A functional assay using human whole blood and flow cytometry analysis to evaluate cytotoxicity and immunomodulatory effect of anti-Trypanosoma cruzi drugs.

The discovery and development of new drugs for the treatment of Chagas disease is urgent due to the high toxicity and low cure efficacy, mainly during the chronic phase of this disease. Other chemotherapeutic approaches for Chagas disease treatment are being researched and require screening assays suitable for evaluating the effectivity of new biologically active compounds. This study aims to evaluate a functional assay using the internalization of epimastigotes forms of Trypanosoma cruzi by human peripheral blood leukocytes from healthy volunteers and analyses by flow cytometry of cytotoxicity, anti-T. cruzi activity, and immunomodulatory effect of benznidazole, ravuconazole, and posaconazole. The culture supernatant was used to measure cytokines (IL-1-ß, IL-6, INF-γ, TNF and IL-10) and chemokines (MCP-1/CCL2, CCL5/RANTES and CXCL8/IL-8). The data showed a reduction in the internalization of T. cruzi epimastigote forms treated with ravuconazole, demonstrating its potential anti-T. cruzi activity. In addition, an increased amount of IL-10 and TNF cytokines was observed in the supernatant of cultures upon the addition of the drug, mainly IL-10 in the presence of benznidazole, ravuconazole and posaconazole, and TNF in the presence of ravuconazole and posaconazole. Moreover, the results revealed a decrease in the MCP-1/CCL2 index in cultures in the presence of benznidazole, ravuconazole, and posaconazole. A decrease in the CCL5/RANTES and CXCL8/IL-8 index in cultures with BZ, when compared to the culture without drugs, was also observed. In conclusion, the innovative functional test proposed in this study may be a valuable tool as a confirmatory test for selecting promising compounds identified in prospecting programs for new drugs for Chagas disease treatment.

Transcription factor SOX3 upregulated pro-apoptotic genes expression in human breast cancer.

BACKGROUND: Sex-determining region Y-box 3 (SOX3) protein, a SOX transcriptions factors group, has been identified as a key regulator in several diseases, including cancer. Downregulation of transcriptions factors in invasive ductal carcinoma (IDC) can interfere in neoplasia development, increasing its aggressiveness. We investigated SOX3 protein expression and its correlation with apoptosis in the MDA-MB-231 cell line, as SOX3 and Pro-Caspase-3 immunoexpression in paraffin-embedded invasive ductal carcinoma tissue samples from patients (n = 27). Breast cancer cell line MDA-MD-231 transfected with pEF1-SOX3 + and pEF1-Empty vector followed by cytotoxicity assay (MTT), Annexin-V FITC PI for apoptosis percentage assessment by flow cytometry, qPCR for apoptotic-related gene expression, immunofluorescence, and immunohistochemistry to SOX3 immunolocalization in culture cells, and paraffin-embedded invasive ductal carcinoma tissue samples. RESULTS: Apoptotic rate was higher in cells transfected with pEF1-SOX3 + (56%) than controls (10%). MDA-MB-231 transfected with pEF1-SOX3 + presented upregulation of pro-apoptotic mRNA from CASP3, CASP8, CASP9, and BAX genes, contrasting with downregulation antiapoptotic mRNA from BCL2, compared to non-transfected cells and cells transfected with pEF1-empty vector (p < 0.005). SOX3 protein nuclear expression was detected in 14% (4/27 cases) of ductal carcinoma cases, and pro-Caspase-3 expression was positive in 50% of the cases. CONCLUSION: Data suggest that SOX3 transcription factor upregulates apoptosis in breast cancer cell line MDA-MB-231, and has a down nuclear expression in ductal carcinoma cases, and need to be investigated as a tumor suppressor protein, and its loss of expression and non-nuclear action turn the cells resistant to apoptosis. Further studies are necessary to understand how SOX3 protein regulates the promoter regions of genes involved in apoptosis.

Pamidronate, a promising repositioning drug to treat leishmaniasis, displays antileishmanial and immunomodulatory potential.

Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum (L. infantum). Currently, there are no vaccines and/or prophylactic therapies against VL, and the recentpharmacological approaches come from the drug repositioning strategy. Here, we evaluated the anticancer drug pamidronate (PAM) to identify a new therapeutic option for the treatment of human VL. We assessed its in vitro antileishmanial activity against the promastigote and amastigote forms of L. infantum by evaluating cell cytotoxicity. The antileishmanial and immunomodulatory activities were assessed using human peripheral blood leukocytes ex vivo. PAM induced the formation of vacuoles in the cytoplasm of the promastigotes and alterations in the morphology of the kinetoplast and mitochondria in vitro, which indicates anti-promastigote activity. PAM also reduced the number of infected macrophages and intracellular amastigotes in a concentration-dependent manner, with cell viability above 70%. In ex vivo, PAM reduced the internalized forms of L. infantum in the classical monocyte subpopulation. Furthermore, it enhanced IL-12 and decreased IL-10 and TGF-ß by monocytes and neutrophils. Increased IFN-γ and TNF levels for CD8- and CD8+ T lymphocytes and B lymphocytes, respectively, were observed after the treatment with PAM, as well as a reduction in IL-10 by the lymphocyte subpopulations evaluated. Taken together, our results suggest that PAM may be eligible as a potential therapeutic alternative for drug repurposing to treat human visceral leishmaniasis.

Acute toxicity and antitumor potential of 1,3,4-trisubstituted-1,2,3-triazole dhmtAc-loaded liposomes on a triple-negative breast cancer model.

For the first time, compounds developed from the 1,2,3-triazole scaffold were evaluated as novel drugs to treat triple-negative breast cancer (TNBC). Four organic salts were idealized as nonclassical bioisosteres of miltefosine, which is used in the topical treatment for skin metastasizing breast carcinoma. Among them, derivative dhmtAc displayed better solubility and higher cytotoxicity against the human breast adenocarcinoma cell line and mouse 4T1 cell lines, which are representatives of TNBC. In vitro assays revealed that dhmtAc interferes with cell integrity, confirmed by lactate dehydogenase leakage. Due to its human peripheral blood mononuclear cell (PBMC) toxicity, dhmtAc in vivo studies were carried out with the drug incorporated in a long-circulating and pH-sensitive liposome (SpHL-dhmtAc), and the acute toxicity in BALB/c mice was determined. Free dhmtAc displayed cardiac and pulmonary toxicity after the systemic administration of 5 mg/kg doses. On the other hand, SpHL-dhmtAc displayed no toxicity at 20 mg/kg. The in vivo antitumor effect of SpHL-dhmtAc was investigated using the 4T1 heterotopic murine model. Intravenous administration of SpHL-dhmtAc reduced the tumor volume and weight, without interfering with the body weight, compared with the control group and the dhmtAc free form. The incorporation of the triazole compound in the liposome allowed the demonstration of its anticancer potential. These findings evidenced 1,3,4-trisubstituted-1,2,3-triazole as a promising scaffold for the development of novel drugs with applicability for the treatment of patients with TNBC.

Morita-Baylis-Hillman adducts derived from thymol: synthesis, in silico studies and biological activity against Giardia lamblia.

Giardiasis is a neglected disease, and there is a need for new molecules with less side effects and better activity against resistant strains. This work describes the evaluation of the giardicidal activity of thymol derivatives produced from the Morita-Baylis-Hillman reaction. Thymol acrylate was reacted with different aromatic aldehydes, using 1,4-diazabicyclo[2.2.2]octane (DABCO) as a catalyst. Eleven adducts (8 of them unpublished) with yields between 58 and 80% were obtained from this reaction, which were adequately characterized. The in silico prediction showed theoretical bioavailability after oral administration as well as antiparasitic activity against Giardia lamblia. Compound 4 showed better biological activity against G. lamblia. In addition to presenting antigiardial activity 24 times better than thymol, this MBHA was obtained in a short reaction time (3 h) with a yield (80%) superior to the other investigated molecules. The molecule was more active than the precursors (thymol and MBHA 12) and did not show cytotoxicity against HEK-293 or HT-29 cells. In conclusion, this study presents a new class of drugs with better antigiardial activity in relation to thymol, acting as a basis for the synthesis of new bioactive molecules. Molecular hybridization technique combined with the Morita-Baylis-Hillman reaction provided new thymol derivatives with giardicidal activity superior to the precursor molecules.

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.

Triethylphosphinegold(I) Complexes with Secnidazole-Derived Thiosemicarbazones: Cytotoxic Activity against HCT-116 Colorectal Cancer Cells under Hypoxia Conditions.

