II WORKSHOP

Jueves 22 de octubre 2015 – Aula 10 Edificio Bibliotecas

Viernes 23 de octubre 2015 – Salón de actos del CIMA

Coordinador: Dr. Paul Nguewa

Secretaria: Arantza Cia Laura Juampérez

Colaboran: Facultades de Medicina, Farmacia y Ciencias

Programa

Programa jueves 22 de octubre

15:45 Apertura de la sesión por parte del Dr. Paul Nguewa, director del ISTUN SESIÓN 1: Moderadoras Dras. Esther Moreno y Celia Fernández 16:00 – 16:20 Verónica Alcolea Devesa Novel heteroaryl selenocyanates and diselenides as potent antileishmanial

agents 16:20 – 16:40 Gabriela González Espinosa Interaction of the complement system with Brucella 16:40 – 17:00 Andrés Vacas Discovery of a Trypanosomatid serine/threonine kinase protein (“Jean3”).

Molecular analysis and applications 17:00 – 17:30 Carlos Chaccour Endectocidas para la eliminación de la malaria. Avances y retos 17:30 – 18:00 Pausa café y visita pósters SESIÓN 2: Moderadoras Dras. Elsa Moreno y Amaia Zúñiga 18:00 – 18:20 Mery Jhenny Santivañez Estudio de posibles mecanismos de acción de nuevos derivados para la

enfermedad de Chagas 18:20 – 18:40 Miguel A. Quiliano Meza Exploring the scope of new arylamino alcohol derivatives: Synthesis,

antiplasmodial evaluation, mechanistic and toxicological studies 18:40 – 18:55 Estrella Martínez Gómez Searching for a new-tagged brucellosis vaccine and an associated test that

allows the differentiation between infected and vaccinated animals, by creating new lipopolysaccharide associated epitopes

18:55 – 19:10 Miriam Salvador Bescós Characterization of mutants in genes putatively involved in Brucella

lipopolysaccharide synthesis

Programa viernes 23 de octubre

SESIÓN 3: 08:45 – 09:25 Dra. María Font Arellano Molecular descriptors calculation as a tool in the analysis of the

antileishmanial activity achieved by two series of diselenide derivatives. An insight into its potential action mechanism

09:25 – 10:00 Dra. Silvia Pérez Silanes Avances en el estudio de nuevos derivados para el tratamiento de la

enfermedad de Chagas 10:00 – 10:35 Dra. Celia Fernández Rubio Caracterización de YinP en Leishmania spp., una nueva diana terapéutica 10:35 – 11:00 IZASA (CYTATION 5 imaging reader) Miguel Ángel Formoso

The State of the art in Microplate Reader & Image Technology 11:00 – 11:30 Pausa café SESIÓN 4: 11:30 – 12:00 Proteómica y Bioinformática. Dr. Fernando Corrales 12:00 – 12:35 Dra. Esther Moreno Amatria Topical formulations for the treatment of cutaneous leishmaniasis 12:35 – 13:10 Dra. Amaia Zúñiga Bacterial pathologies (Brucellosis) 2015 update 13:10 – 13:45 Dra. Silvia Galiano Ruiz Desde las quinoxalinas y arilaminoalcoholes a los nuevos hits: Nuevas

aproximaciones en la búsqueda de antimaláricos 13:45 – 14:00 Comunicación ISTUN. Laura Jaumpérez “¿Quién comunica en el ISTUN?”

Servicio Bibliotecas. Montserrat Royo Investigando con la biblioteca 13:45 – 14:00 Clausura

Presentaciones orales

Presentación oral

Novel heteroaryl selenocyanates and diselenides as potent antileishmanial agents

Ylenia Baquedanoa,b, Verónica Alcoleaa,b, Miguel Ángel Toroc, Killian Jesús Gutiérrezc, Paul Nguewab, María Fonta,b, Esther Morenoa,b, Socorro Espuelasb, Antonio Jiménez-Ruizc, Juan

Antonio Palopa,b, Daniel Planoa,b, Carmen Sanmartína,b aDepartamento de Química Orgánica y Farmacéutica, Universidad de Navarra bInstituto de Salud Tropical, Universidad de Navarra

cDepartamento de Bioquímica y Biología Molecular, Universidad de Alcalá

Para este trabajo se sintetizaron 22 nuevos selenocianatos y diseleniuros unidos a heterociclos con demostrada actividad antiparasitaria como quinolina, quinoxalina, acridina, furano, isoxazol… Los compuestos fueron ensayados contra amastigotes de L. infantum y células THP-1 humanas para determinar también su selectividad. Cabe destacar que muchos de los compuestos fueron activos en el rango micromolar. Los compuestos 1h, 2d, 2e y 2f fueron escogidos como los compuestos líderes de esta serie por mostrar mejores resultados en actividad (IC50 entre 0,45 y 1,27 μM) y selectividad (índice de selectividad >25) que los fármacos de referencia. Estos derivados fueron probados contra macrófagos infectados y se evaluó su capacidad para inhibir la enzima tripanotión reductasa como una primera aproximación a su posible mecanismo de acción. Los compuestos 1h y 2d mostraron una buena asociación entre la inhibición de dicha enzima y su actividad antileishmanicida. Destaca el compuesto 1h como la molécula líder más prometedora para el diseño de un nuevo fármaco leishmanicida por sus excelentes propiedades ADME teóricas. Actualmente se están llevando a cabo los ensayos preclínicos de los compuestos líderes.

Presentación oral

Interaction of the complement system with Brucella Gabriela González Espinoza1, Rodolfo Gutiérrez Fernandez1, Esteban Lizano, Elías Barquero Calvo2, Esteban Chaves Olarte1, Carlos Chacón Díaz1, Ignacio Moriyón3 , Edgardo Moreno2

1 Centro de Investigación en Enfermedades Tropicales, Facultad de microbiología Universidad de Costa Rica, San José, Costa

Rica. 2 Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional, Heredia, Costa Rica. 3Departamento de Microbiología e Instituto de Salud Tropical, Universidad de Navarra, Instituto de Investigación

Sanitaria de Navarra, Pamplona, España.

