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Instituto de Parasitología y Biomedicina
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ABC GENES IN PROTOZOAN PARASITES



Jefe de grupo
    • Santiago Castanys Cuello     
        email: castanys (@ipb.csic.es)
        Tlf: 958181666

    • Francisco Gamarro Conde     
        email: gamarro (@ipb.csic.es)
        Tlf: 958181667



    Personal Investigador Postdoctoral
    • Raquel García Hernández     
    • Verónica Gómez Pérez     
    • José Ignacio Manzano González     
    • María Mercedes Pérez Sánchez-Cañete     


    Personal Investigador Predoctoral
    • Ana Perea Martínez     

     

    LINEAS DE INVESTIGACIÓN


     

    ABC GENES IN PROTOZOAN PARASITES

     

    Our group is included at the Department of Biochemistry and Molecular Pharmacology, Instituto de Parasitología "Lopez-Neyra", Spanish Research Council. The group has experience in the area of Biology and Molecular and Cellular Pharmacology of the protozoan parasites of the family Trypanosomatidae, specifically Trypanosoma cruzi and Leishmania.









     

    Mechanisms of drug resistance in parasites. Search for drug resistance markers.

     

    Presently, we are focusing on the study of miltefosine(hexadecylphosphocholine) (MIL) resistance, the first oral drug active visceral leishmaniasis. We have found that miltefosine resistant parasites are deficient in the inward-directed translocation across the plasma membrane of both MIL and glycerophospholipids, independently of their polar head group, in a process that is protein, energy and temperature dependent, without modification of drug metabolism or endocytic activity. Recently, we have described in Leishmania donovani that resistance to MIL and other phospholipid derivatives can be mediated through a mutation in the MIL transporter (LdMT), a new member of the P-type ATPase aminophospholipid translocase subfamily expressed at the plasma membrane. LdMT mediates the translocation of MIL and glycerophospholipids in Leishmania. Transfection experiments demonstrated that LdMT was able to rescue inward translocation and thus to increase uptake of MIL in resistant parasites. The absence of deletions or rearrangements at the LdMT locus in resistant parasites and the presence of similar expression levels in both mutant and wild-type lines suggested the presence of stable mutations of the gene in the MIL resistant parasites. PCR-based approach identified single but different point mutations in both alleles that are able to inactivate LdMT function. Similarly, in Leishmania resistant lines obtained by mutagenesis, we observed not only a similar phenotype but also a similar genetic basis for the acquisition of resistance: inactivating point mutations within LdMT. Also, we have found that the sensitivity to MIL in Leishmania was directly related with the level of expression of LdMT. We have obtained double knock-out for LdMT and these experiments support that LdMT is essential for the uptake and potency of MIL in L. donovani. Also, the intracellular forms of the parasite obtained from the above mutants, shows similar resistance level to MIL. Thus, these results suggest that the generation of MIL-resistant clinical isolates bearing specific mutations in LdMT is likely to occur. Our results represent the first description of one phospholipid transporter in parasites, whose biological role was related with the maintenance of lipid asymmetry at the plasma membrane. We have established a direct correlation between the sensitivity of these Leishmania species and their drug accumulation profile. Interestingly, L. braziliensis, refractory to the activity of the drug, is unable to accumulate MIL compared with other Leishmania species, indicating that MIL will be only useful in geographical areas where sensitive species are prevalent. We continue with the functional characterization of LdMT and its involvement in phospholipid trafficking. We are interested to know the minimal complex for the functionality of this transporter; if it is required another accessory protein for the transport and activity of LdMT. Considering that aminophospholipid translocase subfamily in Leishmania include 4 different transporters, we will continue with the functional characterization of other members of this subfamily. Considering the increasing problem of drug resistance in Leishmania, we shall explore possible molecular mechanisms of resistance to different leishmanicidal drugs, among others: alkyl-lysophospholipids (miltefosine), sitamaquine and paramomycin, using pharmacoproteomic analysis. This study will give us knowledge of drug resistance markers that could be used in clinical samples with therapeutic failure. All this information will be used to drug design that will be crucial for the control of Leishmaniasis which incidence represents a World Health problem in addition to have a great impact in the economy of the populations affected.









