Semisynthesis of ω-Hydroxyalkylcarbonate Derivatives of Hydroxytyrosol as Antitrypanosome Agents.

Several lipophilic ω-hydroxyalkylcarbonate hydroxytyrosol derivatives and also their corresponding dimeric derivatives have been synthesized, coupling the primary hydroxy group of this phenolic compound with several terminal diols of different chain lengths, by the use of a carbonate linker. The trypanocidal activity and cytotoxicity of these ω-hydroxyalkylcarbonate derivatives of hydroxytyrosol and known alkylcarbonate derivatives of hydroxytyrosol were assessed. Three of the hydroxytyrosol alkylcarbonate derivatives were active against Trypanosoma brucei: two with an alkyl chain of average size (0.2 and 0.5 µM) and another with a double bond in the alkyl chain (0.4 µM). These values suggest an increase in activity with respect to hydroxytyrosol (264-, 90-, and 116-fold, respectively). Furthermore, these compounds showed high selectivity indices against MRC-5, a nontumor human cell line (62, 71, and 39, respectively). Some other ω-hydroxyalkylcarbonate and alkylcarbonate derivatives of hydroxytyrosol were also active against T. brucei within a low micromolar range (about 1 µM).

Synthesis, Binding Properties, and Differences in Cell Uptake of†G-Quadruplex Ligands Based on Carbohydrate Naphthalene Diimide Conjugates.

The G-quadruplexes (G4s) are currently being explored as therapeutic targets in cancer and other pathologies. Six carbohydrate naphthalene diimide conjugates (carb-NDIs) have been synthesized as G4 ligands to investigate their potential selectivity in G4 binding and cell penetration. Carb-NDIs have shown certain selectivity for G4 structures against DNA duplexes, but different sugar moieties do not induce a preference for a specific G4 topology. Interestingly, when monosaccharides were attached through a short ethylene linker to the NDI scaffold, their cellular uptake was two- to threefold more efficient than that when the sugar was directly attached through its anomeric position. Moreover, a correlation between more efficient cell uptake of these carb-NDIs and their higher toxicity in cancerous cell lines has been observed. Carb-NDIs seem to be mainly translocated into cancer cells through glucose transporters (GLUT), of which GLUT4 plays a major role.

Sitamaquine sensitivity in Leishmania species is not mediated by drug accumulation in acidocalcisomes.

Sitamaquine (WR6026), an 8-aminoquinoline derivative, is a new antileishmanial oral drug. As a lipophilic weak base, it rapidly accumulates in acidic compartments, represented mainly by acidocalcisomes. In this work, we show that the antileishmanial action of sitamaquine is unrelated to its level of accumulation in these acidic vesicles. We have observed significant differences in sitamaquine sensitivity and accumulation between Leishmania species and strains, and interestingly, there is no correlation between them. However, there is a relationship between the levels of accumulation of sitamaquine and acidotropic probes, acidocalcisomes size, and polyphosphate levels. The Leishmania major AP3delta-null mutant line, in which acidocalcisomes are devoid of their usual polyphosphate and proton content, is unable to accumulate sitamaquine; however, both the parental strain and the AP3delta-null mutants showed similar sensitivities to sitamaquine. Our findings provide clear evidence that the antileishmanial action of sitamaquine is unrelated to its accumulation in acidocalcisomes.

Characterization of an ABCG-like transporter from the protozoan parasite Leishmania with a role in drug resistance and transbilayer lipid movement.

Leishmaniasis treatment is hampered by the increased appearance of treatment failure. ATP-binding cassette (ABC) transporters are usually involved in drug resistance both in tumor cells and in microorganisms. Here we report the characterization of an ABCG-like transporter, LiABCG6, localized mainly at the plasma membrane in Leishmania protozoan parasites. When overexpressed, this half-transporter confers significant resistance to the leishmanicidal agents miltefosine and sitamaquine. This resistance phenotype is mediated by a reduction in intracellular drug accumulation. LiABCG6 also reduces the accumulation of short-chain fluorescent phospholipid analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. As a whole, these results suggest that LiABCG6 could be implicated in phospholipid trafficking and drug resistance.

G-Quadruplex Identification in the Genome of Protozoan Parasites Points to Naphthalene Diimide Ligands as New Antiparasitic Agents.