Brucellosis is a worldwide-extended zoonosis caused by species of Brucella genus, most of which show host preference. Brucella is an intracellular facultative pathogen that has the ability to avoid the host innate immune response. The complement system is an essential component of the innate immune system and one of its main functions is to kill pathogenic microorganisms. Nevertheless, some pathogenic bacteria including Brucella have developed mechanisms to resist complement mediated killing. To investigate the complement evasion strategy used by Brucella, we first incubated Brucella abortus, Salmonella typhimurium (a bacterium known to be complement resistant) and Ochrobactrum anthropi (phylogenetic related to Brucella) with non-immune human serum and measured bacterial survival and complement activation. The results demonstrated that, although B.abortus was resistant to complement mediated killing, it induced low activation of the complement cascade. Then, we infected C´3 depleted mice and observed that, in contrast with S.typhimurium, B.abortus infection was not lethal. However, C´3 depleted mice showed significantly lower spleen B.abortus counts than the control group, meaning that the lack of C´3 favors the clearance of the bacteria in a Brucella infection. These observations suggest the existence of B.abortus-complement interactions that modulate the system favoring an escape of the bacterium from innate immunity. To investigate this hypothesis, we developed a protocol to obtain the proteins that become attached to bacterial membranes upon incubation with non-immune serum. By proteomic analysis, we identified a variety of proteins that selectively binds B.abortus but not O.anthropi or S.typhimurium. Among these proteins, we identified acute phase proteins, complement factors and regulators of the complement cascade. Interestingly, this interaction was different among different Brucellae species. These findings show the importance of investigating (a) the role of the regulatory proteins in the protection from complement activation; (b) the potential variations in the interactions between complement and Brucella according to the specific characteristics of their natural hosts and (c) the role of Brucella surface molecules in the interaction. References: Eisenschenk,F., Houle, J and Marker, E. (1999). Mechanism of serum resistance among Brucella abortus isolates. Veterinary Microbiology 68 (3-4): 235-44. Fernández C., Nikolich, M., Vemulapalli, R., Sriranganathan, N., Boyle, S., Schurig, G., Hadfield, T and Hoover, D. (2001). Deletion of wboA Enhances Activation of the Lectin Pathway of Complement in Brucella abortus and Brucella melitensis. Infection and immunity 69 (7) : 4407-4416.

Presentación oral

Discovery of a Trypanosomatid serine/threonine kinase protein (“Jean3”). Molecular analysis and applications

Andrés Vacas, Celia Fernández-Rubio, Miriam Algarabel-Olona, Conor Sugder, Óscar Velasco-

Rodríguez, Paul Nguewa

Instituto de Salud Tropical University of Navarra (ISTUN) / Navarra Institute for Health Research (IdiSNA) / Department of Microbiology and Parasitology. Pamplona, Spain

Leishmaniasis, Sleeping sickness and Chagas disease are neglected diseases caused by intra-cellular parasites that belong to the Trypanosomatidae family. These pathologies affect several million people around the world. The transmission of the parasites between invertebrate vectors and mammalian hosts, involves morphological and physiological changes needed to acquire virulence and to adapt to the environment. This mechanism of differentiation is called metacyclogenesis. We identified a number of genes apparently involved during this process. One of our objectives is the validation of these genes as therapeutic targets. After screening the Leishmania and Trypanosoma genomes, we found plausible candidates involved in the parasites differentiation and adaptation phenomena. We mainly focus on protein kinases as promising druggable molecules described against different pathologies. After identifying “Jean3” as a therapeutic target candidate in several trypanosomatids, the analysis of its structure and the prediction of its function were assessed by several bioinformatic tools in T. cruzi and L. major. We also demonstrated that Jean3 is differentially expressed during the stages of growth and proliferation. In addition, the infective forms of these parasites exhibit high gene expression levels of this protein kinase. With the use of the novel pXG-mCherry12 fusion protein plasmid we confirmed Jean3 localization to be cytosolic. We have continued to evaluate Jean3 implication in different processes such as treatment resistance, cell cycle and viability, and parasites virulence acquisition phenomena.

Presentación oral

Endectocidas para la eliminación de la malaria. Avances y retos

Carlos Chaccour1,2,3, Regina Rabinovich3, José Luis Del Pozo1,2

1Clínica Universidad de Navarra, 2Instituto de Medicina Tropical Universidad de Navarra, 3ISGlobal, Barcelona Institute for Global

Health.

Los endectocidas son drogas con actividad ante endoparásitos (como los helmintos intestinales) y también contra los ectoparásitos (como las garrapatas y los piojos). De ellos, la ivermectina es el más conocido y utilizado. En el mercado veterinario es una de las drogas más vendidas. En la medicina humana, ha ayudado a prevenir la ceguera en millones de personas que viven en áreas endémicas de oncocercosis. Los responsables del descubrimiento y desarrollo de la ivemectina, William Campbell y Satoshi Omura, han ganado el premio Nobel de Medicina y Fisiología 2015, por la gran repercusión de esta droga en la salud global. La ivermectina también mata mosquitos en el laboratorio y en el campo. Este efecto anti-mosquito hace de la ivermectina una atractiva herramienta potencial para el control de los mosquitos que transmiten la malaria. Su papel sería complementario a las medidas de control normalmente usadas (mosquiteras impregnadas e insecticida residual). Estas medidas habituales son muy efectivas, pero tienen efecto selectivo ante los mosquitos que se alimentan dentro de las casas y de noche. Lo que hace atractiva la ivermectina en administración masiva, es que afectaría a todos los mosquitos, sin importar donde y cuando se alimenten de las personas tratadas. Para esto debería administrarse a poblaciones enteras (http://www.malariajournal.com/content/12/1/153). Para el desarrollo de esta potencial herramienta es necesario hacer investigación con la vista puesta en el tema regulatorio y así generar la evidencia suficiente para que la OMS pueda emitir una recomendación de política sanitaria en el tema.