     

    Functional characterization of ABC (ATP-binding cassette) transporters.

     

    The ABC superfamily of transporters is thought to be one of the largest protein families present throughout evolution. They transport a range of structurally unrelated compounds using ATP hydrolysis as their energy source. These transporters are composed of two transmembrane domains and two nucleotide binding domains that contain the conserved Walker A and Walker B motifs as well as the ABC signature. This superfamily include 7 different subfamilies: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF y ABCG; these subfamilies are divided into half-transporters, which contain one ATPase domain and one transmembrane domain (ABCD and ABCG subfamilies, and some members of the ABCB subfamily) and full transporters which contain paired modules in tandem (ABCA, ABCB and ABCC subfamilies). The ABCE and ABCF subfamilies contain proteins, which have ATP-binding domains that are clearly derived from others ABC transporters, but have no transmembrane domain and are not know to be involved in any membrane transport functions. In last years, we have been characterizing the functionality of some of these subfamilies in the protozoan parasites Leishmania and Trypanosoma cruzi. Thus, the Leishmania Pgp-MDR1 transporter (included in the ABCB subfamily) is responsible of a multidrug resistant phenotype to different structurally and functionality unrelated drugs. Additionally, this transporter is able to translocate different glycerophospholipids, whose function could be related with a floppase activity energy-dependent, consequently conferring resistance to different phospholipid analogues such as alkyl-lysophospholipids. Similarly, we have characterized the ABCA subfamily in Leishmania and T. cruzi; this subfamily appears to be involved in lipid movements across the plasma membrane of the parasite since overexpression reduces the accumulation of fluorescent phospholipid analogues. Also, this activity may influence the vesicular trafficking, since secreted acid phosphatase (SAP) activity was significantly lower in parasites overexpressing this transporter, as well as the infectivity of parasites. Recently, we are interested in the functional characterization of the half transporters included in the subfamily ABCG in the parasites Leishmania and Trypanosoma brucei. The ABCG or White subfamily of transporters is the latest drug efflux ABC transporters discovered. Among the subfamily, ABCG2 is a well-know efflux ABC transporter identified by different approaches, receiving different and equivalent names such as placenta-specific ABC (ABCP), a breast cancer resistance protein (BCRP) and a mitoxantrone resistance associated protein (MXR). The members of this subfamily have the ATPase catalytic domain located N-terminal to the transmembrane domain. It is generally accepted that ABC half-transporters function as homo- or heterodimers. The members of this subfamily ABCG2 has been shown to be amplified and overexpressed in human cancer cells resistance to different hydrophobic drugs such as mitoxantrone, topotecan,daunorubicin, doxorubicin, methotrexate, camptothecins, flavopiridol, among others. In Leishmania, the ABCG subfamily includes at least 4 different transporters; the overexpression of ABCG15 and ABCG36 in Leishmania confers resistance to hydrophobic drugs such as miltefosine, edelfosine and perifosine. As described in mammalian cells, ABCG transporters function as floppases, able to translocate fluorescent phospholipid analogues. We continue with the characterization of ABCG transporters and its involvement on lipid trafficking and their influence in the biology of the protozoan parasites Leishmania and Trypanosoma brucei.









     

    Reversal agents of drug resístance phenotype mediated by ABC transporters in Leishmania and tumoral cells.

     

