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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.

11p.-6 fig.-1 tab.-1 graph. abst., The recruitment of eosinophils into Leishmania lesions is frequently associated with a favorable evolution. A feasible effector for this process is eosinophil cationic protein (ECP, RNase 3), one of the main human eosinophil granule proteins, endowed with a broad spectrum of antimicrobial activity, including parasites. ECP was active on Leishmania promastigotes and axenic amastigotes (LC50’s = 3 and 16 μM, respectively) but, in contrast to the irreversible membrane damage caused on bacteria and reproduced by its N-terminal peptides, it only induced a mild and transient plasma membrane destabilization on Leishmania donovani promastigotes. To assess the contribution of RNase activity to the overall leishmanicidal activity of ECP, parasites were challenged in parallel with a single-mutant version, ECP-H15A, devoid of RNase activity, that fully preserves the conformation and liposome permeabilization ability. ECP-H15A showed a similar uptake to ECP on promastigotes, but with higher LC50’s (>25 μM) for both parasite stages. ECP-treated promastigotes showed a degraded RNA pattern, absent in ECP-H15A-treated samples. Moreover ECP, but not ECP-H15A, reduced more than 2-fold the parasite burden of infected macrophages. Altogether, our results suggest that ECP enters the Leishmania cytoplasm by an endocytic pathway, ultimately leading to RNA degradation as a key contribution to the leishmanicidal mechanism. Thus, ECP combines both membrane destabilization and enzymatic activities to effect parasite killing. Taken together, our data highlight the microbicidal versatility of ECP as an innate immunity component and support the development of cell-penetrating RNases as putative leishmanicidal agents., This work was supported by MCIN/AEI/ 10.13039/501100011033-FEDER grant numbers PID2019-108166GB-I00 (E.B.), SAF2011-24899 (D.A.), by MCIN Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER RD16/0027/0010 (L.R.), by CSIC PIE 201620E038 (L.R.), and by Generalitat de Catalunya grant 2009SGR492 to D.A. V.A.S. was a recipient of a Francisco Caldas-Colciencias predoctoral fellowship. We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI).

35 p.-5 fig.-1 tab.-1 schem., The human protozoan parasites Leishmania donovani and L. infantum are the causative agents of visceral leishmaniasis, as such, responsible for approximately 30,000 deaths annually. The available chemotherapeutic treatments are reduced to a few drugs whose effectiveness is limited by rising drug resistance/therapeutic failure, and noxious side-effects. Therefore, new therapeutic hits are needed. Compounds displaying the imidazo[2,1-a]isoindole skeleton have shown antichagasic, anti-HIV, antimalarial and anorectic activities. Here, we report the leishmanicidal activity of thirty one imidazo[2,1-a]isoindol-5-ol derivatives on promastigotes and intracellular amastigotes of L. donovani. Eight out of thirty one assayed compounds showed EC50 values ranging between 1 and 2 μM with selectivity indexes from 29 to 69 on infected THP-1 cells. Six compounds were selected for further elucidation of their leishmanicidal mechanism. In this regard, compound 29, the imidazoisoindolol with the highest activity on intracellular amastigotes, induced an early decrease of intracellular ATP levels, as well as mitochondrial depolarization, together with a partial plasma membrane destructuration, as assessed by transmission electron microscopy. Consequently, the inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound, even when other additional targets cannot be ruled out. In all, the results supported the inclusion of the imidazoisoindole scaffold for the development of new leishmanicidal drugs., Financial support from FIS-PI-060782, FIS-PI-052026, MINECO: RETOS (AGL2016-79813-C2-2, to EO), the Spanish Ministerio de Ciencia, Innovación y Universidades (RTI2018-097210-B-I00 to FG), ISCIII-RICET-FEDER: RD16/0027/0018 (EO) and RD16/0027/0010 (LR), and SAF2015-65740 (LR), as the CSIC grant PIE 201620E038 are acknowledged.