Presentación oral

Estudio de posibles mecanismos de acción de nuevos derivados para la enfermedad de Chagas

Mery Santivañez-Veliza*, Elsa Moreno-Vigurib, Celia Fernándezb, Paul A. Nguewab, Amaya Azquetac, Mercedes Gonzálezd, Elena Lizarragaa, Manuel Sánchez-Morenoe y Silvia Pérez-

Silanesab

aDepartamento de Química Orgánica y Farmaceútica. bInstituto de Salud Tropical. cDepartamento de Farmacología y Toxicología. Universidad de Navarra. C/Irunlarrea 1, 31008. Pamplona, España. dDepartamento de Química Medicinal. Facultad de Ciencias.

Universidad de la República. Iguá 4225, Montevideo, Uruguay. eDepartamento de Parasitología, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain

Como continuación en el proyecto de nuevos derivados para la enfermedad de Chagas, se ha realizado una estancia en la Universidad de la República de Uruguay donde se ha estudiado el posible mecanismo de acción de una serie de compuestos selecionados en base a su actividad anti-T-cruzi in vitro y su perfil toxicológico. Teniendo en cuenta la estructura química de los derivados, se estudiaron 3 posibles dianas:

1.- En el ensayo de inhibición de cruzipaina a pH 5.5, 12 de los 22 compuestos evaluados mostraron una inhibición moderada de tipo slow binding con valores de IC50 entre 9.3 y 34.6 µM.

2.- Evaluación del cambio en la excreción de metabolitos. Se ha observado que 4 de los 5

compuestos evaluados, mostraron un aumento significativo en los niveles de succinato excretados respecto al control sin tratar. Lo que sugeriría que los compuestos podrían estar actuando sobre alguna ruta bioquímica relacionada con el metabolismo energético del parásito.

3.- Ensayo de la inhibición de la biosíntesis de esteroles de membrana. 9 de los 19

compuestos evaluados mostraron una posible inhibición de la enzima escualeno epoxidasa, observándose la acumulación de escualeno y depleción de lanosterol. Con el objetivo de obtener resultados cuantitativos, se ha puesto a punto un método mediante GC-MS para la cuantificación de estos esteroles y se está trabajando en la validación de este método analítico. Todavía no se dispone de resultados.

En la actualidad se están realizando estudios frente a la inhibición de la tripanotiona reductasa y frente a la superóxido dismutasa en colaboración de la Universidad de Granada, con el objetivo de poder determinar el posible mecanismo de acción de esta familia de compuestos que están presentando un perfil prometedor como potenciales compuestos antichagásicos.

Presentación oral

Synthesis and antiplasmodial evaluation of new arylaminoalcohol derivatives

Miguel Quiliano,†,π Adela Mendoza,† Kim Y. Fong,& Adriana Pabón,# Nathan E. Goldfarb,Ω

Germán Gonzalez,±,£ Ariane Vettorazzi, Isabelle Fabing,∂ Ben M. Dunn,Ω Giovanny Garavito, § David W. Wright,& Eric Deharo,±£ Silvia Perez-Silanes,†,π Ignacio Aldana,†,π Silvia Galiano-

Ruiz†,π (*) †Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Navarra, Pamplona 31008, Spain. πInstitute of Tropical Health (ISTUN), Universidad de Navarra, Pamplona 31008, Spain. &Department of Chemistry, Vanderbilt

University, Station B 351822, Nashville, TN 37235, USA. #Grupo Malaria, Universidad de Antioquía, Medellín, Colombia. ±Université de Toulouse; UPS; UMR 152 Pharma-Dev; Université Toulouse 3, Faculté des Sciences Pharmaceutiques, F-31062 Toulouse cedex 09, France. £Institut de Recherche pour le Développement (IRD); UMR 152 Pharma-Dev; F-31062 Toulouse cedex 09,

France. ∂Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique SPCMIB – UMR5068, CNRS - Université Paul Sabatier, 118, route de Narbonne 31062 Toulouse Cedex 09, France. §Departamento de Farmacia, Facultad de Ciencias,

Universidad Nacional de Colombia (DFUNC), Bogotá D.C., Colombia.

Some 1-aryl-3 substituted propanol derivatives were synthesized for their ability to inhibit the growth of Plasmodium falciparum chloroquine-resistant FCR-3 strain in culture. Four of tested compounds inhibited 50% of parasite growth at doses < 0.5 µM. The structure of the central arylamine seems to influence the cytotoxicity in Vero cells. Compounds with aminopiperidine as the central amine showed the best antiplasmodial activity. The most active compounds were evaluated for in vivo activity against P. berghei in a 4-day suppressive test.

Presentación oral

Searching for a new-tagged brucellosis vaccine and an associated test that allows the differentiation between infected and vaccinated animals, by creating new lipopolysaccharide associated epitopes

Estrella Martínez-Gómez1, Yolanda Gil-Ramírez1, Amaia Zúñiga-Ripa1 Ignacio Moriyón1, Raquel

Conde-Álvarez 1*and Maite Iriarte1*.

1 Instituto de Salud Tropical. Departamento de Microbiología y Parasitología. Universidad de Navarra. España. (*These authors contributed equally to this work)

Brucellosis is a zoonosis caused by Brucella, a bacterium that infects livestock causing heavy economic losses and that is readily transmitted to humans. A complete lipopolysaccharide O-chain, is critical for Brucella virulence and is the main antigen used for diagnosis. Control and eradication of brucellosis need animal vaccination. The only useful vaccines are two live smooth attenuated strains: B. melitensis Rev1 in sheep and goats, and B. abortus S19 in cattle. However, both induce false positive reactions in serological diagnostic tests, which are mainly based in the immune response against the lipopolysaccharide, and hampers the differentiation between infected and vaccinated animals (DIVA problem). To solve this problem, we introduced in Brucella an acetyltransferase (WbdR) that modifies the lipopolysaccharide O-chain, creating new epitopes. First, we introduced wbdR in an expression plasmid. We have observed that this plasmid is unstable and can be lost. Therefore, to improve the stability of wbdR expression, we have inserted wbdR in an intergenic region of Brucella chromosome. The resulting new strain, Ba::Tn7wbdR, is stable, allows the continuous expression of wbdR, and induces the production of specific antibodies against the acetyl lipopolysaccharide epitopes. These properties are useful for the development of new-tagged vaccines.

