Antileishmanial High-Throughput Drug Screening Reveals Drug Candidates with New Scaffolds

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assay validation was done with Leishmania promastigote forms, including the screening of 4,000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was confirmed against the intramacrophagic amastigote form of the parasite. In vitro microsomal stability and cytochrome P450 (CYP) inhibition of the two most active compounds from this screening effort were assessed to obtain preliminary information on their metabolism in the host. The HTS approach employed here resulted in the discovery of two new antileishmanial compounds, bringing promising candidates to the leishmaniasis drug discovery pipeline

Leishmania-Induced Inactivation of the Macrophage Transcription Factor AP-1 Is Mediated by the Parasite Metalloprotease GP63

Leishmania parasites have evolved sophisticated mechanisms to subvert macrophage immune responses by altering the host cell signal transduction machinery, including inhibition of JAK/STAT signalling and other transcription factors such as AP-1, CREB and NF-κB. AP-1 regulates pro-inflammatory cytokines, chemokines and nitric oxide production. Herein we show that upon Leishmania infection, AP-1 activity within host cells is abolished and correlates with lower expression of 5 of the 7 AP-1 subunits. Of interest, c-Jun, the central component of AP-1, is cleaved by Leishmania. Furthermore, the cleavage of c-Jun is dependent on the expression and activity of the major Leishmania surface protease GP63. Immunoprecipitation of c-Jun from nuclear extracts showed that GP63 interacts, and cleaves c-Jun at the perinuclear area shortly after infection. Phagocytosis inhibition by cytochalasin D did not block c-Jun down-regulation, suggesting that internalization of the parasite might not be necessary to deliver GP63 molecules inside the host cell. This observation was corroborated by the maintenance of c-Jun cleavage upon incubation with L. mexicana culture supernatant, suggesting that secreted, soluble GP63 could use a phagocytosis-independent mechanism to enter the host cell. In support of this, disruption of macrophage lipid raft microdomains by Methyl β-Cyclodextrin (MβCD) partially inhibits the degradation of full length c-Jun. Together our results indicate a novel role of the surface protease GP63 in the Leishmania-mediated subversion of host AP-1 activity

Differential impact of LPG-and PG-deficient Leishmania major mutants on the immune response of human dendritic cells

<div><p>Background</p><p><i>Leishmania major</i> infection induces robust interleukin-12 (IL12) production in human dendritic cells (hDC), ultimately resulting in Th1-mediated immunity and clinical resolution. The surface of <i>Leishmania</i> parasites is covered in a dense glycocalyx consisting of primarily lipophosphoglycan (LPG) and other phosphoglycan-containing molecules (PGs), making these glycoconjugates the likely pathogen-associated molecular patterns (PAMPS) responsible for IL12 induction.</p><p>Methodology/Principal Findings</p><p>Here we explored the role of parasite glycoconjugates on the hDC IL12 response by generating <i>L</i>. <i>major</i> Friedlin V1 mutants defective in LPG alone, (FV1 <i>lpg1-</i>), or generally deficient for all PGs, (FV1 <i>lpg2-</i>). Infection with metacyclic, infective stage, <i>L</i>. <i>major</i> or purified LPG induced high levels of <i>IL12B</i> subunit gene transcripts in hDCs, which was abrogated with FV1 <i>lpg1-</i> infections. In contrast, hDC infections with FV1 <i>lpg2-</i> displayed increased <i>IL12B</i> expression, suggesting other PG-related/<i>LPG2</i> dependent molecules may act to dampen the immune response. Global transcriptional profiling comparing WT, FV1 <i>lpg1-</i>, FV1 <i>lpg2-</i> infections revealed that FV1 <i>lpg1-</i> mutants entered hDCs in a silent fashion as indicated by repression of gene expression. Transcription factor binding site analysis suggests that LPG recognition by hDCs induces IL-12 in a signaling cascade resulting in Nuclear Factor κ B (NFκB) and Interferon Regulatory Factor (IRF) mediated transcription.</p><p>Conclusions/Significance</p><p>These data suggest that <i>L</i>. <i>major</i> LPG is a major PAMP recognized by hDC to induce IL12-mediated protective immunity and that there is a complex interplay between PG-baring <i>Leishmania</i> surface glycoconjugates that result in modulation of host cellular IL12.</p></div

Synaptic plasticity in cephalopods; more than just learning and memory?

The outstanding behavioural capacity of cephalopods is underpinned by a highly sophisticated nervous system anatomy and neural mechanisms that often differ significantly from similarly complex systems in vertebrates and insects. Cephalopods exhibit considerable behavioural flexibility and adaptability, and it might be expected that this should be supported by evident cellular and synaptic plasticity. Here, we review what little is known of the cellular mechanisms that underlie plasticity in cephalopods, particularly from the point of view of synaptic function. We conclude that cephalopods utilise short-, medium-, and long-term plasticity mechanisms that are superficially similar to those so far described in vertebrate and insect synapses. These mechanisms, however, often differ significantly from those in other animals at the biophysical level and are deployed not just in the central nervous system, but also to a limited extent in the peripheral nervous system and neuromuscular junctions.</p

Parasite Burden in Hamsters Infected with Two Different Strains of Leishmania (Leishmania) infantum: “Leishman Donovan Units” versus Real-Time PCR

To develop and test new therapeutics and immune prophylaxis strategies for visceral leishmaniasis (VL), understanding tissue parasitism evolution after experimental infection with Leishmania infantum is important. Experimental infection in a hamster model (Mesocricetus auratus) reproduces several typical aspects of canine and human VL that are closely related to the inoculum’s route. We quantified the parasitism in the liver and spleen of hamsters experimentally infected by various routes (intradermal, intraperitoneal, and intracardiac [IC]) and different strains of L. infantum (MHOM/BR/74/PP75 and Wild) and compared two different methodologies to evaluate tissue parasitism (Leishman Donovan units [LDU] and real-time qPCR). In addition, the quantification of specific total-IgG in the serum of uninfected and infected hamsters was determined by ELISA. The animals were followed for 1, 3, 6 and 9 months post-infection for survival analysis. We found that infection with the Wild strain by the IC route resulted in higher mortality. Positive antibody (IgG) responses were detected with higher peaks at 6 and 9 months in the IC group inoculated with PP75 strain. However, in animals infected with the Wild strain the IgG levels were elevated in all infected groups during all the time evaluated. We also observed by LDU analysis that the IC route lead to higher parasitism in the liver and spleen with both strains. Furthermore, qPCR showed higher sensitivity for identifying animals with low parasitic burden. In conclusion, qPCR can be useful for assessing parasitism in the spleen and liver of a hamster model infected with L. infantum independent of the route of infection, and this technique may become an essential tool for assessing parasite density in the hamster model after experimental treatment or immunization with potential vaccine candidates