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The C-terminal nucleotide-binding domain (NBD2) of a P-glycoprotein-like transporter, encoded by the ltrmdr1 gene in Leishmania tropica and involved in parasite multidrug resistance (MDR), was overexpressed in Escherichia coli as a hexahistidine tagged protein and purified. The L. tropica recombinant domain efficiently bound fluorescent derivatives of ATP, the hydrophobic steroid analogue RU 486, and different classes of flavonoids with the following efficiency: flavone > flavanone > isoflavone > glucorhamnosyl-flavone > chromone. The affinity for flavones was dependent on the presence of hydroxyl groups at positions 5 and 3 and was further increased by a hydrophobic 1,1-dimethylallyl substituent at position 8. When flow cytometry was used to measure daunomycin accumulation in a MDR L. tropica line, a reversing effect was observed with flavones such as dimethylallyl-kaempferide at low concentration or apigenin at higher concentration, but neither with the glucorhamnosyl derivative rutin nor with the isoflavone genistein. The in vivo reversing effect of dimethylallyl-kaempferide was correlated to a high inhibition of MDR cell growth in the presence of daunomycin. The results suggest that flavone inhibition of both daunomycin efflux and parasite growth in the presence of the drug correlates to direct binding of the compound to cytosolic domain of the P-glycoprotein-like transporter.

We selected a Leishmania tropica cell line resistant to daunomycin (DNM) that presents a multidrug-resistant (MDR) phenotype characterized by overexpression of a P-glycoprotein of 150 kDa. The resistant line overexpressed an MDR-like gene, called ltrmdr1, located in an extrachromosomal circular DNA. DNM uptake experiments using laser flow cytometry showed a significant reduction in drug accumulation in the resistant parasites. The initial stages of the interaction of DNM with membranes from wild-type and DNM-resistant parasites were defined by a rapid kinetic stopped-flow procedure which can be described by two kinetic components. On the basis of a previous similar kinetic study with tumor cells, we ascribed the fast component to rapid interaction of DNM with membrane surface components and the slow component to passive diffusion of the drug across the membranes. The results reported here indicate that entrance of DNM into wild-type parasites was facilitated in respect to the resistant ones. We propose that resistance to DNM in L. tropica is a multifactorial event involving at least two complementary mechanisms. an altered drug membrane permeability and the overexpression of a protein related to P-glycoprotein that regulates drug efflux.

We have selected for a Leishmania infantum cell line resistant to high levels of methotrexate (MTX). The resulting cells were 1233-fold more resistant than wild-type and contained amplified H-region circles. Homologous genes to the antifolate resistant ltdh gene and to the P-glycoprotein ltpgpA gene of Leishmania tarentolae were observed to be contained within the amplicon. In order to invoke additional mechanisms of resistance, we examined possible variations in MTX accumulation. Resistance was not correlated with a decreased uptake of MTX. On the contrary, the resistant line presented a 3-fold increase in the steady-state accumulation of drug with regard to the wild-type line. Northern blot analysis using gene specific probes, showed that the ltdh probe and the ltpgpA probe recognized single transcripts of 1 kb and 5 kb respectively which were both overexpressed only approx. 5-fold in resistant cells. We propose that amplification of the antifolate resistance gene, homologue to the ltdh gene of L. tarentolae, is apparently the only mechanism involved in resistance to the cytotoxic drug MTX in L. infantum resistant to 1000 microM of MTX.

A methotrexate (MTX)-resistant Leishmania tropica line develops a stable drug-resistant phenotype in which the resistance mechanism is associated with a significant reduction in MTX accumulation. After a 2 hr exposure to [3H]MTX, a L. tropica line resistant to 1000 microM of MTX did not accumulate more than 3% of the amount of drug incorporated by wild-type cells. The same resistant cell line was found to be cross-resistant to several unrelated drugs. The monoclonal antibody C219, directed against the cytoplasmic domain of mammalian P-glycoproteins, recognized a putative P-glycoprotein of 240 kDa overexpressed in the resistant line. Also, this resistant line showed the overexpression of the putative homolog of the ltpgpE gene, as determined by northern blot analysis using gene-specific probes for the P-glycoprotein genes of Leishmania tarentolae. This overexpression was not correlated with a proportional increase in the copy number of the gene, but Southern blot analysis suggested that the ltpgpE homolog was overexpressed as a consequence of gene rearrangement. This would be considered as an epiphenomenon that probably does not arise from the same MTX-resistant mechanism.