Presentación oral

Characterization of mutants in genes putatively involved in Brucella lipopolysaccharide synthesis

Miriam Salvador Bescós1, Maite Iriarte Cilveti, Ignacio Moriyón Uría, Raquel Conde Álvarez.

Universidad de Navarra – Departamento de Microbiología y Parasitología, Instituto de Salud Tropical. 1msalvador.1@alumni.unav.es

Brucellosis is a zoonotic disease caused by Brucella. The lipopolysaccharide (LPS) of Brucella plays a major role in its virulence as impairs normal recognition by the immune system and allows Brucella to multiply (Moriyón and Berman, 1982). It is known that mutants in glycosyltransferases involved in the synthesis of a LPS core ramification are attenuated, escape recognition by the innate immune system and are good vaccine candidates against brucellosis (Conde-Álvarez et al., 2012). The chemical structure of the Brucella core LPS suggests that other glycosyltransferases, apart from the already identified, should also be implicated in its biosynthesis. Thus, the study of those genes could help to improve the generation of new vaccines against brucellosis. We used a bioinformatics search to identify the ORFs putatively encoding core glycosyltransferases. We constructed mutants in 7 of these ORFs and observed that all of them kept the O-chain in their LPS. Interestingly, a mutant in ORF BAB1_0953 lost reactivity against polyclonal and monoclonal antibodies that recognize the core section. A mutant in BAB1_0953 was more sensitive than the parental strain to innate immune system components such as the complement present in new-born bovine serum and bactericidal peptides. Since this mutant keeps the O-chain, these preliminary results suggest that BAB1_0953 (named wadD) is a new glycosyltransferase probably adding a sugar to the core ramification of Brucella LPS that is not linked to the O-chain. In vivo studies should be done in order to confirm its role in virulence. References: - Conde-Álvarez,R.; Arce-Gorvel,V.; Iriarte,M.; Mancek-Keber,M.; Barquero-Calvo,E.; Palacios-Chaves,L.; Chacon-Diaz,C.; Chaves-Olarte,E.; Martirosyan,A.; von Bargen,K.; Grillo,M.J.; Jerala,R.; Brandenburg,K.; Llobet,E.; Bengoechea,J.A.; Moreno,E.; Moriyón,I.; Gorvel,J.P. (2012). The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition. PLoS Pathog., 8, 5, e1002675. - Moriyón, I., Berman, D. T. (1982). Effects of nonionic, ionic, and dipolar ionic detergents and EDTA on the Brucella cell envelope. J Bacteriol 152: 822-828.

Presentación oral

Molecular descriptors calculation as a tool in the analysis of the antileishmanial activity achieved by two series of diselenide derivatives. An insight into its potential action mechanism

María Fonta,b, Ylenia Baquedanob,c, Daniel Planob,c, Esther Morenob, Socorro Espuelasb,

Carmen Sanmartínb,c, Juan Antonio Palopb,c

Journal of Molecular Graphics and Modelling 60 (2015) 63–78

aSección de Modelización Molecular, Departamento de Química Orgánica y Farmacéutica, bInstituto de Salud Tropical, cSección de

Síntesis, Departamento de Química Orgánica y Farmacéutica, University of Navarra, Irunlarrea, 1. E-31008 Pamplona, Spain

A molecular modeling study has been carried out on two previously reported series of symmetric diselenide derivatives that show remarkable antileishmanial in vitro activity against Leishmania infantum intracellular amastigotes and in infected macrophages (THP-1 cells), in addition to showing favorable selectivity indices. Series 1 consists of compounds that can be considered as central scaffold constructed with a diaryl/dialkylaryl diselenide central nucleus, decorated with different substituents located on the aryl rings. Series 2 consists of compounds constructed over a diaryl diselenide central nucleus, decorated in 4 and 4’ positions with an aryl or heteroaryl sulfonamide fragment, thus forming the diselenosulfonamide derivatives. With regard to the diselenosulfonamide derivatives (2 series), the activity can be related, as a first approximation, with (a) the ability to release bis(4-aminophenyl)diselenide, the common fragment which can be ultimately responsible for the activity of the compounds. (b) the anti-parasitic activity achieved by the sulfonamide pharmacophore present in the analyzed derivatives. The data that support this connection include the topography of the molecules, the conformational behavior of the compounds, which influences the bond order, as well as the accessibility of the hydrolysis point, and possibly the hydrophobicity and polarizability of the compounds.

Presentación oral

Avances en el estudio de nuevos derivados para el tratamiento de la enfermedad de Chagas

Elsa Moreno-Viguri, Mery Jhenny Santiváñez, Rocío Paucar y Silvia Pérez-Silanes

Universidad de Navarra. Departamento de Química Orgánica. Instituto de Salud Tropical

