Synthesis and Antileishmanial Activity of 1,2,4,5-Tetraoxanes against Leishmania donovani

A chemically diverse range of novel tetraoxanes was synthesized and evaluated in vitro against intramacrophage amastigote forms of Leishmania donovani. All 15 tested tetraoxanes displayed activity, with IC50 values ranging from 2 to 45 µm. The most active tetraoxane, compound LC140, exhibited an IC50 value of 2.52 ± 0.65 µm on L. donovani intramacrophage amastigotes, with a selectivity index of 13.5. This compound reduced the liver parasite burden of L. donovani-infected mice by 37% after an intraperitoneal treatment at 10 mg/kg/day for five consecutive days, whereas miltefosine, an antileishmanial drug in use, reduced it by 66%. These results provide a relevant basis for the development of further tetraoxanes as effective, safe, and cheap drugs against leishmaniasis.This research was funded by Fundação para a Ciência e a Tecnologia (FCT), and FEDER/COMPETE 2020-UE, through projects UID/Multi/04326/2019 (Centre of Marine Sciences-CCMAR) and PTDC/MAR-BIO/4132/2014.info:eu-repo/semantics/publishedVersio

Chitosan Contribution to Therapeutic and Vaccinal Approaches for the Control of Leishmaniasis.

The control of leishmaniases, a complex parasitic disease caused by the protozoan parasite Leishmania, requires continuous innovation at the therapeutic and vaccination levels. Chitosan is a biocompatible polymer administrable via different routes and possessing numerous qualities to be used in the antileishmanial strategies. This review presents recent progress in chitosan research for antileishmanial applications. First data on the mechanism of action of chitosan revealed an optimal in vitro intrinsic activity at acidic pH, high-molecular-weight chitosan being the most efficient form, with an uptake by pinocytosis and an accumulation in the parasitophorous vacuole of Leishmania-infected macrophages. In addition, the immunomodulatory effect of chitosan is an added value both for the treatment of leishmaniasis and the development of innovative vaccines. The advances in chitosan chemistry allows pharmacomodulation on amine groups opening various opportunities for new polymers of different size, and physico-chemical properties adapted to the chosen routes of administration. Different formulations have been studied in experimental leishmaniasis models to cure visceral and cutaneous leishmaniasis, and chitosan can act as a booster through drug combinations with classical drugs, such as amphotericin B. The various architectural possibilities given by chitosan chemistry and pharmaceutical technology pave the way for promising further developments

Cross-study analysis of genomic data defines the ciliate multigenic epiplasmin family: strategies for functional analysis in Paramecium tetraurelia

<p>Abstract</p> <p>Background</p> <p>The sub-membranous skeleton of the ciliate <it>Paramecium</it>, the epiplasm, is composed of hundreds of epiplasmic scales centered on basal bodies, and presents a complex set of proteins, epiplasmins, which belong to a multigenic family. The repeated duplications observed in the <it>P. tetraurelia </it>genome present an interesting model of the organization and evolution of a multigenic family within a single cell.</p> <p>Results</p> <p>To study this multigenic family, we used phylogenetic, structural, and analytical transcriptional approaches. The phylogenetic method defines 5 groups of epiplasmins in the multigenic family. A refined analysis by Hydrophobic Cluster Analysis (HCA) identifies structural characteristics of 51 epiplasmins, defining five separate groups, and three classes. Depending on the sequential arrangement of their structural domains, the epiplasmins are defined as symmetric, asymmetric or atypical. The EST data aid in this classification, in the identification of putative regulating sequences such as TATA or CAAT boxes. When specific RNAi experiments were conducted using sequences from either symmetric or asymmetric classes, phenotypes were drastic. Local effects show either disrupted or ill-shaped epiplasmic scales. In either case, this results in aborted cell division.</p> <p>Using structural features, we show that 4 epiplasmins are also present in another ciliate, <it>Tetrahymena </it><it>thermophila</it>. Their affiliation with the distinctive structural groups of <it>Paramecium </it>epiplasmins demonstrates an interspecific multigenic family.</p> <p>Conclusion</p> <p>The epiplasmin multigenic family illustrates the history of genomic duplication in <it>Paramecium</it>. This study provides a framework which can guide functional analysis of epiplasmins, the major components of the membrane skeleton in ciliates. We show that this set of proteins handles an important developmental information in <it>Paramecium </it>since maintenance of epiplasm organization is crucial for cell morphogenesis.</p

