Cyclobenzaprine raises ROS levels in Leishmania infantum and reduces parasite burden in Infected mice

11 p.-5 fig. Ferreira Cunha-Júnior, Edézio et al.Background The leishmanicidal action of tricyclic antidepressants has been studied and evidences have pointed that their action is linked to inhibition of trypanothione reductase, a key enzyme in the redox metabolism of pathogenic trypanosomes. Cyclobenzaprine (CBP) is a tricyclic structurally related to the antidepressant amitriptyline, differing only by the presence of a double bond in the central ring. This paper describes the effect of CBP in experimental visceral leishmaniasis, its inhibitory effect in trypanothione reductase and the potential immunomodulatory activity.Methodology/Principal Findings In vitro antileishmanial activity was determined in promastigotes and in L. infantum-infected macrophages. For in vivo studies, L. infantum-infected BALB/c mice were treated with CBP by oral gavage for five days and the parasite load was estimated. Trypanothione reductase activity was assessed in the soluble fraction of promastigotes of L. infantum. For evaluation of cytokines, L. infantum-infected macrophages were co-cultured with BALB/c splenocytes and treated with CBP for 48 h. The supernatant was analyzed for IL-6, IL-10, MCP-1, IFN-γ and TNF-α. CBP demonstrated an IC50 of 14.5±1.1μM and an IC90 of 74.5±1.2 μM in promastigotes and an IC50 of 12.6±1.05 μM and an IC90 of 28.7±1.3 μM in intracellular amastigotes. CBP also reduced the parasite load in L. infantum-infected mice by 40.4±10.3% and 66.7±10.5% in spleen at 24.64 and 49.28 mg/kg, respectively and by 85.6±5.0 and 89.3±4.8% in liver at 24.64 and 49.28mg/kg, after a short-term treatment. CBP inhibited the trypanothione reductase activity with a Ki of 86 ± 7.7 μM and increased the ROS production in promastigotes. CBP inhibited in 53% the production of IL-6 in infected macrophages coculture.Conclusion/Significance To the best of our knowledge, this study is the first report of the in vivo antileishmanial activity of the FDA-approved drug CBP. Modulation of immune response and induction of oxidative stress in parasite seem to contribute to this efficacy.This work was supported by Programa Estratégico de Apoio à Pesquisa em Saúde,FIOCRUZ/Conselho Nacional de Desenvolvimento Científico e Tecnológico - www.cnpq.br, (PAPES/ CNPq 407680/2012-8 to ECTS and 407590/2012-9 to EEAA), Fundação de Apoio a Pesquisa do Estado do Rio de Janeiro ± www.faperj.br (Fellow and grant E-26/010.001828/2016 to EFCJ), Conselho Nacional de Desenvolvimento CientõÂfico e TecnoloÂgico (CNPq/Universal grant 470627/2013-1 to EEAA), São Paulo Research Foundation - www.fapesp.br (FAPESP 2015/23403-9 to AGT),Programa Estatal de Investigación, Desarrollo e Innovación Orientada a los Retos de la Sociedad FEDER - www.idi.mineco.gob.es/ (SAF2015-65740-R) and Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER -www.isciii.es/ (RD12/0018/0007) (to LR).Peer reviewe

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes

Leishmania infantum modulates host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis

24 p.-8 fig.-9 fig.supl.-1 tab.sup. Diana Moreira et alt.Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.This work was funded by FEDER funds through the Operational Competitiveness Programme - COMPETE and by National Funds through FCT - Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124- FEDER-011054 (PTDC/SAU-FCF/100749/2008) and PTDC/BIA-MIC/118644/2010. The research leading to these results has also received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED). DM and VR were supported bySFRH/BD/91543/2012 and SFRH/BD/64064/2009, respectively. LR was supported by PN de I+D+I 2008-2011, PI12-02706 and VI PN de I+D+I 2008-2011,ISCIII -Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER (RICET RD12/0018/0007). ER was supported by a project grant of the Spanish Ministerio de Economía y Competitividad(SAF2010-20256). JE was supported by an ANR grant (LEISH-APO, France) and a Partenariat Hubert Curien (PHC) (program Volubilis, MA/11/262). JE is also supported by the Canada Research Chair programme. ML was supported by a fellowship fromANR. RS was supported by Programa Ciência - financed by Programa Operacional Potencial Humano POPH - QREN - Tipologia 4.2 - Promocão do Emprego Científico, co-funded by Fundo Social Europeu and National funding from Ministry of Science, Technology and Higher Education (MCTES).Peer reviewe