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, is considered a neglected tropical disease. CD is one of the most important medical problems in rural areas of South America, an estimated 7-8 million persons are infected in the world and the remaining 30-40% of patients will develop a determinate forma of chronic disease. Moreover, migration and travel have extended the distribution to other continents including North America, Europe and Western Pacific, where significant numbers of CD sufferers can now be found. Despite numerous advances published in recent years, benznidazole (Bzn) and nifurtimox (Nfx) remain the only available drugs for the treatment of disease (None of them are authorized by the FDA). Many side effects and therapeutic deficiencies of these drugs are still justifying the urgent need to continue with the research for the discovery of novel therapeutic alternatives. Our research group decided to test our in-house compound library for their in vitro anti T. cruzi activity. Fruit of this preliminary screening we found anti T-cruzi promising activity in a group of ketone derivatives. Thanks to our huge experience in the field of Neglected Diseases and Medicinal Chemistry and considering these promising results, the research group recognized the great chance to start a new project with the aim of discovering potential agents against T.cruzi. More than hundred new derivatives have been obtained by simple and cheap synthetically routes and their pharmacological profile have been evaluated considering potency and preliminary toxicological studies. We have selected 5 out of 14 derivatives with in vitro IC50<10 M and SIcomp/SIBnz>50 in amastigote and trypomastigote forms in three different T. cruzi strains. Moreover, none of the compounds showed genotoxicity in the SOS/umu screening test; meanwhile both Nfx and Bzn showed genotoxicity in this level. Preliminary in vivo assays have revealed the quick ability of the lead compounds to decrease the parasitemia in a murine mice model at dosses of 50 mg/kg and they will be continue for further testing. These promising in vivo results make the new derivatives valid as drug candidates.

Presentación oral

Characterization of Yin P from Leishmania spp., a new therapuetic target

Celia Fernández-Rubio1, Miriam Algarabel Olona1, Andrés Vacas Oleas1, Esther Moreno

Amatria2, Socorro Espuelas2, Paul Nguewa1

1Instituto de Salud Tropical Universidad de Navarra (ISTUN)/Department of Microbiology and Parasitology/ IdiSNA, Navarra Institute for Health Research. Pamplona, Spain. 2Instituto de Salud Tropical Universidad de Navarra (ISTUN)/Department of Pharmacy and

Pharmaceutical Technology, University of Navarra, Pamplona, Spain.

The World Health Organization (WHO) estimates that over one thousand million people are affected by at least one of the 17 Neglected Tropical Diseases (NTDs) such as schistosomiasis, river blindness (onchocerciasis), African trypanosomiasis, Chagas disease or visceral leishmaniasis. Leishmaniasis is a group of clinic manifestations produced by Leishmania spp. At least 20 species of Leishmania are responsible for the three clinical forms of the disease: visceral, cutaneous and mucocutaneous. The WHO estimates that Leishmaniasis is prevalent in 98 countries on 5 continents, and exhibits an incidence of 1.3 million new cases annually, of which 300,000 are visceral and 1 million are cutaneous or mucocutaneous. Leishmania spp. are heteroxenous parasites with a digenetic life-cycle. They need two hosts to complete its life cycle: an invertebrate vector, sandfly of the genus Phlebotomus (Old World strains) or Lutzomyia (American strains); and a vertebrate definitive host (dogs, wild mammals or humans). Leishmania parasites are pleomorphic organisms with two main different morphological forms during their life cycle: intracellular amastigotes in the mammalian host and motile promastigotes in the sand fly vector. Current treatment is toxic, expensive and its effectiveness has decreased due to the emergence of resistances. These disadvantages prompted the search of new therapeutic targets, as useful tools to develop new drugs in order to improve current treatments. Some virus and parasitic infections are oncogenic inducing tumors, but the implicated molecules are still unknown. Recent studies identified in Leishmania potential therapeutic targets similar to the ones described in different tumors (mTOR, PARP, tyrosine-kinase receptors…). In addition, there are several antitumoral drugs that have demostrated antileishmanial activity. The availability to sequence the complete genome of some Leishmania species allowed us to identify the homologue of an oncogene in Leishmania called Yin P. Yin P protein has an oncogenic domain related to cell cyle and DNA repair. Yin P is involved in several cellular processes, such as: embrionary progress, ribosomal biogenesis, cellular proliferation, genetic transcription and carcinogenesis. Yin P silencing lead to the decrease of the proliferation and cellular death in colon and breast cancer. Several data link Yin P to the regulation of genetic and proteic expression, and c-Jun, JunD or c-Fos seem to participate in Yin P expression. Recently, a relation between virulence and disemination in vivo has been related with the homologue of Yin P in Candida albicans. Recent data suggest that Yin P may be a therapeutic target against tumors, however its involvement in Leishmania biology is not known yet. In this project, we aim to characterize functionally the homologue of Yin P in Leishmania and analize its potential as therapeutic target against leishmaniasis.

Presentación oral

Topical formulations for the treatment of cutaneous leishmaniasis

Esther Morenoa,b,*, Juana Schwartza,c, Esther Larreaa,b, Iosune Condec, María Fonta,b,d, Paul A. Nguewaa,b, Carmen Sanmartína,b,d, Juan M. Iracheb,c and Socorro Espuelasa,d,c,*

aInstitute of Tropical Health, University of Navarra, bInstituto de Investigación Sanitaria de Navarra (IdISNA), cPharmacy and

Pharmaceutical Technology Department, University of Navarra and dOrganic and Pharmaceutical Chemistry Department, University of Navarra, 31008 Pamplona, Spain *E-mails: emorenoa@unav.es and sespuelas@unav.es

Currently, topical treatment of cutaneous leishmaniasis (CL) is limited to the least severe forms of the disease, which do not disseminate. Patients affected by CL need a safe, topical and well tolerated treatment that cures the lesions without leaving deep scars. A topical formulation for the treatment of CL should be able to promote drug penetration into the skin and reach the parasites located in the dermis. This ability depends on the physicochemical properties of the drug, the type of vehicle and the status of the skin [1]. Besides, a compound with a good intrinsic activity against Leishmania is required. On the other hand, nanoparticles (NP) are of great interest for the topical application of drugs because they can enhance their penetration and extend their residence time in the skin. These general principles justify the development of chitosan NP for the topical application of β-lapachone (β-LP). In this study, we proposed the loading of β-LP in lecithin-chitosan NP for targeting the drug to the dermis, where infected macrophages reside, and promote wound healing. The loading of β-LP in lecithin-chitosan NP was critical to achieve important drug accumulation in the dermis and permeation through the skin. In addition, it did not influence the drug antileishmanial activity. When topically applied in L. major infected BALB/c mice, β-LP NP achieved no parasite reduction but they stopped the lesion progression. Immuno-histopatological assays in CL lesions and quantitative mRNA studies in draining lymph nodes confirmed that β-LP exhibited anti-inflammatory activity leading to the downregulation of IL-1β and COX-2 expression and a decrease of neutrophils infiltrate.