GDP-Mannose Pyrophosphorylase: A Biologically Validated Target for Drug Development Against Leishmaniasis

Leishmaniases are neglected tropical diseases that threaten about 350 million people in 98 countries around the world. In order to find new antileishmanial drugs, an original approach consists in reducing the pathogenic effect of the parasite by impairing the glycoconjugate biosynthesis, necessary for parasite recognition and internalization by the macrophage. Some proteins appear to be critical in this way, and one of them, the GDP-Mannose Pyrophosphorylase (GDP-MP), is an attractive target for the design of specific inhibitors as it is essential for Leishmania survival and it presents significant differences with the host counterpart. Two GDP-MP inhibitors, compounds A and B, have been identified in two distinct studies by high throughput screening and by a rational approach based on molecular modeling, respectively. Compound B was found to be the most promising as it exhibited specific competitive inhibition of leishmanial GDP-MP and antileishmanial activities at the micromolar range with interesting selectivity indexes, as opposed to compound A. Therefore, compound B can be used as a pharmacological tool for the development of new specific antileishmanial drugs

clag9 Is Not Essential for PfEMP1 Surface Expression in Non-Cytoadherent Plasmodium falciparum Parasites with a Chromosome 9 Deletion

BACKGROUND: The expression of the clonally variant virulence factor PfEMP1 mediates the sequestration of Plasmodium falciparum infected erythrocytes in the host vasculature and contributes to chronic infection. Non-cytoadherent parasites with a chromosome 9 deletion lack clag9, a gene linked to cytoadhesion in previous studies. Here we present new clag9 data that challenge this view and show that surface the non-cytoadherence phenotype is linked to the expression of a non-functional PfEMP1. METHODOLOGY/PRINCIPAL FINDINGS: Loss of adhesion in P. falciparum D10, a parasite line with a large chromosome 9 deletion, was investigated. Surface iodination analysis of non-cytoadherent D10 parasites and COS-7 surface expression of the CD36-binding PfEMP1 CIDR1α domain were performed and showed that these parasites express an unusual trypsin-resistant, non-functional PfEMP1 at the erythrocyte surface. However, the CIDR1α domain of this var gene expressed in COS-7 cells showed strong binding to CD36. Atomic Force Microscopy showed a slightly modified D10 knob morphology compared to adherent parasites. Trafficking of PfEMP1 and KAHRP remained functional in D10. We link the non-cytoadherence phenotype to a chromosome 9 breakage and healing event resulting in the loss of 25 subtelomeric genes including clag9. In contrast to previous studies, knockout of the clag9 gene from 3D7 did not interfere with parasite adhesion to CD36. CONCLUSIONS/SIGNIFICANCE: Our data show the surface expression of non-functional PfEMP1 in D10 strongly indicating that genes other than clag9 deleted from chromosome 9 are involved in this virulence process possibly via post-translational modifications

Squelette membranaire chez Paramecium tetraurelia : caractérisation d'une nouvelle famille multigénique et analyse par les approches GFP et RNAi