58 p.-6 fig.-1 tab.-5 schem.-1 graph.abst, A new class of quinoline derivatives, bearing amino chains at C-4 and a styryl group at C-2, were tested on Leishmania donovani promastigotes and axenic and intracellular Leishmania pifanoi amastigotes. The introduction of the C-4 substituent improves the activity, which is due to interference with the mitochondrial activity of the parasite and its concomitant bioenergetic collapse by ATP exhaustion. Some compounds show a promising antileishmanial profile, with low micromolar or submicromolar activity on promastigote and amastigote forms and a good selectivity index., This research was supported by the following grants; (a) MLB: MIUR, PRIN 201274BNKN_003; (b) JCM: Universidad Complutense (GR3/14) and MINECO (CTQ2015-68380-R); (c) LR: RD16/0027/0010, CSIC PIE-201020E054 and MINECO SAF2015-65740-R. A predoctoral contracts to MS from Universidad Complutense and a FPI contract to MP from MINECO (BES-2016–076410P) are also gratefully acknowledged.

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17 p.-6 fig.-2 tab., BACKGROUND:Antimicrobial peptides are on the first line of defense against pathogenic microorganisms of many living beings. These compounds are considered natural antibiotics that can overcome bacterial resistance to conventional antibiotics. Due to this characteristic, new peptides with improved properties are quite appealing for designing new strategies for fighting pathogenic bacteria., METHODS:Sixteen designed peptides were synthesized using Fmoc chemistry; five of them are new cationic antimicrobial peptides (CAMPs) designed using a genetic algorithm that optimizes the antibacterial activity based on selected physicochemical descriptors and 11 analog peptides derived from these five peptides were designed and constructed by single amino acid substitutions. These 16 peptides were structurally characterized and their biological activity was determined against Escherichia coli O157:H7 (E. coli O157:H7), and methicillin-resistant strains of Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were determined., RESULTS:These 16 peptides were folded into an α-helix structure in membrane-mimicking environment. Among these 16 peptides, GIBIM-P5S9K (ATKKCGLFKILKGVGKI) showed the highest antimicrobial activity against E. coli O157:H7 (MIC=10µM), methicillin-resistant Staphylococcus aureus (MRSA) (MIC=25µM) and Pseudomonas aeruginosa (MIC=10 µM). Peptide GIBIM-P5S9K caused permeabilization of the bacterial membrane at 25 µM as determined by the Sytox Green uptake assay and the labelling of these bacteria by using the fluoresceinated peptide. GIBIM-P5S9K seems to be specific for these bacteria because at 50 µM, it provoked lower than 40% of erythrocyte hemolysis., CONCLUSION:New CAMPs have been designed using a genetic algorithm based on selected physicochemical descriptors and single amino acid substitution. These CAMPs interacted quite specifically with the bacterial cell membrane, GIBIM-P5S9K exhibiting high antibacterial activity on Escherichia coli O157:H7, methicillin-resistant strains of Staphylococcus aureus and P. aeruginosa., This project was founded by COLCIENCIAS (Project Number: 110265740828) and MINECO ((Spain project number CTQ2013-41507-R).LR was supported by grants from Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER, RICET RD12/0018/0007, and RD16/0027/0010

42 p.-8 fig. Drug repurposing affords the implementation of new treatments at a moderate cost and under a faster time-scale. Most of the clinical drugs against Leishmania share this origin. The antidepressant sertraline has been successfully assayed in a murine model of visceral leishmaniasis. Nevertheless, sertraline targets in Leishmania were poorly defined. In order to get a detailed insight into the leishmanicidal mechanism of sertraline on Leishmania infantum, unbiased multiplatform metabolomics and transmission electron microscopy were combined with a focused insight into the sertraline effects on bioenergetics metabolism of the parasite. Sertraline induced respiration uncoupling, a significant decrease of intracellular ATP level, and oxidative stress in L. infantum promastigotes. Metabolomics evidenced an extended metabolic disarray caused by sertraline. This encompasses a remarkable variation of the levels of thiol-redox and polyamine biosynthetic intermediates, as well as shortage of intracellular amino acids used as metabolic fuel by Leishmania Sertraline killed Leishmania through a multitarget mechanism of action, tackling essential metabolic pathways of the parasite. As such, sertraline is a valuable candidate for visceral leishmaniasis treatment under a drug repurposing strategy. This work was supported by grants of the Fondo de Investigaciones Sanitarias-ISCIII-FEDER (PI12-02706), and (RD16/0027/0010, Red de Enfermedades Tropicales, subprogram RETICS del Plan Estatal de I+D+i (2013-2016) co-financed with FEDER funds, SAF2015-65740-R, and CSIC PIE 201620E038 to L. R., São Paulo State Research Foundation (FAPESP 2015/23403-9 to A. G.T.); EADS-CASA/ Brazilian Air Force (FAB) mobility program to M.L.L., and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to M.L.L.