An ex vivo multiparametric flow cytometry assay using human whole blood to simultaneously measure cytotoxicity and leishmanicidal activities.

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.

Synthesis of novel 2,3,4-trisubstituted-oxazolidine derivatives and in vitro cytotoxic evaluation.

We have previously reported the discovery of cytotoxic and pro-apoptotic hit compound 1,1-dimethylethyl (S)- 2,2-dimethyl-4-[(3-nitrophenoxy)methyl]-3-oxazolidinecarboxylate 1 against leukemia cells. In the present work we describe the synthesis of 25 derivatives of this hit varying the substituent at ring or stereochemistry of the oxazolidine ring and evaluated them against human cancer cells lines. Six compounds exerted significant activity against HL60 promyelocytic leukemia cells with IC50 in low micromolar range (4-18 µM) and three compounds displayed activity against MDA-MB231 breast cancer cells (25-37 µM). In vitro cytotoxicity on normal cells PBMC (human peripheral blood mononuclear cells) was also evaluated. Compounds 7e (p-NO2, S) and 7m (p-COOCH3, S) showed good antiproliferative activity against HL60 (4 and 5 µM) and MDA-MB231 (37 and 25 µM) without affecting lymphocyte proliferation in PBMC, indicating low toxicity to normal cells. Besides, compound 7e induced DNA fragmentation on about 100% of HL60 cells at 50 µM. In this case, it was more potent than 7m and lead 1. This indicated that compound 7e has a great pro-apoptotic potential.

Cytotoxic activity, albumin and DNA binding of new copper(II) complexes with chalcone-derived thiosemicarbazones.

[Cu(HL)Cl2] complexes of chalcone-derived thiosemicarbazones were obtained with 3-phenyl-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone (HPyCTPh), complex (1), 3-(4-chlorophenyl)-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone (HPyCT4ClPh), complex (2), 3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone (HPyCT4BrPh), complex (3), and 3-(4-nitrophenyl-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone (HPyCT4NO2Ph), complex (4). 1-3 showed interaction with bovine serum albumin (BSA) and deoxyribonucleic acid from calf thymus (CT-DNA). The cytotoxic activities of the thiosemicarbazones and complexes (1-4) were tested against HL60 (wild type human promyelocytic leukemia), Jurkat (human immortalized line of T lymphocyte), MDA-MB 231 (human breast carcinoma) and HCT-116 (human colorectal carcinoma) tumor cell lineages. Upon coordination to copper(II) cytotoxicity significantly increased in Jurkat, MDA-MB 231 and HCT-116 cells. Unlike the free thiosemicarbazones, 1-4 induced DNA fragmentation in solid tumor cells indicating their pro-apoptotic potential.