A BODIPY-embedding miltefosine analog linked to cell-penetrating Tat(48-60) peptide favors intracellular delivery and visualization of the antiparasitic drug

Therapeutic application of many drugs is often hampered by poor or denied access to intracellular targets. A case in point is miltefosine (MT), an orally active antiparasitic drug, which becomes ineffective when parasites develop dysfunctional uptake systems. We report here the synthesis of a fluorescent BODIPY-embedding MT analogue with appropriate thiol functionalization allowing linkage to the cell-penetrating Tat(48-60) peptide through disulfide or thioether linkages. The resulting constructs are efficiently internalized into the otherwise MT-invulnerable R40 Leishmania strain, resulting in fast parasite killing, and hence successful avoidance of the resistance. In the disulfide-linked conjugate, an additional fluoro tag on the Tat moiety allows to monitor its reductive cleavage within the cytoplasm. Terminally differentiated cells such as peritoneal macrophages, impervious to MT unless infected by Leishmania, can uptake the drug in its Tat-conjugated form. The results afford proof-of-principle for using CPP vectors to avert drug resistance in parasites, and/or for tackling leishmaniasis by modulating macrophage uptake. © 2014 Springer-Verlag Wien.Peer Reviewe

Study of the antiparasitary activity of new synthetic peptides against Leishmania sp.

Trabajo presentado en el 31 Congreso Latinoamericano de Química, celebrado en Lima (Perú) del 14 al 17 de octubre de 2014.La leishmaniasis es una enfermedad parasitaria endémica en el mundo. Más de 350 millones de personas en 88 países se encuentran en riesgo de infección a causa de esta enfermedad tropical desatendida. Además, no existen vacunas eficaces disponibles contra la leishmaniasis, y el control de la enfermedad se basa exclusivamente en los tratamientos terapéuticos con fármacos tóxicos, algunos de los cuales fueron desarrollados en la década de 1940. Como la resistencia del parásito se vuelve más frecuente, existe una creciente preocupación de que los fármacos que se utilizan actualmente pronto se convertirán en tratamientos ineficaces. En consecuencia, existe una necesidad urgente de desarrollar nuevas clases de compuestos que sean activos contra cepas resistentes a los medicamentos de Leishmania (L.) (Marr y col., 2012). En este contexto, los Péptidos Antimicrobianos (PAM) son moléculas efectoras clave en la inmunidad innata de los organismos. La anfipaticidad de los péptidos les permite interactuar con las membranas cargadas negativamente, causando la directa desestabilización de la superficie de las membranas con formación de poros y posterior lisis de la célula. Debido a todas estas propiedades, es que se han iniciado estudios considerando la posibilidad de utilizar esta clase de moléculas como nuevos fármacos en diferentes tipos de enfermedades infecciosas y, en particular, en el tratamiento de la Leishmaniasis, sustentados en el amplio espectro antimicrobiano que presentan los PAMs (Torrent y col., 2012). En este estudio, se sintetizaron 18 PAM (P1 a P18) y se evaluó su efecto sobre hematíes y actividad antileishmanial en promastigotes de L. donovani y amastigotes de L. pifanoi. Los PAM fueron más efectivos sobre la forma de amastigotes con valores de EC50 entre 7.8 y 50 µM. y LC50 entre 0.45 y 5.7 µM. En la forma promastigotes sólo el PAM P17 presentó un efecto leishmanicida considerable a largo plazo (72 h), con un valor del 10% de células viables. La actividad hemolítica de todos los PAM fue mayor a 50 µM para todos los casos. El mecanismo de acción de los PAM se evaluó usando las sondas de Sytox Green y Bisoxonol y se observó que todos los PAMs, a excepción del P10, causaron permeabilización de la membrana plasmática del promastigote y del amastigote. Adicionalmente, se evaluó también la concentración de ATP intracelular en promastigotes de L. donovani 3-Luc tras la adición de diferentes concentraciones de péptido. La disminución de luminiscencia fue dependiente de la concentración de péptido. Por último, se determinó la citotoxicidad de los PAM en macrófagos peritoneales, donde el P6 se destacó por presentar un 100% de células viables a 5 µM. En consecuencia, a partir de este estudio proponemos cinco PAM como prometedores candidatos leishmanicidas.Los autores deseamos agradecer al Instituto de Salud Carlos III de España por el financiamiento de los proyectos FIS PS12-02706 y RD 12/0018/0007, MINECO CTQ2013-41507-R de España, Vicerrectoría de Investigación y Extensión (VIE) de la Universidad Industrial de Santander y Colciencias de Colombia por el proyecto número 1102-5453-1671Peer Reviewe

