Virtual screening and in vitro assays of novel hits as promising DPP-4 inhibitors.

Diabetes is a metabolic disorder that presents hyperglycemia and vascular complications due to the non-production of insulin or its inappropriate use by the body. One of the strategies to treat diabetes is the inhibition of dipeptidyl peptidase-4 (DPP-4) and it is interesting to conduct virtual screening studies to search for new inhibitors of the DPP-4 enzyme. This study involves a virtual screening using the crystallographic structure of DPP-4 and a compound subset from the ZINC database. To filter this compound subset, we used some physicochemical properties, positioning at the three DPP-4 binding sites, molecular interactions, and ADME-Tox properties. The conformations of ligands obtained from AutoDock Vina were analyzed using a consensus with other algorithms (AutoDock and GOLD). The compounds selected from virtual screening were submitted to biological assays using the "DPPIV-Glo™ protease assay". Cytotoxicity tests were also performed. One promising compound (ZINC1572309) established interactions with important residues at the binding site. The results of the ADME-Tox prediction for ZINC1572309 were compared with a reference drug (sitagliptin). The cytotoxicity of sitagliptin and ZINC1572309 were evaluated using the XTT short-term cytotoxic assay, including normal and tumor cell lines to observe the cellular response to inhibitor treatment at different genetic bases. Both compounds (ZINC1572309 and the reference drug - sitagliptin) also inhibited DPP-4 activity, suggesting interesting biological effects of the selected compound at non-cytotoxic concentrations. Therefore, from in silico and in vitro studies, a potential hit as DPP-4 inhibitor was discovered and it can be structurally optimized to achieve suitable activity and pharmacokinetic profiles.

New antifungal ent-labdane diterpenes against Candida glabrata produced by microbial transformation of ent-polyalthic acid.

Candida glabrata, the most common non-albicans Candida species and one of the primary causes of candidemia, exhibits decreased susceptibility to azoles and more recently to echinocandins. Polyalthic acid 1, a furan diterpene, has been shown promising biological potential and in this study ent-polyalthic acid derivatives with antifungal activity against Candida glabrata were produced by microbial transformation. Incubation of 1 with Aspergillus brasiliensis afforded two known (compounds 5 and 10) and eight new derivatives (compounds 2-4, 6-9 and 11). The most common reaction was hydroxylation, but isomerization of the double bond and acetylation were also detected. None of the tested compounds showed cytotoxicity against HeLa, MCF-7 and MCF-10A cell lines showing IC50 values ranging from 62.6 µM to > 500 µM. Compounds 1, 5, 6, 8 and 11 showed fungistatic effects (ranging from 34.1 µM to 39.5 µM) on C. glabrata at lower concentrations than fluconazole (163.2 µM). Compounds 1, 6 and 8 were more potent fungicides (ranging from 79.0 to 143.6 µM) than fluconazole, which showed fungicidal effect at concentrations higher than 163.2 µM. These results suggest that ent-polyalthic acid and some of its derivatives could be used as lead compounds to develop new antifungal agents.

Comparative study of cytotoxicity and genotoxicity of commercial Jeffamines® and polyethylenimine in CHO-K1 cells.

Jeffamines® are a family of polymers containing primary amine groups attached to the extremities of polyether backbone which can be used as biomaterials. They have been used in combination with polyethylenimine (PEI) to improve biocompatibility in drug and gene delivery systems. Despite these facts, very few studies have been done on cytotoxicity and genotoxicity of pure Jeffamines® or compared with PEI. The present study aimed to evaluate and compare the cytotoxic and genotoxic effects of Jeffamines® and PEI in CHO-K1 cells. Specifically, polypropylene oxide 2000 (PPO 2000, Jeffamine® D series), polyethylene oxide 1900 (PEO 1900, Jeffamine® ED series), branched 25 kDa PEI, and linear 20 kDa PEI were evaluated at different concentrations. Cell viability and proliferation were assessed by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and 5-bromo-2'-deoxyuridine (BrdU) assays, respectively. Genotoxicity was evaluated using single cell gel electrophoresis assay and the cytokinesis-blocked micronucleus assay. PPO 2000 was the most cytotoxic Jeffamine® , whereas PEO 1900 did not caused significant cell death at any tested concentration. Branched PEI was more cytotoxic than linear PEI (LPEI) and both were more cytotoxic than Jeffamines® . Only PPO 2000 induced DNA damage when evaluated in comet assay probably due to its cytotoxicity. PPO 2000, PEO 1900, and PEI did not increase the frequency of micronuclei when tested at sub-cytotoxic concentrations. This work provides new insights about biocompatibility of Jeffamines® and PEI and suggests the genotoxicological safety for further investigations of PEO 1900 in drug and gene delivery systems. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 742-750, 2018.

Inactivation of ß-Lapachone Cytotoxicity by Filamentous Fungi that Mimic the Human Blood Metabolism.

