Dipeptidyl nitrile derivatives suppress the Trypanosoma cruzi in vitro infection.

Chagas disease affects several countries around the world with health and sanitation problems. Cysteine proteases are essential for the virulence and replication of the Trypanosoma cruzi, being modulated by dipeptidyl nitriles and derivatives. Here, four dipeptidyl nitrile derivatives were assayed in three T. cruzi morphologies and two strains (Tulahuen and Y) using a set of assays: (i) analysis of the inhibitory activity against cysteine proteases; (ii) determination of the cytotoxic activity and selectivity index; (iii) verification of the inhibition of the trypomastigote invasion in the host cell. These compounds could inhibit the activity of cysteine proteases using the selective substrate Z-FR-MCA for the trypomastigote lysate and extracellular amastigotes. Interestingly, these compounds did not present relevant enzymatic inhibition for the epimastigote lysate. Most of the substances were also cytotoxic and selective against the trypomastigotes and intracellular amastigotes. The best compound of the series (Neq0662) could reduce the enzymatic activity of the cysteine proteases for the trypomastigotes and amastigotes. It was equipotent to the benznidazole drug in the cytotoxic studies using these two parasite forms. Neq0662 was also selective for the parasite, and it inhibited the invasion of the mammalian host cell in all conditions tested at 10 µM. The stereochemistry of the trifluoromethyl group was an important factor for the bioactivity when the two diastereomers (Neq0662 and Neq0663) were compared. All-in-all, these results indicate that these compounds could move further in the drug development stage because of its promising bioactive profile.

Withaferin A and Withanolide D Analogues with Dual Heat-Shock-Inducing and Cytotoxic Activities: Semisynthesis and Biological Evaluation.

Withanolides constitute a valuable class of bioactive natural products because some members of the class are known to exhibit cytotoxic activity and also induce a cytoprotective heat-shock response. In order to understand the relationship between their structures and these dual bioactivities of the withanolide scaffold, we obtained 25 analogues of withaferin A (WA) and withanolide D (WD) including 17 new compounds by semisynthesis involving chemical and microbial transformations. Hitherto unknown 16ß-hydroxy analogues of WA and WD were prepared by their reaction with triphenylphosphine/iodine, providing unexpected 5ß-hydroxy-6α-iodo analogues (iodohydrins) followed by microbial biotransformation with Cunninghamella echinulata and base-catalyzed cyclization of the resulting 16ß-hydroxy iodohydrins. Evaluation of these 25 withanolide analogues for their cytotoxicity and heat-shock-inducing activity (HSA) confirmed the known structure-activity relationships for WA-type withanolides and revealed that WD analogues were less active in both assays compared to their corresponding WA analogues. The 5ß,6ß-epoxide moiety of withanolides contributed to their cytotoxicity but not HSA. Introduction of a 16ß-OAc group to 4,27-di- O-acetyl-WA enhanced cytotoxicity and decreased HSA, whereas introduction of the same group to 4- O-acetyl-WD decreased both activities.

Biological Activity and Physicochemical Properties of Dipeptidyl Nitrile Derivatives Against Pancreatic Ductal Adenocarcinoma Cells.

BACKGROUND: Pancreatic cancer is one of the most aggressive types with high mortality in patients. Therefore, studies to discover new drugs based on cellular targets have been developed to treat this disease. Due to the importance of Cysteine Protease (CP) to several cellular processes in cancer cells, CP inhibitors have been studied as novel alternative approaches for pancreatic cancer therapy. OBJECTIVE: The cytostatic potential of new CP inhibitors derived from dipeptidyl nitriles is analyzed in vitro using pancreatic cancer (MIA PaCa-2) cells. METHODS: The cytotoxic and cytostatic activities were studied using MTT colorimetric assay in 2D and 3D cultures. Colony formation, migration in Boyden chamber and cell cycle analysis were applied to further study the cytostatic activity. The inhibition of cysteine proteases was evaluated with Z-FR-MCA selective substrate, and ROS evaluation was performed with DCFH-DA fluorophore. Permeability was investigated using HPLC-MS to obtain log kw. Combination therapy was also evaluated using the best compound with gemcitabine. RESULTS: The inhibition of intracellular CP activity by the compounds was confirmed, and the cytostatic effect was established with cell cycle retention in the G1 phase. CP inhibitors were able to reduce cell proliferation by 50% in the clonogenic assay, and the same result was achieved for the migration assay, without any cytotoxic effect. The Neq0554 inhibitor was also efficient to increase the gemcitabine potency in the combination therapy. Physicochemical properties using an artificial membrane model quantified 1.14 ≥ log Kw ≥ 0.75 for all inhibitors (also confirmed using HPLC-MS analysis) along with the identification of intra and extracellular metabolites. Finally, these dipeptidyl nitrile derivatives did not trigger the formation of reactive oxygen species, which is linked to genotoxicity. CONCLUSION: Altogether, these results provide a clear and favorable picture to develop CP inhibitors in pre-clinical assays.