References: [1] Moreno E et al. Exp. Op. Drug Deliv. 2014, 11 (579-597).

Acknowledgements: We would like to thank the FIMA Foundation, the Institute of Tropical Health (University of Navarra) and the Ministry of Economy and Competitiveness of the Spanish Government (Subprograma: Torres Quevedo) for their support.

Presentación oral

Bacterial pathologies (Brucellosis). 2015 Update

Amaia Zúñiga-Ripa, Raquel Conde-Álvarez, Maite Iriarte, Ignacio Moriyón

Departamento de Microbiología y Parasitología. Instituto de Salud Tropical. Universidad de Navarra

The brucellae cause brucellosis, a zoonosis affecting livestock and humans. B. abortus and B. melitensis infect ruminants, and B. suis swine and wildlife. Moreover, sheep are infected by B. ovis, a rough (R) species. Vaccination is essential to control brucellosis but the only vaccines useful are attenuated strains (B. melitensis Rev1 in small ruminants, and B. abortus S19 in cattle) that induce false positive reactions in serological tests hampering differentiation of infected and vaccinated animals (DIVA problem). Moreover, they are not completely safe or stable, and S19 does not induce optimal protection. Rev1 is the only vaccine against B. ovis, and when discontinued after B. melitensis eradication, B. ovis becomes an emerging problem in several EU regions. Porcine brucellosis by B. suis biovar 2 is also an emerging problem in the EU, particularly in outdoor breeding systems because wild boars are infected by biovar 2. There is no vaccine against B. suis. Thus, new and better vaccines are necessary. Several developments by the Brucellosis research group concerning B. melitensis and B. abortus have been either marketed or taken up for further development and/or application by interested parties and companies. Therefore, our present work is aimed to exploit these previous findings focusing on the needs identified on B. ovis and B. suis brucellosis. Attenuation and improved immunogenicity on both backgrounds have been achieved on the bases of previous findings on lipopolysaccharide-core structure disruption. Moreover, for B. suis, an extension of previous studies on metabolism guarantees a proper balance of immunogenicity/attenuation. Concerning B. ovis, one of the main difficulties to develop a specific vaccine is the CO2-dependence of this species. Since a CO2-independent strain would be an invaluable tool for the development of a new vaccine, we are working on the mechanisms underlying this CO2 dependence. In addition to research, a main aim of our group is the training of people working on Brucellosis diagnosis and control in countries where the disease is endemic. During this year, two foreign visitors have come to our laboratory. Gabriela González, a visiting student from “Escuela de Medicina Veterinaria, Universidad Nacional, Costa Rica” is working on the interaction of the Brucella membrane with the immune system, and Mammar Khames (Department of Biology, University of Medea, Algeria) is performing a phenotypic and genotypic characterization of Brucella strains isolated from autochthonous livestock in Algeria. Also, there is ongoing cooperation with researchers and clinicians from Africa and Asia by providing them with logistic and diagnostic support. In this context, we conducted a one-week training course on Brucellosis diagnosis and control at Makerere University (Uganda) in June. This course was attended by medical and veterinary practitioners and students from different African countries (Uganda, Sudan, Burundi and Eritrea).

Presentación oral

Desde las quinoxalinas y arilaminoalcoholes a los nuevos hits: Nuevas aproximaciones en la búsqueda de antimaláricos

Silvia Galiano-Ruiz,†,π Miguel Quiliano,†,π Kim Y. Fong,& Adriana Pabón,# Isabelle Fabing,∂ Ben

M. Dunn,Ω Giovanny Garavito,§ David W. Wright,& Eric Deharo,±£ Ignacio Aldana†,π

†Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad de Navarra, Pamplona 31008, Spain. πInstitute of Tropical Health (ISTUN), Universidad de Navarra, Pamplona 31008, Spain. &Department of Chemistry, Vanderbilt

University, Station B 351822, Nashville, TN 37235, USA. #Grupo Malaria, Universidad de Antioquía, Medellín, Colombia. ΩDepartment of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, USA. ±Université de Toulouse; UPS; UMR 152 Pharma-Dev; Université Toulouse 3, Faculté des Sciences Pharmaceutiques, F-31062 Toulouse cedex 09, France.

£Institut de Recherche pour le Développement (IRD); UMR 152 Pharma-Dev; F-31062 Toulouse cedex 09, France. ∂Laboratoire de Synthèse et Physicochimie de Molécules d’Intérêt Biologique SPCMIB – UMR5068, CNRS - Université Paul Sabatier, 118, route de

Narbonne 31062 Toulouse Cedex 09, France. §Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia (DFUNC), Bogotá D.C., Colombia.