Triethylphosphinegold(I) complexes [Au(HL1)P(CH2CH3)3]PF6 (1), [Au(HL2)P(CH2CH3)3]PF6 (2), and [Au(HL3)P(CH2CH3)3]PF6 (3) were obtained with (E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ylidene)hydrazinecarbothioamide (HL1), (E)-N-methyl-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ylidene)hydrazinecarbothioamide (HL2), and (E)-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ylidene)-N-phenylhydrazinecarbothioamide (HL3). All compounds were assayed for their cytotoxic activities against HCT-116 colorectal carcinoma cells under normoxia and hypoxia conditions and against nonmalignant HEK-293 human embryonic kidney cells under normoxia conditions. The thiosemicarbazone ligands HL1-HL3 were inactive against HCT-116 cells under hypoxia but while HL3 was inactive, HL1 and HL2 proved to be cytotoxic to both cell lineages under normoxia conditions. Complexes (1-3) and the triethylphosphinegod(I) precursor proved to be active against both cell lineages in normoxia as well as in hypoxia. While 1 and 3 revealed to be active against HEK-293 and HCT-116 cells, being approximately as active against HCT-116 cells in normoxia as under hypoxia, complex (2) proved to be more active against HCT-116 cells under hypoxia than under normoxia conditions, and more active against HCT-116 cells than against the nonmalignant HEK-293 cells, with the selectivity index, calculated as SI = IC50HEK-293/IC50HCT-116hypoxia, equal to 3.7, similar to the value obtained for the control drug tirapazamine (tirapazamine (TPZ), SI = 4). Although the compounds showed distinct cytotoxic activities, the electrochemical behaviors of HL1-HL3 were very similar, as were the behaviors of complexes (1-3). Complex (2) deserves special interest since it was significantly more active under hypoxia than under normoxia conditions. Hence, in this case, selective reduction of the nitro group in a low oxygen pressure environment, resulting in toxic reactive oxygen species (ROS) and damage to DNA or other biomolecules, might operate, while for the remaining compounds, other modes of action probably occur.

In vitro leishmanicidal, antibacterial and antitumour potential of anhydrocochlioquinone A obtained from the fungus Cochliobolus sp.

The bioassay-guided fractionation of the ethyl acetate extract of the fungus Cochliobolus sp. highlighted leishmanicidal activity and allowed for anhydrocochlioquinone A (ANDC-A) isolation. MS, 1D and 2D NMR spectra of this compound were in agreement with those published in the literature. ANDC-A exhibited leishmanicidal activity with EC 50 value of 22.4 microgram/mL (44 mu M) and, when submitted to the microdilution assay against Gram-ositive and Gram-negative bacteria, showed a minimal inhibitory concentration against Staphylococcus aureus ATCC 25295 of 128 microgram/mL (248.7 mu M). It was also active against five human cancer cell lines, showing IC50 values from 5.4 to 20.3 mu M. ANDC-A demonstrated a differential selectivity for HL-60 (SI 5.5) and THP-1 (SI 4.3) cell lines in comparison with Vero cells and was more selective than cisplatin and doxorubicin against MCF-7 cell line in comparison with human peripheral blood mononuclear cells. ANDC-A was able to eradicate clonogenic tumour cells at concentrations of 20 and 50 mu M and induced apoptosis in all tumour cell lines at 20 mu M. These results suggest that ANDC-A might be used as a biochemical tool in the study of tumour cells biochemistry as well as an anticancer agent with durable effects on tumours.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts.

This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.

Design, synthesis and structure-activity relationship studies of a novel focused library of 2,3,4-substituted oxazolidines with antiproliferative activity against cancer cell lines.

In the present work we describe the synthesis and antiproliferative evaluation of a focused library of 30 novel oxazolidines designed by modification of N-substituent, by ring variation, by alkyl variation or by extension of the structure. It was noted that carbamate and N,O-aminal groups were essential for activity. In general, replacement of the phenyl ring with pyridinyl was not tolerated. However, the introduction of a second phenyl ring with an appropriate spacer at the 3- or 4-position of the first phenyl ring generally enhanced the cytotoxic profile. Among all the prepared compounds, 24 was the most potent compound found in this class, being active on four of five cancer cell lines and it was 5-fold and 10-fold more potent than the lead compounds against HL60 and JURKAT cells, respectively. In addition, it showed relevant activity against MCF-7 and HCT-116 cells, which were resistant to lead. Moreover, 24 showed little antiproliferative activity against VERO, indicating low toxicity to normal cells. Thus, this compound has the potential to be developed as an anticancer agent.