    During last years, we have been interested on the search of new natural compounds able to modulate the activity of ABC transporters both in Leishmania parasites and cancer cells overexpressing the P-glycoprotein MDR1. Among the active compounds identified in plant extracts of the Celastraceae family are sesquiterpenes, which constitute a wide family of natural compounds with a considerable range of bioactive properties and with potential clinical application as MDR reversal agents in cancer cells and in the protozoan parasite Leishmania. We have characterized a total of 58 and 79 different dihydro-beta-agarofuran sesquiterpenes as reversal agents of Leishmania and cancer MDR phenotype, respectively. The results have allowed us to identify the most potent sesquiterpenes and to assess the specific interaction with the P-glycoprotein. Similar experiments using MRP (ABCC) and BCRP (ABCG)-expressing cells showed that sesquiterpenes do not modify the activities of these ABC transporters. Direct molecular interaction of active sesquiterpenes with plasma membranes from cells overexpressing P-glycoprotein and purified protein, and photoaffinity labelling confirm that sesquiterpenes interact with transmembrane domains of P-glycoprotein. Moreover, the identification of P-glycoprotein as the cellular target of sesquiterpenes and the knowledge about their molecular mechanism of action has stimulated us to start the studies directed to a computer-assisted quantitative-structrural activity relationship model (QSAR) that will allow the rational design of new reversal agents more potents and less toxics. Moreover, we continue the characterization of the mechanism of action of sesquiterpenes in Leishmania and mammalian cells such as: influence on protein expression levels, activity on cytochrome P450, and induction of apoptosis on MDR overexpressing cells. Additionally, we have started the search for reversal natural compounds specific for ABCC (MRP) and ABCG (BCRP) transporters of Leishmania and cancer cells.









     


    ORGANISMOS FINANCIADORES ÚLTIMOS 5 AÑOS

    - ANALISIS DE AISLADOS CLINICOS DE LEISHMANIA INFANTUM PROCEDENTES DE FALLO TERAPEUTICO: BUSQUEDA DE MARCADORES DE RESISTENCIA.. PROYECTO, PN2015 - PROY I+D+I - PRG. RETOS DE LA SOCIEDAD, Ref: SAF2015-68042-R, (2016 - 2018).

    - TRATAMIENTO DE LEISHMANIASIS: ESTUDIO DE LA COMBINATORIAL DE FARMACOS Y BUSQUEDA DE MARCADORES DE RESISTENCIA.. PROYECTO, , Ref: P11-CTS-7282, (2013 - 2016).

    - IMPLICACIÓN DE LOS TRANSPORTADORES ABCG Y OTROS TRANSPORTADORES ABC ESPECÍFICOS DE TRIPANOSOMATIDOS EN LA INFECTIVIDAD Y RESISTENCIA A FÁRMACOS EN LEISHMANIA. PROYECTO, PLAN NACIONAL 2012, Ref: SAF2012-34267, (2013 - 2015).

    - Tratamiento de la leishmaniasis: estudio de la combinatorial de fármacos y búsqueda de marcadores de resistencia. PROYECTO, PROYECTOS INTRAMURALES, Ref: 201220E051, (2012 - 2013).

    - MECANISMO DE RESISTENCIA A MILTEFOSINA EN LEISHMANIA: CARACTERIZACION DEL COMPLEJO DE TRANSPORTE DEL FARMACO. PROYECTO, PN2011 - I.F.N.O.- BIOMEDICINA, Ref: SAF2011-28102, (2012 - 2014).

    - Estudio de la resistencia a la combinatorial de fármacos en el parásito Leishmania. PROYECTO, PROYECTOS INTRAMURALES, Ref: 201120E029, (2011 - 2012).

    - Caracterización de nuevos transportadores ABC en Leishmania: su implicación en infectividad y resistencia a fármacos. PROYECTO, INV.FUND.NO ORIENTADA.- BIOMEDICINA, Ref: SAF2009-07440, (2010 - 2012).

    - Caracterización de nuevos compuestos revertidores de la multirresistencia a fármacos mediada por la glicoproteína-P en células tumorales. PROYECTO, PLAN ANDALUZ DE INVESTIGACION, Ref: P08-CTS-03625, (2009 - 2012).

