Immucillins Impair Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis Multiplication In Vitro

Abstract

Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05–500 μM and, when needed, 0.01–50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.

Immucillins ImmA and ImmH Are Effective and Non-toxic in the Treatment of Experimental Visceral Leishmaniasis

“…Sonicates were centrifuged and their supernatants separated for purification by column chromatography using Ni-NTA Superflow resin (Qiagen, USA). The column was eluted with a 50mM potassium phosphate, 300 mM NaCl buffer, pH: 8.0, using a 50 to 300mM imidazole gradient [ 29 ]. The fraction containing NH36 recombinant antigen was recovered, dyalized and preserved at -80°C.…”

“…(L . ) donovani recombinant NH36 enzymatic activity [ 29 ]. IA and IH inhibited the NH36 enzymatic activity with Ki = 0.080 μM for IA and 0.019 μM for IH.…”

“…infantum chagasi amastigotes in vitro causing no apparent damage to macrophage viability. IA and IH were less toxic and more potent than Glucantime [ 29 ].…”

“…Our results of the in vitro model suggested that IA, IH and SMIH might provide new chemotherapy agents for leishmaniasis [ 29 ]. Here we tested their efficacy and toxicity compared to those induced by the standard treatment with Glucantime, on BALB/c mice and CB hamsters infected with L .…”

In vitro effects of purine and pyrimidine analogues on Leishmania donovani and Leishmania infantum promastigotes and intracellular amastigotes

“…Although resistance mechanisms to purine starvation allow parasite survival, this pathway is a target of choice for rational drug design (De Koning et al 2005, Vincent andBarrett 2015). For instance, the nucleoside analogues immucillins reduced in vitro the replication of L. infantum intramacrophagic amastigotes (Freitas et al 2015). The pyrimidine nucleoside analogue 5-fluorouracil is converted in vivo into the active metabolite 5fluoroxyuridine monophosphate (F-UMP) that incorporates into RNA, thereby inhibiting cell growth.…”