BackgroundAntimony resistance complicates the treatment of infections caused by the parasite Leishmania.Methodology/Principal FindingsUsing next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion.Conclusions/SignificanceThis is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites.

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Livers were evaluated for hepatocytes histological alterations, peroxidase activity, and apoptosis. Increased proportions of swollen and apoptotic hepatocytes were observed in animals treated with Glu compared to animals treated with saline or MA. The peroxidase activity was also enhanced in the liver of animals that received Glu. Cotreatment with AA reduced the extent of histological changes, the apoptotic index, and the peroxidase activity to levels corresponding to the control group. Moreover, the association with AA did not affect the hepatic uptake of Sb and the ability of Glu to reduce the liver and spleen parasite loads in infected mice. In conclusion, our data supports the use of pentavalent antimonials with low residue of Sb(III) and the association of pentavalent antimonials with AA, as effective strategies to reduce side effects in antimonial therapy.

Antimonial compounds have been used for more than a century in the treatment of the parasitic disease leishmaniasis. Although pentavalent antimonials are still first-line drugs in several developing countries, this class of drugs is no longer recommended in the Indian sub-continent because of the emergence of drug resistance. The precise mechanisms involved in the resistance of leishmania parasites to antimony are still subject to debate. It is now well documented that drug resistance in leishmania parasites is a multifactorial phenomenon involving multiple genes whose expression pattern synergistically leads to the resistance phenotype. The reduction of intracellular antimony accumulation is a frequent change observed in resistant leishmania cells; however, no comprehensive transport model has been presented so far to explain this change and its contribution to Leishmania resistance. The present review firstly covers the actual knowledge on the metabolism of antimonial drugs, the mechanisms of their transmembrane transport and intracellular processing in Leishmania. It further describes both the functional and molecular changes associated with Sb resistance in this organism. Possible transport models based on the actual knowledge are then presented, as well as their functional implications. Biophysical and pharmacological strategies are finally proposed for the precise identification of the transport pathways.

Two novel trivalent antimony(III) and bismuth(III) complexes with the nitrogen-donor heterocyclic ligand dipyrido[3,2-a:2',3'-c]phenazine (dppz) were synthesized and characterized as [Sb(dppz)Cl3]∙H2O∙CH3OH and [Bi(dppz)Cl3]. The crystal structure of Sb(III) complex was determined by X-ray crystallography. These complexes were evaluated for their activity against the promastigote form of Sb(III)-sensitive and -resistant Leishmania infantum chagasi and Leishmania amazonensis strains. Both complexes were more effective than dppz alone in inhibiting the growth of Leishmania promastigotes and were at least 77 and 2,400 times more active than potassium antimonyl tartrate in Sb(III)-sensitive and -resistant Leishmania, respectively. The cytotoxicity of dppz and its complexes against mouse peritoneal macrophages occurred at dppz concentrations at least 6-fold greater than those found to be active against Leishmania promastigotes.To investigate the role of the metal in the improved antileishmanial activity of dppz, the activity of the Sb(III) complex was compared between the Sb-resistant mutants and their respective parental sensitive strains. The lack of cross-resistance to the Sb(III)-dppz complex together with the much lower activity of antimonyl tartrate, SbCl3 and BiCl3 strongly support the model that the metal is not active by itself but improves the activity of dppz through complexation.

Two bismuth(III) porphyrins-5,10,15,20-tetrakis(phenyl)porphyrinatobismuth(III) nitrate, [Bi(III)(TPP)]NO3, and the unprecedent 5,10,15,20-tetrakis(4-carbomethoxyphenyl)porphyrinatobismuth(III) nitrate, [Bi(III)(T4CMPP)]NO3, and two unprecedented antimony(V) porphyrins dichlorido(5,10,15,20-tetrakis(phenyl)porphyrinato)antimony(V) bromide, [Sb(V)(TPP)Cl2]Br, and dibromido(5,10,15,20-tetrakis(4-carbomethoxyphenyl)porphyrinato)antimony(V) bromide, [Sb(V)(T4CMPP)Br2]Br,-were synthesized by reacting the corresponding porphyrin ligand with Bi(NO3)3·5H2O or SbCl3. All compounds were characterized by UV-vis, (1)H NMR spectroscopy, and mass spectrometry. The new compounds were also characterized by elemental analysis. Because antimony and bismuth compounds have been widely applied in medicine, the activity of these complexes was tested against Sb-sensitive and -resistant Leishmania amazonensis parasites. [Sb(V)(T4CMPP)Br2]Br was more active against the promastigote form of Sb-resistant mutant strain as compared to the sensitive parental strain, with IC50 in the micromolar range. These data contrasted with those obtained using the Sb(III) drug potassium antimony tartrate, which displayed IC50 of 110 μmol L(-1) against the Sb-sensitive parasite and was almost inactive against the Sb-resistant strain. The H2T4CMPP ligand also showed antileishmanial activity against Sb-resistant and -sensitive strains, but with IC50 at least tenfold greater than that of the complex. The Sb(V)-porphyrin complex was also active against intracellular amastigotes and showed a higher selectivity index than the conventional Sb(V) drug glucantime, in both Sb-sensitive and -resistant strains. The greater antileishmanial activity of this complex could be attributed to an increased cellular uptake of Sb. Thus, [Sb(V)(T4CMPP)Br2]Br constitutes a new antileishmanial drug candidate.

Antimonial drugs have been used for a century in the therapy of the parasitic disease leishmaniasis. Even though pentavalent antimonials are still first-line drugs, they exhibit several limitations, including severe side effects, the need for daily parenteral administration and drug resistance. The molecular structure of pentavalent antimonials, their metabolism and mechanism of action, are still being investigated. Previous studies suggest that pentavalent antimony acts as a prodrug which is converted to the active and more toxic trivalent antimony. Other works support the direct involvement of pentavalent antimony. Recent data indicate that thiols and ribose-containing biomolecules may mediate the pharmacological action of these drugs. Trypanothione reductase and zinc-finger proteins were identified as possible molecular targets. This review summarizes the progress achieved to date on the chemistry of antimonial drugs in biological systems.

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