDEs leads to a fatal increase and imbalance in cellular cAMP levels.…”