The cortex of most ciliated protozoa encloses a membrane skeleton including the epiplasm, a sub-membranous layer extending underneath the cytoplasmic face of the inner alveolar membrane. In Paramecium, epiplasmic scales are centered around ciliary units and are composed of a complex set of proteins named epiplasmins. In the first part of this study, using the sequence of two epiplasmins (EPI-1 and EPI-2), we have contributed to the annotation of Paramecium tetraurelia macronuclear genome and identified 39 additional sequences. Two orthologous sequences were found in the Tetrahymena thermophila genome. Structural analysis of the 43 sequences indicates that the hallmark of this new multigenic family is a 79 amino acids domain flanked by two Q, P and V-rich stretches of sequence that are much more variable in amino-acid composition. A phylogenetic analysis confirmed the existence of 3 sub-families (on a total of 4) that were previously determined on the basis of distinct biochemical properties. The expression of GFP-tagged epiplasmin 1 showed significant labelling of epiplasmic scales and oral structures as well. The second part concerns the functional analysis of epiplasmins using an RNA interference approach. Following the ingestion of bacteria producing double-stranded RNA homologous to the EPI-1 and/or EPI-2 coding sequences, we obtained a phenotype named "boomerang" characterized by repeated blockages of cytokinesis leading to monstrous organisms which are finally lysed after 72 hours of induction. The analysis of induced cells by fluorescence microscopy revealed multiple abnormalities in cortical morphogenesis and karyokinesis processes despite the normal progression of some mechanisms like oral apparatus duplication. Finally, the production of a novel antibody raised against 2 peptides (1 and 2) included in epiplasmins 1 and 2 sequences, and decorating the epiplasm of Paramecium, allowed us to evidence the co-suppression of a sub-family of epiplasmins following the "boomerang" phenotype induction. The epitope of the monoclonal antibody (CTS-32) which labels the overall family of epiplasmins is not contained in peptides 1 and 2.Chez la paramécie, l'épiplasme se présente sous forme d'écailles indépendantes disposées autour de chaque appareil ciliaire et composées d'une multitude de protéines appelées épiplasmines. Dans une première partie, nous avons contribué à l'annotation du génome macronucléaire de Paramecium tetraurelia à partir de la séquence de 2 épiplasmines (EPI-1 et EPI-2) et identifié 39 gènes supplémentaires. L'analyse des séquences indique que la signature de cette nouvelle famille est un domaine de 79 acides aminés. L'expression de l'épiplasmine 1 couplée à la GFP confirme la localisation de cette protéine au niveau des écailles épiplasmiques. La deuxième partie porte sur l'analyse fonctionnelle d'épiplasmines à partir d'une approche ARN interférence. Nous avons obtenu un phénotype caractérisé par un blocage répété de la cytocinèse conduisant à des organimes monstrueux qui sont finalement lysés après 72 h d'induction. L'analyse en microscopie à fluorescence a révélé l'absence de sillon de division

Squelette menbranaire chez Paramecium tetraurelia (caractérisation d'une nouvelle famille multigénique et analyse par les approche GFP et RNAi)

Chez la paramécie, l'épiplasme se présente sous forme d'écailles indépendantes disposées autour de chaque appareil ciliaire et composées d'une multitude de protéines appelées épiplasmines. Dans une première partie, nous avons contribué à l'annotation du génome macronucléaire de Paramecium tetraurelia à partir de la séquence de 2 épiplasmines (EPI-1 et EPI-2) et identifié 39 gènes supplémentaires. L'analyse des séquences indique que la signature de cette nouvelle famille est un domaine de 79 acides aminés. L'expression de l'épiplasmine 1 couplée à la GFP confirme la localisation de cette protéine au niveau des écailles épiplasmiques. La deuxième partie porte sur l'analyse fonctionnelle d'épiplasmines à partir d'une approche ARN interférence. Nous avons obtenu un phénotype caractérisé par un blocage répété de la cytocinèse conduisant à des organimes monstrueux qui sont finalement lysés après 72 h d'induction. L'analyse en microscopie à fluorescence a révélé l'absence de sillon de divisionCLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

In vitro evaluation of antimicrobial agents on Acanthamoeba sp. and evidence of a natural resilience to amphotericin B