Synthesis, characterization and evaluation of synthetic antimicrobial peptides designed against Escherichia coli 0157: H7 and Methicillin-Resistant Staphylococcus aureus (MRSA)

Trabajo presentado en el 31 Congreso Latinoamericano de Química, celebrado en Lima (Perú) del 14 al 17 de octubre de 2014.Actualmente, existe un gran interés por desarrollar nuevos antibióticos frente a bacterias patógenas. Esto se debe, esencialmente, a la creciente resistencia bacteriana. Por esta razón, en las últimas décadas, ha surgido la necesidad de obtener nuevos compuestos antimicrobianos efectivos en el tratamiento de enfermedades infecciosas de alta prevalencia e infecciones sistémicas de difícil manejo terapéutico ( Xu, Flavin, y Flavin, 2014). Recientemente, los péptidos antimicrobianos (PAM) obtenidos sintéticamente han surgido como una alternativa promisoria para el tratamiento de infecciones microbianas y muestran propiedades multifuncionales con implicaciones como agentes terapéuticos (Cruz y col., 2014). Por lo anterior, en este trabajo de investigación se llevó a cabo el diseño y síntesis de una serie de 18 péptidos con potencial actividad antibiótica. Inicialmente, se estudió la estructura de los PAM utilizando herramientas de bioinformática como: i) ¿Design and Prediction Antimicrobial Peptides¿ (DEPRAMS, por sus siglas en inglés), ii) ClustalW y iii) CPH models, entre otras, para determinar los residuos importantes asociados a la actividad antimicrobiana. Posteriormente, se sintetizaron nuevas secuencias peptídicas en fase sólida empleando la estrategia Fmoc (9-fluorenilmetoxicarbonilo). Los PAM fueron denominados como P1 a P18. Las secuencias bases y los análogos sintéticos se obtuvieron con una pureza del 95% y un perfil típico de ¿-hélice, de acuerdo con los espectros de dicroísmo circular (CD). Además, la estructura primaria de los PAM fue confirmada por espectrometría de masas MALDI-TOF. La actividad de los PAM fue evaluada en cepas patógenas de Escherichia coli 0157:H7 (E. coli O157:H7) y Staphylococcus aureus resistente a meticilina (SARM) y se obtuvieron valores de 1.5 y 2.5 µM para el péptido P17, respectivamente. La hemólisis de los PAM frente a eritrocitos de carnero en todos los ensayos no superó el 40%. Además, el mecanismo de acción de los PAM fue estudiada con la sonda aniónica de Sytox-Green, con la cual se demostró que, en general, la formación de poros transmembrana de los PAM es la forma de actuación más común, excepto para el PAM P10. Finalmente, pensamos que estos PAM pueden proponerse como una alternativa terapéutica para las infecciones causadas por SARM y E. coli O157:H7 que se podrían desarrollar y aplicar como antibióticos por sistemas de liberación controlada.Los autores deseamos agradecer al Instituto de Salud Carlos III de España por el financiamiento de los proyectos FIS PS12-02706 y RD 12/0018/0007, MINECO CTQ2013-41507-R de España, Vicerrectoría de Investigación y Extensión (VIE) de la Universidad Industrial de Santander y Colciencias de Colombia por el proyecto número 1102-5453-1671.Peer Reviewe

Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.This work was supported by grants of Sao Paulo State Research Foundation (FAPESP 2019/10434-4 to S.E.T.B. and 2021/04464-8 and 2017/50333-7 to A.G.T.); Subdirección General de Redes y Centros de Investigación Cooperativa - FEDER (RICET RD16/0027/0010, and Ministerio de Ciencia e Innovación España. Plan Estatal de Investigación Científica y Técnica y de Innovación 2017-2020-FEDER PID2019- 108166GB-100 /AEI/10.13039/501100011033 to L. R.), EADS-CASA/Brazilian Air Force (FAB) mobility program to M.L.L., and Coordenaçao de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to M. L.L.Peer reviewe

Influence of the side chain structure on the anti-leishmanial effect of methotrexate conjugates with polymeric branched chain polypeptides

1 p. 4 fig. 1 tab.Peer reviewe