     

     

    PUBLICACIONES ÚLTIMOS 5 AÑOS

    -Manzano, J.I.; Lecerf-schmidt, F.; Lespinasse, M.; Di Pietro, A.; Castanys, S.; Boumendjel, A.; Gamarro, F., Identification of specific reversal agents for Leishmania ABCI4-mediated antimony resistance by flavonoid and trolox derivative screening, Journal of Antimicrobial Chemotherapy, 2014, Vol. 69: 664-672, ARTÍCULO

    -Annang, F.; Pérez-Moreno, G.; García-Hernández, R.; Cordon-Obras, C.; Martín , J. ; Tormo, J.R.; Rodríguez, L.; De Pedro, N.; Gómez-Pérez, V.; Valente, M.; Reyes, F.; Genilloud, O.; Vicente, F.; Castanys, S.; Ruiz-Pérez, L.M.; Navarro, M.; Gamarro, F.; González-Pacanowska, D., High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: Human African trypanosomiasis, leishmaniasis, and chagas disease, Journal of Biomolecular Screening, 2014, Vol. 20: 82-91, ARTÍCULO

    -García-Sánchez, S.; Sánchez-Cañete, M.P.; Gamarro, F.; Castanys, S., Functional role of evolutionarily highly conserved residues, N-glycosylation level and domains of the Leishmania miltefosine transporter-Cdc50 subunit, Biochemical Journal, 2014, Vol. 459: 83-94, ARTÍCULO

    -Gómez-Pérez, V.; Manzano, J.I.; García-Hernández, R.; Castanys, S.; Gamarro, F.; Campos, J.M., Design, synthesis and anti-leishmanial activity of novel symmetrical bispyridinium cyclophanes, European Journal of Medicinal Chemistry, 2014, Vol. 89: 362-369, ARTÍCULO

    -Gómez-Perez, V.; Manzano, J.I.; Garciá-Hernández, R.; Castanys, S.; Rosa, J.M.C.; Gamarro, F., 4-Amino bis-pyridinium derivatives as novel antileishmanial agents, Antimicrobial Agents and Chemotherapy, 2014, Vol. 58: 4103-4112, ARTÍCULO

    -Campos-Salinas, J.; León-Guerrero, D.; González-Rey, E.; Delgado, M.; Castanys, S.; Pérez-Victoria, J.M.; Gamarro, F., LABCG2, a New ABC Transporter Implicated in Phosphatidylserine Exposure, Is Involved in the Infectivity and Pathogenicity of Leishmania, PLoS Neglected Tropical Diseases, 2013, Vol. 7: null-, ARTÍCULO

    -Manzano, J.I.; García-Hernández, R.; Castanys, S.; Gamarro, F., A new ABC half-transporter in leishmania major is involved in resistance to antimony, Antimicrobial Agents and Chemotherapy, 2013, Vol. 57: 3719-3730, ARTÍCULO

    -Caballero, E.; Manzano, J.I.; Puebla, P.; Castanys, S.; Gamarro, F.; San Feliciano, A., Oxazolo[3,2-a]pyridine. A new structural scaffold for the reversal of multi-drug resistance in Leishmania, Bioorganic and Medicinal Chemistry Letters, 2012, Vol. 22: 6272-6275, ARTÍCULO

    -García-Hernández, R.; Manzano, J.I.; Castanys, S.; Gamarro, F., Leishmania donovani Develops Resistance to Drug Combinations, PLoS Neglected Tropical Diseases, 2012, Vol. 6: null-, ARTÍCULO

    -Pérez-Victoria, J.M., Bavchvarov, B.I., Torrecillas, I.R., Martínez-García, M., López-Martín, C., Campillo, M., Castanys, S., Gamarro, F., Sitamaquine overcomes ABC-mediated resistance to miltefosine and antimony in Leishmania, Antimicrob Agents Chemother, 2011, Vol. 55: 3838-3844, ARTÍCULO

    -Manzano, J.I.; Carvalho, L.; García-Hernández, R.; Poveda, J.A.; Ferragut, J.A.; Castanys, S.; Gamarro, F.; Garcia-Hernandez, R., Uptake of the antileishmania drug tafenoquine follows a sterol-dependent diffusion process in Leishmania. , Journal of Antimicrobial Chemotherapy, 2011, Vol. 66: 2562-2565, ARTÍCULO

    -Carvalho, L.; Luque-Ortega, J.R.; López-Martín, C.; Castanys, S.; Rivas, L.; Gamarro, F., The 8-aminoquinoline analogue sitamaquine causes oxidative stress in Leishmania donovani promastigotes by targeting succinate dehydrogenase., Antimicrobial Agents and Chemotherapy, 2011, Vol. 55: 4204-4210, ARTÍCULO