The free-living amoeba (FLA) Acanthamoeba sp. is an opportunistic pathogen that can cause amoebic keratitis (AK) or granulomatous amoebic encephalitis (GAE). While current treatments of AK are long with some relapses, no consensus therapy has been developed for GAE remaining lethal in 90% of the cases. In this context, efficient antiacanthamoebal drugs have to be identified. In this work, 15 drugs used in the treatment of AK or GAE or in other parasitic diseases were evaluated for their in vitro activity on A. castellanii. Hexamidine, voriconazole and clotrimazole exhibited the highest activities with IC50 values at 0.05 μM, 0.40 μM and 0.80 μM, respectively, while rifampicin, metronidazole and cotrimoxazole were inactive. Among 15 drug associations evaluated, no synergistic effect was observed, and one antagonism was determined between hexamidine and chlorhexidine. Interestingly, amphotericin B was the only drug presenting an increase of IC50 as a function of treatment duration. The amoebae susceptibility to amphotericin B cultured in the presence of 250 μM of the drug was similar to the one of a naive control, revealing that no resistant strain could be selected. However, the amoebae susceptibility always returned to an initial level at each passage. This natural and non-acquired adaptation to amphotericin B, qualified as resilience, was observed in several strains of A. castellanii and A. polyphaga. Using a pharmacological approach with effectors of different cellular mechanisms or transports, and an ultrastructural analysis of amphotericin B-treated amoebae, the involvement of several mitochondria-dependent pathways as well as multidrug resistant transporters was determined in amphotericin B resilience. Based on the observations from this study, the relevance of using amphotericin B in GAE treatments may be reconsidered, while the use of some other drugs, such as rifampicin or cotrimoxazole, is not relative to intrinsic antiacanthamoebal activity. Keywords: Acanthamoeba sp., Antimicrobial agents, Amphotericin B, Resilienc

Drugs used for the treatment of cerebral and disseminated infections caused by free‐living amoebae

Abstract Free‐living amoebae (FLAs) are protozoa developing autonomously in diverse natural or artificial environments. The FLAs Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri represent a risk for human health as they can become pathogenic and cause severe cerebral infections, named granulomatous amoebic encephalitis (GAE), Balamuthia amoebic encephalitis (BAE), and primary amoebic meningoencephalitis (PAM), respectively. Additionally, Acanthamoeba sp. can also rarely disseminate to diverse organs, such as the skin, sinuses, or bones, and cause extracerebral disseminated acanthamebiasis (EDA). No consensus treatment has been established for cerebral FLA infections or EDA. The therapy of cerebral and disseminated FLA infections often empirically associates a large diversity of drugs, all exhibiting a high toxicity. Nevertheless, these pathologies lead to a high mortality, above 90% of the cases, even in the presence of a treatment. In the present work, a total of 474 clinical cases of FLA infections gathered from the literature allowed to determine the frequency of usage, as well as the efficacy of the main drugs and drug combinations used in the treatment of these pathologies. The efficacy of drug usage was determined based on the survival rate after drug administration. The most efficient drugs, drug combinations, and their mechanism of action were discussed in regard to the present recommendations for the treatment of GAE, EDA, BAE, and PAM. At the end, this review aims to provide a useful tool for physicians in their choice to optimize the treatment of FLA infections

Minor Impact of A258D Mutation on Biochemical and Enzymatic Properties of Leishmania infantum GDP-Mannose Pyrophosphorylase

Background: Leishmaniasis, a vector-borne disease caused by the protozoan parasite from the genus Leishmania, is endemic to tropical and subtropical areas. Few treatments are available against leishmaniasis, with all presenting issues of toxicity, resistance, and/or cost. In this context, the development of new antileishmanial drugs is urgently needed. GDP-mannose pyrophosphorylase (GDP-MP), an enzyme involved in the mannosylation pathway, has been described to constitute an attractive therapeutic target for the development of specific antileishmanial agents. Methods: In this work, we produced, purified, and analyzed the enzymatic properties of the recombinant L. infantum GDP-MP (LiGDP-MP), a single leishmanial GDP-MP that presents mutation of an aspartate instead of an alanine at position 258, which is also the single residue difference with the homolog in L. donovani: LdGDP-MP. Results: The purified LiGDP-MP displayed high substrate and cofactor specificities, a sequential random mechanism of reaction, and the following kinetic constants: Vm at 0.6 &micro;M&middot;min&minus;1, Km from 15&ndash;18 &micro;M, kcat from 12.5&ndash;13 min&minus;1, and kcat/Km at around 0.8 min&minus;1&micro;M&minus;1. Conclusions: These results show that LiGDP-MP has similar biochemical and enzymatic properties to LdGDP-MP. Further studies are needed to determine the advantage for L. infantum of the A258D residue change in GDP-MP