BACKGROUND AND OBJECTIVES: ß-Lapachone is a drug candidate in phase II clinical trials for treatment of solid tumors. The therapeutic efficacy of ß-lapachone is closely related to its metabolism, since this o-naphthoquinone produces cytotoxic effect after intracellular bioreduction by reactive oxygen species formation. The aim of this study was to produce ß-lapachone human blood phase I metabolites to evaluate their cytotoxic activities. METHODS: The biotransformation of ß-lapachone was performed using Mucor rouxii NRRL 1894 and Papulaspora immersa SS13. The metabolites were isolated and their chemical structures determined from spectrometric and spectroscopic data. Cell cytotoxicity assays were carried out with ß-lapachone and its metabolites using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. RESULTS: Microbial transformation of ß-lapachone by filamentous fungi resulted in the production of five metabolites identical to those found during human blood metabolism, a novel metabolite and a product stated before only in a synthetic procedure. The analysis of the results showed that ß-lapachone metabolites were not cytotoxic for the neoplastic cell line SKBR-3 derived from human breast cancer and the normal human fibroblast cell line GM07492-A. The cytotoxic activity assay against the neoplastic cell line SKBR-3 revealed that the lowest half-maximal inhibitory concentration (IC50) values of these ß-lapachone metabolites were 33- to 52-fold greater than IC50 values of ß-lapachone. CONCLUSIONS: The cytotoxic activity of ß-lapachone in vivo may be reduced due to its swift conversion in blood.

Microbial Metabolism of Atovaquone and Cytotoxicity of the Produced Phase I Metabolite.

BACKGROUND AND OBJECTIVES: Atovaquone is a hydroxynaphthoquinone with selective action in the mitochondrial respiratory chain of malaria parasite. It is employed for both the treatment and prevention of malaria, in a combination with proguanil. The aim of this study was to elucidate the in vitro metabolites from atovaquone and to evaluate their cytotoxic activities. METHODS: The biotransformation of atovaquone was performed using Mucor rouxii NRRL 1894, Cunninghamella echinulata var. elegans ATCC 8688a and C. elegans ATCC 10028b, which have been reported as microbial models of mammalian drug metabolism. Experiments were also carried out with two probiotic strains from the human intestinal tract: Bifidobacterium sp. and Lactobacillus acidophilus. The phase I metabolite was isolated, its chemical structure was elucidated and its toxicity was evaluated using the neoplastic cell line SKBR-3 derived from human breast cancer and normal human fibroblast cell line GM07492-A. Cell cytotoxicity assays were also carried out with atovaquone. RESULT: Only the fungi were able to convert atovaquone to metabolite trans-3-[4'-(4″-chlorophenyl)cyclohexyl)-1,2-dioxo-dihydro-1H-indene-3-carboxylic acid. The metabolite displayed 50 % inhibitory concentration (IC50) values of 110.20 ± 2.2 and 108.80 ± 1.5 µmol/L against breast cancer cell line SKBR-3 and fibroblasts cell line GM07492-A, respectively. The IC50 values of atovaquone were 282.30 ± 1.8 and 340.50 ± 1.4 µmol/L against breast cancer and normal fibroblasts cell lines, respectively. CONCLUSIONS: The produced metabolite was more toxic than atovaquone and was not selective to normal or cancer cell lines. The present study is the first to report the production of atovaquone metabolite.

The X-ray repair cross complementing protein 1 (XRCC1) rs25487 polymorphism and susceptibility to cirrhosis in Brazilian patients with chronic viral hepatitis.

INTRODUCTION: The progression of hepatic disease in chronic viral hepatitis is accompanied by an increased production of reactive oxygen species (ROS), as well as an accumulation of oxidative DNA damage, which is primarily repaired through base excision repair. XRCC1 (X-ray repair cross complementing protein 1) is one of the most important proteins involved in this repair pathway. The present study was carried out to verify the possible association of the XRCC1 rs25487 polymorphism with cirrhosis in patients from Central-West Brazil. MATERIAL AND METHODS: A total of 227 individuals with viral hepatitis, 53 cirrhotic and 174 non-cirrhotic, were genotyped for the XRCC1 rs25487 polymorphism using PCR-RFLP. RESULTS: There were significantly higher frequencies of both the Arg/Gln genotype and of individuals with at least one Gln allele (Arg/ Gln+Gln/Gln) among cirrhotic patients (56.6% and 69.8%) compared with non-cirrhotic patients (25.8% and 37.9%). Both conditions were significantly associated with cirrhosis, independent of age, sex, alcohol intake or tobacco use (adjusted OR = 3.5, CI = 1.7-7.4, p = 0.001 and adjusted OR = 3.1, CI = 1.5-6.3, p = 0.002, respectively). Similar results were obtained for a group of HCV-infected patients but not for HBV-infected patients. CONCLUSIONS: The XRCC1 rs25487 polymorphism may influence the development of cirrhosis in viral hepatitis patients, and additional investigation will be necessary.

Genotoxic effect of Physalis angulata L. (Solanaceae) extract on human lymphocytes treated in vitro.

Physalis angulata L (Solanaceae) is a medicinal plant from North of Brazil, whose different extracts and infusions are commonly used in the popular medicine for the treatment of malaria, asthma, hepatitis, dermatitis and rheumatism. However, the genotoxic effects of P. angulata on human cells is not well known. The main purpose of the present study was to evaluate the in vitro genotoxic effects of aqueous extract of P. angulata using the comet assay and the micronucleus assay in human lymphocytes provided from 6 healthy donors. Treatments with P. angulata extracts were performed in vitro in order to access the extent of DNA damage. The comet assay has shown that treatments with P. angulata at 0.5, 1.0, 2.0, 3.0 and 6.0 microg/mL in culture medium were genotoxic. Lymphocytes treated with P. angulata at the concentrations of 3.0 and 6.0 microg/mL in culture medium showed a statistically significant increase in the frequency of micronucleus (p<0.05), however, the cytokinesis blocked proliferation index (CBPI) was not decreased after P. angulata treatment. In conclusion, the present work demonstrated the genotoxic effects of P. angulata extract on human lymphocytes in vitro.