    -Kennedy, M.L.; Llanos, G.G.; Castanys, S.; Gamarro, F.; Bazzocchi, I.L.; Jiménez, I.A., Terpenoids from maytenus species and assessment of their reversal activity against a multidrug-resistant leishmania tropica line, Chemistry and Biodiversity, 2011, Vol. 8: 2291-2298, ARTÍCULO

    -Pérez-Victoria, J.M.; Bavchvarov, B.I.; Torrecillas, I.R.; Martínez-García, M.; López-Martín, C.; Campillo, M.; Castanys, S.; Gamarro, F., Sitamaquine overcomes ABC-mediated resistance to miltefosine and antimony in Leishmania. , Antimicrobial Agents and Chemotherapy, 2011, Vol. 55: 3838-3844, ARTÍCULO

    -Perestelo, N.R.; Sánchez-Cañete, M.P.; Gamarro, F.; Jiménez, I.A.; Castanys, S.; Bazzocchi, I.L., Overcoming human P-glycoprotein-dependent multidrug resistance with novel dihydro-ß-agarofuran sesquiterpenes., European Journal of Medicinal Chemistry, 2011, Vol. 46: 4915-4923, ARTÍCULO

    -Pérez-Victoria, I.; Pérez-Victoria, F.J.; Roldán-Vargas, S.; García-Hernández, R.; Carvalho, L.; Castanys, S.; Gamarro, F.; Morales, J.C.; Pérez-Victoria, J.M., Non-reducing trisaccharide fatty acid monoesters: Novel detergents in membrane biochemistry, Biochimica et Biophysica Acta - Biomembranes, 2011, Vol. 1808: 717-726, ARTÍCULO

    -Kennedy, M.L.; Cortés, F.; Piñero, J.E.; Castanys, S.; López-Arencibia, A.; Gamarro, F.; Bazzocchi, I.L.; Jiménez, I.A., Leishmanicidal and reversal multidrug resistance constituents from Aeonium lindleyi, Planta Medica, 2011, Vol. 77: 77-80, ARTÍCULO

    -Manzano, J.I.; Carvalho, L.; Pérez-Victoria, J.M.; Castanys, S.; Gamarro, F., Increased glycolytic ATP synthesis is associated with tafenoquine resistance in Leishmania major, Antimicrobial Agents and Chemotherapy, 2011, Vol. 55: 1045-1052, ARTÍCULO

    -Campos-Salinas, J.; Cabello-Donayre, M.; García-Hernández, R.; Pérez-Victoria, I.; Castanys, S.; Gamarro, F.; Pérez-Victoria, J.M., A new ATP-binding cassette protein is involved in intracellular haem trafficking in Leishmania. , Molecular Microbiology, 2011, Vol. 79: 1430-1444, ARTÍCULO


     

     

    TESIS DOCTORALES ÚLTIMOS 5 AÑOS

     

    2016

    David León Guerrero

    Caracterización funcional del transportador ABCG2 de Leishmania major

    IPBLN CSIC

     

     
    2016

    Sebastián García Sánchez

    Mecanismo de resistencia a miltefosina en Leishmania: Caracterización de la subunidad proteica LiRos3

    IPBLN CSIC

     

     
    2014

    José Ignacio Manzano González

    Implicación del transportador ABCI4 de Leishmania major en resistencia a fármacos e infectividad

    Instituto de Parasitología y Biomedicina "López-Neyra" CSIC-Universidad de Granada

     

     
    2013

    Verónica Gómez Pérez

    NUEVOS PIRIDOFANOS SIMETRICOS CON ACTIVIDAD ANTIPROLIFERATIVA Y LEISHMANICIDA

    Instituto de Parasitología y Biomedicina "López-Neyra" CSIC-Universidad de Granada

     

     

     

    PATENTES ÚLTIMOS 5 AÑOS

     

    - DERIVADOS CICLOFANICOS DE BIS-PIRIDINIO COMO FARMACOS ANTI-PROTOZOARIOS. PCT/ES13/070683, 2013

     


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