title: 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.
authors.affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, United States of America.
authors.forename: Elisangela Oliveira
authors.initials: EO
authors.lastname: Freitas
authors.affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Dirlei
authors.initials: D
authors.lastname: Nico
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, United States of America.
authors.forename: Rong
authors.initials: R
authors.lastname: Guan
authors.affiliation: Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: José Roberto
authors.initials: JR
authors.lastname: Meyer-Fernandes
authors.affiliation: The Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Keith
authors.initials: K
authors.lastname: Clinch
authors.affiliation: The Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Gary B
authors.initials: GB
authors.lastname: Evans
authors.affiliation: The Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Tyler
authors.affiliation: Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, New York, United States of America.
authors.forename: Vern L
authors.initials: VL
authors.lastname: Schramm
authors.affiliation: Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Clarisa B
authors.initials: CB
authors.lastname: Palatnik-de-Sousa
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D000241:Adenosine / Q000031:analogs & derivatives; D000818:Animals; D000981:Antiprotozoal Agents / Q000737:chemistry / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D018314:Leishmania infantum / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D007894:Leishmania mexicana / Q000187:drug effects* / Q000254:growth & development / Q000648:ultrastructure; D016773:Leishmaniasis, Cutaneous / Q000188:drug therapy; D007898:Leishmaniasis, Visceral / Q000188:drug therapy; D051379:Mice; D008807:Mice, Inbred BALB C; D046529:Microscopy, Electron, Transmission; D009699:N-Glycosyl Hydrolases / Q000037:antagonists & inhibitors; D011684:Purine Nucleosides / Q000737:chemistry / Q000494:pharmacology; D011744:Pyrimidinones / Q000737:chemistry / Q000494:pharmacology; D011758:Pyrroles / Q000737:chemistry / Q000494:pharmacology; D011759:Pyrrolidines / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D011684:Purine Nucleosides; D011744:Pyrimidinones; D011758:Pyrroles; D011759:Pyrrolidines; C113100:immucillin G; C113101:forodesine; D009699:N-Glycosyl Hydrolases; D000225:Adenine; D000241:Adenosine; C517546:galidesivir