Flavonoids represent a potential source of new antitrypanosomatidic leads. Starting from a library of natural products, we combined target-based screening on pteridine reductase 1 with phenotypic screening on Trypanosoma brucei for hit identification. Flavonols were identified as hits, and a library of 16 derivatives was synthesized. Twelve compounds showed EC50 values against T. brucei below 10 μM. Four X-ray crystal structures and docking studies explained the observed structure-activity relationships. Compound 2 (3,6-dihydroxy-2-(3-hydroxyphenyl)-4H-chromen-4-one) was selected for pharmacokinetic studies. Encapsulation of compound 2 in PLGA nanoparticles or cyclodextrins resulted in lower in vitro toxicity when compared to the free compound. Combination studies with methotrexate revealed that compound 13 (3-hydroxy-6-methoxy-2-(4-methoxyphenyl)-4H-chromen-4-one) has the highest synergistic effect at concentration of 1.3 μM, 11.7-fold dose reduction index and no toxicity toward host cells. Our results provide the basis for further chemical modifications aimed at identifying novel antitrypanosomatidic agents showing higher potency toward PTR1 and increased metabolic stability.

Regulatory pathways that drive early hematogenous dissemination of tumor cells are insuffi ciently defi ned. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to defi ne patients with early disseminated breast cancer and identifi ed low retinoic acid-induced 2 (RAI2) expression to be signifi cantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. SIGNIFICANCE:We identifi ed downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specifi ed the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer. Cancer Discov;5(5);

Intracellular pathogens belonging to the genus Leishmania have developed effective strategies that enable them to survive within host immune cells. Immunostimulatory compounds that counteract such immunological escape mechanisms represent promising treatment options for diseases. Here, we demonstrate that a lipopeptidephosphoglycan (LPPG) isolated from the membrane of a protozoan parasite, Entamoeba histolytica (Eh), shows considerable immunostimulatory effects targeted against Leishmania (L.) major, a representative species responsible for cutaneous leishmaniasis (CL). Treatment led to a marked reduction in the number of intracellular Leishmania parasites in vitro, and ameliorated CL in a mouse model. We next designed and synthesized analogs of the phosphatidylinositol anchors harbored by EhLPPG; two of these analogs reproduced the anti-leishmanial activity of the native compound by inducing production of pro-inflammatory cytokines. The use of such compounds, either alone or as a supportive option, might improve the currently unsatisfactory treatment of CL and other diseases caused by pathogen-manipulated immune responses.

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The majority of PCR-based detection systems for Leishmania spp. and Trypanosoma cruzi aim at high sensitivity and specificity, rather than an accurate parasite load quantification required for experimental infections in basic research and drug development. Here, we describe the use of a dual-labelled probe qPCR to detect and quantify intracellular Old World Leishmania spp. and T. cruzi amastigotes after in vitro and in vivo infection experiments. We show that quantification of parasite actin gene DNA relative to the host cell actin gene DNA accurately reflects the parasite load relative to the host cells and that qPCR quantification is highly sensible to drug-induced cell death. Furthermore, qPCR allows to determine parasite loads even after host cell detachment and/or rupture, important when comparing untreated versus drug-treated samples. The method is also suitable for the quantification of parasites from infected mouse tissue, making it suitable for drug testing and mutant phenotype analysis.

With an estimated number of new cases annually of approximately 1.4 million, leishmaniasis belongs to the most important parasitic diseases in the world. Nevertheless, existing drugs against leishmaniasis in general have several drawbacks that urgently necessitate new drug development. A glycolipid molecule of the intestinal protozoan parasite Entamoeba histolytica and its synthetic analogs previously showed considerable immunotherapeutic effects against Leishmania major infection. Here, we designed and synthesized a series of new immunostimulatory compounds derived from the phosphatidylinositol b anchor of Entamoeba histolytica (EhPIb) subunit of the native compound and investigated their antileishmanial activity in vitro and in vivo in a murine model of cutaneous leishmaniasis. The new synthetic EhPIb analogs showed almost no toxicity in vitro. Treatment with the analogs significantly decreased the parasite load in murine and human macrophages in vitro. In addition, topical application of the EhPIb analog Eh-1 significantly reduced cutaneous lesions in the murine model, correlating with an increase in the production of selected Th1 cytokines. In addition, we could show in in vitro experiments that treatment with Eh-1 led to a decrease in mRNA expression of arginase-1 (Arg1) and interleukin 4 (IL-4), which are required by the parasites to circumvent their elimination by the immune response. The use of the host-targeting synthetic EhPIb compounds, either alone or in combination therapy with antiparasitic drugs, shows promise for treating cutaneous leishmaniasis and therefore might improve the current unsatisfactory status of chemotherapy against this infectious disease.

Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.

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