Effects of two 6-quinolinyl chalcones on the integrity of plasma membrane of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, yet few therapeutic options exist. Our aim was to search for new compounds with high efficacy, low toxicity, shorter treatment time and affordable cost. We studied two synthetic 6-quinolinyl chalcones, 3b and 3e, to determine their effects on VERO cells, antifungal activity, survival curve, interaction with other drugs and phenotypic effects against several isolates of Paracoccidioides spp. In this study, we verified that the compounds were not toxic, exhibited superior in vitro activity compared with that shown by trimethoprim-sulfamethoxazole, and after 5 days of treatment, decreased the fungal cell viability by approximately 70%. Additionally, no interactions were observed between the tested compounds and other drugs. We also found that these compounds induced morphological changes, such as shriveling of cells, fragmentation of the plasma membrane and cytoplasmic disorganization in vitro. The changes observed by microscopy assays corroborate the observation made with propidium iodide, where the number of cells stained with the compounds was higher than that observed after amphotericin B treatment. We observed an increase in the efflux of K+ and a loss of intracellular contents in cells treated with 3b and 3e, confirming their effects on fungal membranes. However, damage to the membrane was not associated with a decrease in membrane ergosterol levels. The experimental evidences showed no direct indications of cellular wall damage caused by these compounds. Thus, these results confirm the antifungal potential of 3b and 3e against Paracoccidioides spp. with possible action on the membrane.

Cytotoxic and antimicrobial effects of indium(iii) complexes with 2-acetylpyridine-derived thiosemicarbazones.

Complexes [In(2Ac4oClPh)Cl2(MeOH)] (1), [In(2Ac4pFPh)Cl2(MeOH)] (2), [In(2Ac4pClPh)Cl2(MeOH)] (3) and [In(2Ac4pIPh)Cl2(MeOH)] (4) were obtained with N(4)-ortho-chlorophenyl-2-acetylpyridine thiosemicarbazone (H2Ac4oClPh), N(4)-para-fluorophenyl-2-acetylpyridine thiosemicarbazone (H2Ac4pFPh), N(4)-para-chlorophenyl-2-acetylpyridine thiosemicarbazone (H2Ac4pClPh) and N(4)-para-iodophenyl-2-acetylpyridine thiosemicarbazone (H2Ac4pIPh). Theoretical studies suggested that the coordinated methanol molecule can be easily replaced by DMSO used in the preparation of stock solutions, with the formation of [In(L)Cl2(DMSO)] (HL = thiosemicarbazonate ligand), and that the replacement of DMSO by water is unfavorable. However, for all complexes the displacement of one or two chloride ligands by water in aqueous solution is extremely favorable. The cytotoxic activity of the compounds was evaluated against HL-60, Jurkat and THP-1 leukemia and against MDA-MB-231 and HCT-116 solid tumor cell lines, as well as against Vero non-malignant cells. The cytotoxicity and selectivity indexes (SI) increased in several cases for the indium(iii) complexes in comparison with the free thiosemicarbazones. The antimicrobial activity of the compounds was investigated against Candida albicans, Candida dubliniensis, Candida lusitaniae and Candida parapsilosis. In many cases complexation resulted in a substantial increase of the antifungal activity. Complexes (1-4) were revealed to be very active against C. lusitaniae and C. dubliniensis. Structure-activity relationship (SAR) studies were carried out to identify the physico-chemical properties that might be involved in the antifungal action, as well as in the cytotoxic effect of the compounds against HL-60 cells. In both cases, correlations between the bioactivity and physico-chemical properties did not appreciably change when the chloride ligands in [In(L)Cl2(DMSO)] were replaced by water molecules, suggesting [In(L)Cl(H2O)(DMSO)]+ or [In(L)(H2O)2(DMSO)]2+ to be the species that interact with the biological media.

Cytotoxicity and bacterial membrane destabilization induced by Annona squamosa L. extracts

ABSTRACT This study aimed to further investigate the cytotoxicity against tumor cell lines and several bacterial strains of Annona squamosa and its mode of action. Methanol extracts of A. squamosa leaves (ASL) and seeds (ASS) were used. ASL showed significant antibacterial activity against S. aureus, K. pneumoniae and E. faecalis with MIC values of 78, 78 and 39 µg/mL respectively. Moreover, ASL exhibited significant biofilm disruption, rapid time dependent kinetics of bacterial killing, increased membrane permeability and significantly reduced the cell numbers and viability. Regarding the cytotoxicity against tumor cell lines, ASS was more active against Jurkat and MCF-7 cells, with CI50 1.1 and 2.1 µg/mL, respectively. ASL showed promising activity against Jurkat and HL60, with CI50 4.2 and 6.4 µg/mL, respectively. Both extracts showed lower activity against VERO cells and reduced the clonogenic survival at higher concentrations (IC90) to MCF-7 and HCT-116 lineages. The alkaloids anonaine, asimilobine, corypalmine, liriodenine nornuciferine and reticuline were identified in extracts by UPLC-ESI-MS/MS analysis. This study reinforced that A. squamosa presents a remarkable phytomedicinal potential and revealed that its antimicrobial mechanism of action is related to bacterial membrane destabilization.

