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

PLoS One. 2015 Apr 24;10(4):e0124183. doi: 10.1371/journal.pone.0124183. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / analogs & derivatives
  • Adenine / chemistry
  • Adenine / pharmacology
  • Adenosine / analogs & derivatives
  • Animals
  • Antiprotozoal Agents / chemistry
  • Antiprotozoal Agents / pharmacology*
  • Cell Proliferation / drug effects
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Female
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Leishmania infantum / drug effects*
  • Leishmania infantum / growth & development
  • Leishmania infantum / ultrastructure
  • Leishmania mexicana / drug effects*
  • Leishmania mexicana / growth & development
  • Leishmania mexicana / ultrastructure
  • Leishmaniasis, Cutaneous / drug therapy
  • Leishmaniasis, Visceral / drug therapy
  • Mice
  • Mice, Inbred BALB C
  • Microscopy, Electron, Transmission
  • N-Glycosyl Hydrolases / antagonists & inhibitors
  • Purine Nucleosides / chemistry
  • Purine Nucleosides / pharmacology
  • Pyrimidinones / chemistry
  • Pyrimidinones / pharmacology
  • Pyrroles / chemistry
  • Pyrroles / pharmacology
  • Pyrrolidines / chemistry
  • Pyrrolidines / pharmacology

Substances

  • Antiprotozoal Agents
  • Enzyme Inhibitors
  • Purine Nucleosides
  • Pyrimidinones
  • Pyrroles
  • Pyrrolidines
  • immucillin G
  • forodesine
  • N-Glycosyl Hydrolases
  • Adenine
  • Adenosine
  • galidesivir

Grants and funding

This work has been supported by: EOF, DN, JRMF, CPS: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ, Fellowships 301215-2007-3, 302039/2010-4, 559756/2010-0 and grant 404400/ 2012-4), by Fundação de Amparo à Pesquisa do Estado de Rio de Janeiro (FAPERJ, grants E-26/102733/2008 and E-26/102957/2011 and Fellowships E-26/102415/2010 and E-26/110535/2010). EOF, VLS, RG, CPS: PROLAB Award PABMB/ASBMB PROLAB (Promoting Research Opportunities For Latin American Biochemists (PROLAB) of the American Society for Biochemistry and Molecular Biology). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.