La urgente necesidad de controlar y erradicar la malaria, la enfermedad parasitaria más devastadora a nivel mundial, constituye uno de los mayores desafíos científicos dentro de los objetivos marcados en la Declaración del Milenio de Naciones Unidas del año 2000, siendo la estrategia clave el descubrimiento y desarrollo de nuevos fármacos con un buen perfil de eficacia y seguridad, con nuevos mecanismos de acción eficaces frente a cepas multirresistentes de parásito y mejores propiedades farmacocinéticas. Con este objetivo, iniciamos este proyecto en el que se han diseñado in silico y sintetizado un total de 361 nuevos compuestos, 234 derivados de quinoxalinas y 127 derivados de arilaminoalcoholes. El perfil de eficacia/seguridad de la mayoría de ellos es muy prometedor con una alta eficacia in vitro en cepas Plasmodium falciparum cloroquino-sensibles 3D7 y cloroquino-resistentes FCR-3 con ICS0< 1 M y con baja citotoxicidad en la línea celular HEPG-2 exhibiendo un IS>S0, muy por encima de los criterios establecidos por la MMV. Además, todos ellos resultaron no genotóxicos en el posterior screening SOS/UMU confirmando su seguridad in vitro. En los ensayos in vivo en cepas Plasmodium berghei, dos de los derivados de arilaminoalcoholes mostraron un porcentaje de inhibición de la parasitemia muy por encima del fármaco de referencia que es la cloroquina. Estos resultados son muy prometedores porque se han llevado a cabo en el modelo animal preclínico más exigente y usando la vía oral como vía de administración que es la recomendada para su uso clínico. Se han llevado a cabo también estudios del mecanismo de acción de los compuestos si bien son necesarios confirmarlos con otros ensayos complementarios. Consecuencia de estos importantes resultados, es la solicitud de una patente internacional, la publicación de cinco artículos científicos, cuatro publicados y uno de ellos enviado y de S comunicaciones en Congresos Internacionales dentro del área de la Química Médica así como la formación de un nuevo grupo de investigación con expertos en diferentes áreas proporcionando una dimensión multidisciplinar e internacional al proyecto.

Presentaciones pósters

Presentación póster

Desarrollo de una vacuna frente a Shigelosis

A. I. Camacho1, J. Matías1, Y. Pastor1, M. J. Dávila1, J.M. Irache2, A. Gil3, I. Aizpun3, I. Peñuelas4, R. Ramos-Membrive4, C. Gamazo1

1 Departamento de Microbiología y Parasitología, Universidad de Navarra (Pamplona, España) 2 Departamento de Farmacia y Tecnología Farmacéutica, Universidad de Navarra (Pamplona, España) 3 Departamento de Farmacología y Toxicología,

Universidad de Navarra (Pamplona, España) 4 Departamento de Medicina Nuclear, Clínica Universidad de Navarra (Pamplona, España)

Diarrhea remains the second leading cause of death among children under five years old, globally. The principal bacterial agents of diarrhoeal diseases are: Vibrio cholera; a variety of Salmonella enterica serotypes, including S. Typhi; Campylobacter spp. (especially C. jejuni); a variety of enteropathogenic Escherichia coli serotypes, including the enterotoxigenic ETEC; and, Shigella spp., a leading cause of diarrheal diseases in many countries. In fact, five to fifteen percent of all diarrheal episodes can be attributed to an infection with Shigella. Shigella spp. is the causal agent of Shigellosis. Shigella strains can be divided into 4 species (S. dysenteriae, S. sonnei, S. flexneri and S. boydii). Among them, Shigella flexneri, and specifically the serotype 2a, has got the highest rates being endemic in developing countries. The objective of our group is the development of a safe and effective vaccine against Shigellosis. Currently, our work is focused on Shigella flexneri. Particularly, our group investigates the potential of outer membrane vesicles (OMVs) derived from S. flexneri as an approach for a vaccine candidate against Shigellosis. OMVs are naturally released by various Gram-negative bacteria and predominantly contain outer membrane components. We demonstrated that immunization of mice by mucosal route (e.g., intranasal [i.n.] or intragastric) with OMVs derived from S. flexneri conferred protection against a lethal dose with the homologous strain 56 days after the immunization. These data indicates that OMV possess potential for vaccine development. Our current work includes different aspects: (i) optimization of the antigenic extract in order to increase the yield of production; (ii) toxicity studies of free antigens (Toxicology Department, CIFA); (iii) encapsulation of the free antigen in bioadhesive nanoparticles in order to improve immunological properties of the vaccine formulation (Department of Pharmaceutical Technology); (iv) Biodistribution assays using radiolabelled antigen in order to study the localization and residence time of vaccine formulation (Radiopharmacy Department, CUN) Finally, it is important to highlight that a member of our group has recently stayed in Monkole Hospital (DR Congo) in order to isolate and identify strains responsible for Shigellosis

Presentación póster

Carbonic anhydrases and CO2-dependence in Brucella

Lara Pérez Etayo, Amaia Zúñiga-Ripa, Maite Iriarte and Ignacio Moriyón

Instituto de Salud Tropical / Departamento de Micribiología y Parasitología, Universidad de Navarra

Brucellosis is an important worldwide zoonosis caused by bacteria of the genus Brucella. This genus is divided into several species, including B. abortus, B. melitensis and B. suis that infect humans, and B. ovis that, although non zoonotic, also cause serious economical losses. Animal vaccination is the main way to prevent this disease, and the principal vaccines available are B. abortus S19 and B. melitensis Rev1. However, there is no specific vaccine against B. ovis and protection is achieved using B. melitensis Rev1. Stopping vaccination with Rev1 when B. melitensis has been eradicated leads to a clear increase in the number of infections caused by B. ovis. For this reason, research on B. ovis-specific vaccines is an area of increasing interest. Nevertheless, since the vast majority of B. ovis strains require CO2 for growth, the deve-lopment of a specific B. ovis vaccine would be facilitated if we obtain a B. ovis strain able to grow in CO2 atmospheric conditions. In this work we have analyzed the mechanisms underlying CO2 depen-dence in B. abortus and B. ovis. We have identified the carbonic anhydrases (CAI and CAII) responsible for B. abortus growth in normal CO2 atmospheric conditions. We have also demonstrated that lack of CAII is responsible for CO2-dependent growth of B. abortus strains 292 and 544, and that the introduction of this CAII reverts this CO2-dependence. Construction and analysis of mutants in CAI and CAII are in progress and they will allow us to verify our hypothesis. In the case of B. ovis, both CAI and CAII are disrupted and introduction of both of them seems to be required to grow in normal atmospheric conditions. Finally, we have constructed the key instruments that will permit to generate a B. ovis strain able to grow in normal atmospheric conditions that could be used as a base for the development of new B. ovis specific vaccines and for the production of B. ovis antigens for diagnosis.