Human topoisomerase IB is a target of a thiosemicarbazone copper(II) complex.

The human topoisomerase IB inhibition and the antiproliferative activity of 3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone HPyCT4BrPh alone and its copper(II) complex [Cu(PyCT4BrPh)Cl] was investigated. [Cu(PyCT4BrPh)Cl] inhibits both the DNA cleavage and religation step of the enzyme, whilst the ligand alone does not display any effect. In addition we show that coordination to copper(II) improves the cytotoxicity of HPyCT4BrPh against THP-1 leukemia and MCF-7 breast cancer cells. The data indicate that the copper(II) thiosemicarbazone complex may hit human topoisomerase IB and that metal coordination can be useful to improve cytotoxicity of this versatile class of compounds.

Bismuth(III) complexes with 2-acetylpyridine- and 2-benzoylpyridine-derived hydrazones: Antimicrobial and cytotoxic activities and effects on the clonogenic survival of human solid tumor cells.

Complexes [Bi(2AcPh)Cl2]·0.5H2O (1), [Bi(2AcpClPh)Cl2] (2), [Bi(2AcpNO2Ph)Cl2] (3), [Bi(2AcpOHPh)Cl2]·2H2O (4), [Bi(H2BzPh)Cl3]·2H2O (5), [Bi(H2BzpClPh)Cl3] (6), [Bi(2BzpNO2Ph)Cl2]·2H2O (7) and [Bi(H2BzpOHPh)Cl3]·2H2O (8) were obtained with 2-acetylpyridine phenylhydrazone (H2AcPh), its -para-chloro-phenyl- (H2AcpClPh), -para-nitro-phenyl (H2AcpNO2Ph) and -para-hydroxy-phenyl (H2AcpOHPh) derivatives, as well as with the 2-benzoylpyridine phenylhydrazone analogues (H2BzPh, H2BzpClPh, H2BzpNO2Ph, H2BzpOHPh). Upon coordination to bismuth(III) antibacterial activity against Gram-positive and Gram-negative bacterial strains significantly improved except for complex (4). The cytotoxic effects of the compounds under study were evaluated on HL-60, Jurkat and THP-1 leukemia, and on MCF-7 and HCT-116 solid tumor cells, as well as on non-malignant Vero cells. In general, 2-acetylpyridine-derived hydrazones proved to be more potent and more selective as cytotoxic agents than the corresponding 2-benzoylpyridine-derived counterparts. Exposure of HCT-116 cells to H2AcpClPh, H2AcpNO2Ph and complex (3) led to 99% decrease of the clonogenic survival. The IC50 values of these compounds were three-fold smaller when cells were cultured in soft-agar (3D) than when cells were cultured in monolayer (2D), suggesting that they constitute interesting scaffolds, which should be considered in further studies aiming to develop new drug candidates for the treatment of colon cancer.

Metal complexes of 3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone: cytotoxic activity and investigation on the mode of action of the gold(III) complex.

Complexes [Au(PyCT4BrPh)Cl]Cl (1), [Pt(PyCT4BrPh)Cl]0.5KCl (2), and [Pd(PyCT4BrPh)Cl]KCl (3) were obtained with 3-(4-bromophenyl)-1-pyridin-2-ylprop-2-en-1-one thiosemicarbazone (HPyCT4BrPh). Although complexes (2) and (3) did not exhibit potent cytotoxic activity, HPyCT4BrPh and its gold(III) complex (1) proved to be highly cytotoxic against HL-60 (human promyelocytic leukemia) and THP-1 (human monocytic leukemia) cells, and against MDA-MB 231 and MCF-7 (human breast adenocarcinoma) solid tumor cells. Except for HL-60 cells, upon coordination to gold(III) a 2- to 3-fold increase in the cytotoxic effect was observed. An investigation on the possible biological targets of the gold(III) complex was carried out. Complex (1) but not the free thiosemicarbazone inhibits the enzymatic activity of thioredoxin reductase (TrxR). The affinity of 1 for TrxR suggests metal binding to a selenol residue in the active site of the enzyme. While HPyCT4BrPh was inactive, 1 was able to inhibit topoisomerase IB (Topo IB) activity. Hence, inhibition of TrxR and Topo IB could contribute to the mechanism of cytotoxic action of complex (1).