G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4's including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.

Celastraceae sesquiterpenes as a new class of modulators that bind specifically to human P-glycoprotein and reverse cellular multidrug resistance.

Overexpression of ABCB1 (MDR1) P-glycoprotein, a multidrug efflux pump, is one mechanism by which tumor cells may develop multidrug resistance (MDR), preventing the successful chemotherapeutic treatment of cancer. Sesquiterpenes from Celastraceae family are natural compounds shown previously to reverse MDR in several human cancer cell lines and Leishmania strains. However, their molecular mechanism of reversion has not been characterized. In the present work, we have studied the ability of 28 dihydro-beta-agarofuran sesquiterpenes to reverse the P-glycoprotein-dependent MDR phenotype and elucidated their molecular mechanism of action. Cytotoxicity assays using human MDR1-transfected NIH-3T3 cells allowed us to select the most potent sesquiterpenes reversing the in vitro resistance to daunomycin and vinblastine. Flow cytometry experiments showed that the above active compounds specifically inhibited drug transport activity of P-glycoprotein in a saturable, concentration-dependent manner (K(i) down to 0.24 +/- 0.01 micromol/L) but not that of ABCC1 (multidrug resistance protein 1; MRP1), ABCC2 (MRP2), and ABCG2 (breast cancer resistance protein; BCRP) transporters. Moreover, sesquiterpenes inhibited at submicromolar concentrations the P-glycoprotein-mediated transport of [(3)H]colchicine and tetramethylrosamine in plasma membrane from CH(R)B30 cells and P-glycoprotein-enriched proteoliposomes, supporting that P-glycoprotein is their molecular target. Photoaffinity labeling in plasma membrane and fluorescence spectroscopy experiments with purified protein suggested that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Finally, sesquiterpenes modulated P-glycoprotein ATPase-activity in a biphasic, concentration-dependent manner: they stimulated at very low concentrations but inhibited ATPase activity as noncompetitive inhibitors at higher concentrations. Sesquiterpenes from Celastraceae are promising P-glycoprotein modulators with potential applications in cancer chemotherapy because of their MDR reversal potency and specificity for P-glycoprotein.

Tyrosol and hydroxytyrosol derivatives as antitrypanosomal and antileishmanial agents.

Trypanosomiasis and leishmaniasis keep being a real challenge for health and development of African countries. Existing treatments have considerable side effects and increase resistance of the parasites. We have measured antitrypanosomal and antileishmanial activity of natural phenols, tyrosol (TYR) and hydroxytyrosol (HT) and several of their esters and metabolites. We found significant IC50 values against Trypanosoma brucei for HT decanoate ester and HT dodecanoate ester (0.6 and 0.36 µM, respectively). This represents a large increase in activity with respect to HT (79 and 132 fold, respectively). Moreover, both compounds displayed a high selectivity index against MRC-5, a non-tumoral human cell line (118 and 106, respectively). Then, we synthesized a focused library of compounds to explore structure-activity. We found the ether and thiourea analogs of HT decanoate ester and HT dodecanoate ester also showed IC50 values against T. brucei in the low micromolar range. In conclusion, the di-ortho phenolic ring and medium size alkyl chain are essential for activity whereas the nature of the chemical bond among them seems less important.

The overexpression of a new ABC transporter in Leishmania is related to phospholipid trafficking and reduced infectivity.

This paper reports the characterization of a new ABC transporter (LtrABC1.1), related to the human ABCA subfamily, in the protozoan parasite Leishmania tropica. LtrABC1.1 is a tandem duplicated gene flanked by inverted repeats. LtrABC1.1 is expressed mainly in the flagellar pocket of the parasite. Drug resistance studies in Leishmania overexpressing LtrABC1.1 showed the transporter not to confer resistance to a range of unrelated drugs. LtrABC1.1 appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. The activity of this protein may also affect membrane movement processes since secreted acid phosphatase (SAP) activity was significantly lower in promastigotes overexpressing LtrABC1.1. In vitro infection experiments with macrophages indicated LtrABC1.1-transfected parasites to be significantly less infective. Together, these results suggest that this new ABC transporter could play a role in lipid movements across the plasma membrane, and that its activity might influence vesicle trafficking. This is the first ABCA-like transporter described in unicellular eukaryotes.