Functional expression of a multidrug P-glycoprotein transporter of Leishmania Fernando Corte ´s-Selva a , Francisco Mun ˜ oz-Martı ´nez a , Attila Ilia ´s b , Antonio Ignacio Jime ´nez c , Andra ´s Va ´radi b , Francisco Gamarro a , Santiago Castanys a, * a Instituto de Parasitologı ´a y Biomedicina ‘‘Lo ´ pez-Neyra’’, Consejo Superior de Investigaciones Cientı ´ficas, Parque Tecnolo ´ gico de Ciencias de la Salud, Avda. del Conocimiento s/n, 18100 Armilla, Granada, Spain b Institute of Enzymology, Hungarian Academy of Sciences, Karolina ut 29, 1113 Budapest, Hungary c Instituto Universitario de Bio-Orga ´ nica ‘‘Antonio Gonza ´ lez’’, Universidad de La Laguna, Avenida Astrofı ´sico Francisco Sa ´ nchez, 2, 38206 La Laguna, Tenerife, Spain Received 28 January 2005 Abstract P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Bac- ulovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-b-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line over- expressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents. Ó 2005 Elsevier Inc. All rights reserved. Keywords: ABC transporter; P-glycoprotein; Leishmania; Multidrug resistance; Insect cells; Modulator; Drug transport Leishmaniasis is a worldwide parasitic disease, affect- ing 88 countries. Some 12 million people throughout the world suffer from any of the four major forms of leish- maniasis (cutaneous, diffuse cutaneous, mucocutaneous, and visceral) and 2 million new cases are estimated to occur annually. Leishmaniasis is spreading in several areas of the world as a result of epidemiological changes that sharply increase the overlapping of AIDS and vis- ceral leishmaniasis [1]. The main control strategy is case finding and treatment plus, when feasible, vector con- trol. Pentavalent antimonials are still first-line drugs, after more than six decades of use. But the treatment is long, given systemically, toxic, and expensive. More- over, the appearance of antimonial resistance has chan- ged the pattern of leishmaniasis treatment in the world. Miltefosine (hexadecylphosphocholine) has been estab- lished within the last years as the first effective and safe oral treatment against leishmaniasis, although its leish- manicidal mechanism is not fully understood. The understanding of the molecular drug-resistance mecha- nisms that Leishmania adopts or may adopt in the future is of high clinical relevance. ATP-binding cassette (ABC) transporters are in- volved, at least in vitro, in the resistance to many anti- parasitic drugs [2–4]. In addition, new putative antiparasitic drugs such as anthracyclines [5], taxol [6], and azoles [7,8] are known substrates of ABC transport- ers, and thus could induce a multidrug resistance (MDR) phenotype. Leishmania P-glycoprotein (Pgp) is 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.02.009 * Corresponding author. Fax: +34 958 181633. E-mail address: castanys@ipb.csic.es (S. Castanys). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 329 (2005) 502–507 BBRC