Antifungal activity of extracts from Atacama Desert fungi against Paracoccidioides brasiliensis and identification of Aspergillus felis as a promising source of natural bioactive compounds.

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values ≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, ß-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Antifungal activity of extracts from Atacama Desert fungi againstParacoccidioides brasiliensis and identification ofAspergillus felis as a promising source of natural bioactive compounds

Fungi of the genus Paracoccidioides are responsible for paracoccidioidomycosis. The occurrence of drug toxicity and relapse in this disease justify the development of new antifungal agents. Compounds extracted from fungal extract have showing antifungal activity. Extracts of 78 fungi isolated from rocks of the Atacama Desert were tested in a microdilution assay against Paracoccidioides brasiliensis Pb18. Approximately 18% (5) of the extracts showed minimum inhibitory concentration (MIC) values≤ 125.0 µg/mL. Among these, extract from the fungus UFMGCB 8030 demonstrated the best results, with an MIC of 15.6 µg/mL. This isolate was identified as Aspergillus felis (by macro and micromorphologies, and internal transcribed spacer, β-tubulin, and ribosomal polymerase II gene analyses) and was grown in five different culture media and extracted with various solvents to optimise its antifungal activity. Potato dextrose agar culture and dichloromethane extraction resulted in an MIC of 1.9 µg/mL against P. brasiliensis and did not show cytotoxicity at the concentrations tested in normal mammalian cell (Vero). This extract was subjected to bioassay-guided fractionation using analytical C18RP-high-performance liquid chromatography (HPLC) and an antifungal assay using P. brasiliensis. Analysis of the active fractions by HPLC-high resolution mass spectrometry allowed us to identify the antifungal agents present in the A. felis extracts cytochalasins. These results reveal the potential of A. felis as a producer of bioactive compounds with antifungal activity.

Identification and Optimization of Anthranilic Acid Based Inhibitors of Replication Protein†A.

Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that initiates the DNA damage response pathway through protein-protein interactions (PPIs) mediated by its 70N domain. The identification and use of chemical probes that can specifically disrupt these interactions is important for validating RPA as a cancer target. A high-throughput screen (HTS) to identify new chemical entities was conducted, and 90 hit compounds were identified. From these initial hits, an anthranilic acid based series was optimized by using a structure-guided iterative medicinal chemistry approach to yield a cell-penetrant compound that binds to RPA70N with an affinity of 812 nm. This compound, 2-(3- (N-(3,4-dichlorophenyl)sulfamoyl)-4-methylbenzamido)benzoic acid (20 c), is capable of inhibiting PPIs mediated by this domain.

Annona glabra Flavonoids Act As Antimicrobials by Binding to Pseudomonas aeruginosa Cell Walls.

Pseudomonas aeruginosa is an important pathogen in opportunistic infections in humans. The increased incidence of antimicrobial-resistant P. aeruginosa isolates has highlighted the need for novel and more potent therapies against this microorganism. Annona glabra is known for presenting different compounds with diverse biological activities, such as anti-tumor and immunomodulatory activities. Although other species of the family display antimicrobial actions, this has not yet been reported for A. glabra. Here, we investigated the antimicrobial activity of the ethyl acetate fraction (EAF) obtained from the leaf hydroalcoholic extract of A. glabra. EAF was bactericidal against different strains of P. aeruginosa. EAF also presented with a time- and concentration-dependent effect on P. aeruginosa viability. Testing of different EAF sub-fractions showed that the sub-fraction 32-33 (SF32-33) was the most effective against P. aeruginosa. Analysis of the chemical constituents of SF32-33 demonstrated a high content of flavonoids. Incubation of this active sub-fraction with P. aeruginosa ATCC 27983 triggered an endothermic reaction, which was accompanied by an increased electric charge, suggesting a high binding of SF32-33 compounds to bacterial cell walls. Collectively, our results suggest that A. glabra-derived compounds, especially flavonoids, may be useful for treating infections caused by P. aeruginosa.