Combination of Suboptimal Doses of Inhibitors Targeting Different Domains of LtrMDR1 Efficiently Overcomes Resistance of Leishmania spp. to Miltefosine by Inhibiting Drug Efflux

Abstract

Miltefosine (hexadecylphosphocholine) is the first orally active drug approved for the treatment of leishmaniasis. We have previously shown the involvement of LtrMDR1, a P-glycoprotein-like transporter belonging to the ATP-binding cassette superfamily, in miltefosine resistance in Leishmania. Here we show that overexpression of LtrMDR1 increases miltefosine efflux, leading to a decrease in drug accumulation in the parasites. Although LtrMDR1 modulation might be an efficient way to overcome this resistance, a main drawback associated with the use of P-glycoprotein inhibitors is related to their intrinsic toxicity. In order to diminish possible side effects, we have combined suboptimal doses of modulators targeting both the cytosolic and transmembrane domains of LtrMDR1. Preliminary structure-activity relationships have allowed us to design a new and potent flavonoid derivative with high affinity for the cytosolic nucleotide-binding domains. As modulators directed to the transmembrane domains, we have selected one of the most potent dihydro-␤-agarofuran sesquiterpenes described, and we have also studied the effects of two of the most promising, latest-developed modulators of human P-glycoprotein, zosuquidar (LY335979) and elacridar (GF120918). The results show that this combinatorial strategy efficiently overcomes P-glycoprotein-mediated parasite miltefosine resistance by increasing intracellular miltefosine accumulation without any side effect in the parental, sensitive, Leishmania line and in different mammalian cell lines.

The Oral Antimalarial Drug Tafenoquine Shows Activity against Trypanosoma brucei

“…Transmission electron microscopy (TEM) was performed as described previously (20). Briefly, BSF parasites were incubated as described above with 1 M TFQ and fixed with glutaraldehyde (2.5%) for 4 h at 4°C.…”

Sitamaquine Overcomes ABC-Mediated Resistance to Miltefosine and Antimony inLeishmania

“…The 50% effective concentration (EC 50 ) is the concentration of drug that decreases the rate of cell growth by 50%; the resistance factor is expressed as the ratio of the EC 50 of the MDR line (in the presence or absence of inhibitor) to the EC 50 of the WT line. The effect of the inhibitors on the accumulation of [ 14 C]miltefosine was analyzed as described previously (28). (46), and Staphylococcus aureus Sav1866 multidrug transporter (PDB code: 2HYD) (10), which share a sequence identities with LMDR1 of 24 and 28%, respectively (see Table S1 in the supplemental material), as templates.…”

“…LMDR1 also confers resistance to daunomycin, puromycin, vinblastine, adriamycin, and edelfosine (6,29). Although it cannot be efficiently inhibited by classical inhibitors of human Pgp such as verapamil and cyclosporine (29), we have previously shown that hemisynthetic flavonoids (27,31) and ␤-agarofuran sesquiterpenes (33) are able to inhibit LMDR1 by binding to nucleotide binding domains and to transmembrane domains, respectively (28). These two different targets in LMDR1 have allowed the combination of low concentrations of these compounds to inhibit transporter activity (28), although both their price and difficult preparation could hamper any future clinical use.…”

“…LMDR1, which was the first protein found to confer miltefosine resistance in Leishmania (32), reduces the intracellular accumulation of miltefosine due to active drug efflux (28). LMDR1 also confers resistance to daunomycin, puromycin, vinblastine, adriamycin, and edelfosine (6,29).…”

“…Although it cannot be efficiently inhibited by classical inhibitors of human Pgp such as verapamil and cyclosporine (29), we have previously shown that hemisynthetic flavonoids (27,31) and ␤-agarofuran sesquiterpenes (33) are able to inhibit LMDR1 by binding to nucleotide binding domains and to transmembrane domains, respectively (28). These two different targets in LMDR1 have allowed the combination of low concentrations of these compounds to inhibit transporter activity (28), although both their price and difficult preparation could hamper any future clinical use. We have therefore continued our search for new agents that can reverse miltefosine resistance in Leishmania by considering that the ideal features of a good LMDR1 inhibitor should include, among others requirements, a low toxicity for mammalian cells, an inability to be transported by LMDR1, and, if possible, a specific leishmanicidal effect.…”