Presentación póster

Role of the pyruvate phosphate dikinase, the pyruvate kinase and the Entner-Doudoroff pathway in the synthesis of pyruvate in Brucella suis 5

Leticia Lázaro-Antón1, Amaia Zúñiga-Ripa1, Thibault Barbier2, Jean-Jacques Letesson2, Maite

Iriarte1 and Ignacio Moriyón1

1Departamento de Microbiología e Instituto de Salud Tropical, Universidad de Navarra, Instituto de Investigación Sanitaria de

Navarra, Pamplona, Spain. llazaro@alumni.unav.es 2Research Unit in Biology of Microorganisms — URBM, NARILIS, UNAmur, Namur, Belgium

Bacteria of the genus Brucella are facultative intracellular parasites of mammals causing brucellosis, a worldwide-extended zoonosis. The pathogenicity of these bacteria resides largely in their ability to escape early detection by the innate immune system. Moreover, it has been proposed that the brucellae adapt their metabolism to avoid host cell damage but the metabolism in the intracellular replicative niche is largely unknown. Recently, we have shown that mutants in key gluconeogenic enzymes are not attenuated suggesting that hexoses can be obtained from the host1. Here, we have investigated the use of glucose by B. suis biovar 5 by disrupting genes putatively coding for pyruvate phosphate dikinase (PpdK), pyruvate kinase (Pyk) and 6-phospho-D-gluconate dehydratase (Edd), the first enzyme of the Entner-Doudoroff pathway. Growth was impaired in ppdK or pyk mutants showing that both are required for glucose catabolism. Moreover, the edd mutant showed a growth defect indicating a relevant role of the Entner-Doudoroff pathway. Consistent with these results, the simultaneous deletion of pyk, ppdK and edd inhibited growth on sugars as the only carbon source. This triple mutant is a useful tool for further studies on the use of hexoses during infection.

1Zúñiga-Ripa A, Barbier T, Conde-Álvarez R, Martínez-Gómez E, Palacios-Chaves L, Gil-Ramírez Y, Grilló MJ, Letesson JJ, Iriarte M, Moriyón I. Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1,6-bisphosphatases for full virulence in laboratory models.. J Bacteriol. 2014 Aug 15; 196(16):3045-57.

Presentación póster

Construction of two mCherry plasmids (pXG-mCherry) for transgenic Leishmania: valuable tools for future molecular analysis of the

trypanosomatid biology Andrés Vacas-Oleas, Conor Sugden, Óscar Velasco-Rodríguez, Celia Fernández-Rubio,

Miriam Algarabel-Olona, Paul Nguewa

Instituto de Salud Tropical University of Navarra (ISTUN) / Navarra Institute for Health Research (IdiSNA) / Department of Microbiology and Parasitology. Pamplona, Spain

We have constructed and tested two plasmids (pXG-mCherry12 and pXG-mCherry34) targeting the expression of the monomeric fluorescent red protein mCherry in Leishmania parasites. Using the Leishmania episomal expression vector pXG, the mCherry sequence was inserted from the Trypanosoma cruzi pTREX-mCherry plasmid. An N-terminal polylinker was also added in the plasmid to obtain the vector denominated pXG-mCherry12. We also removed in the plasmid the Stop codon and substituted it with a polylinker (multiple cloning site, MCS) to generate a novel vector named pXG-mCherry34. Both plasmids red florescence was tested after being transfected in Leishmania major parasites. The availability of these two new plasmid constructions, one for N-terminal fusion proteins (pXG-mCherry12), and the second plasmid for C-terminal fusion proteins (pXG-mCherry34) will become a valuable tool for future molecular analysis of the trypanosomatid biology. We are planning to analyze in the promastigotes and particularly the amastigotes, the levels of expression of both novel pXG-mCherry vectors once transfected in Leishmania. Red fluorescent parasites will be useful for drug screening assays, FACS selection, in vivo studies, and protein localization (fluorescent fusion proteins) by fluorescent microscopy.

Presentación póster

Structural and functional characterization of YinP, an oncogene homologue expressed in Leishmania spp.

Miriam Algarabel-Olona1*, Andrés Vacas-Oleas1, Conor Sugden1, Celia Fernández-Rubio1,

Ester Moreno-Amatría2, Socorro Espuelas2, Paul Nguewa1**

1. Instituto de Salud Tropical Universidad de Navarra (ISTUN)/Department of Microbiology and Parasitology/ IdiSNA, Navarra Institute for Health Research. Pamplona, Spain. 2. Instituto de Salud Tropical Universidad de Navarra (ISTUN)/Department of

Pharmacy and Pharmaceutical Technology, University of Navarra, Pamplona, Spain. *malgarabel@alumni.unav.es **panguewa@unav.es

Leishmaniasis is a vector-borne neglected tropical disease caused by members of the genus Leishmania with at least 20 species of Leishmania are responsible for the three clinical forms of disease: visceral, cutaneous and mucocutaneous Leishmania spp. are hetero-xenous parasites with a dige-netic life cycle. They need two hosts to complete their life cycle: an invertebrate vector (sandfly) and a vertebrate as a definitive host. Metacyclogenesis is a biological process whereby Leishmania transforms from poorly infective promastigotes into highly infective promas-tigotes called metacyclic forms. This transformation naturally occurs inside the insect gut. Recently, we identified many genes involved in this process and we performed several analyses in order to validate them as therapeutic targets. One of those genes is YinP, highly conserved from yeast to human and related to biological processes such as cell cycle. The analysis of its structure was firstly assessed by several bioinformatic tools showing that it contains an oncogenic domain. Secondly, after completing several assays, we observed that this gene is highly expressed in the infective metacyclic parasites. Therefore, it may play a role in parasites differentiation and metacyclogenesis processes. Now, we are analyzing its relation with the pathogenicity of Leishmania parasites