Determination of structural features that underpin the pannexin1 channel inhibitory activity of the peptide 10Panx1.

Pannexin1 channels facilitate paracrine communication and are involved in a broad spectrum of diseases. Attempts to find appropriate pannexin1 channel inhibitors that showcase target-selective properties and in vivo applicability remain nonetheless scarce. However, a promising lead candidate, the ten amino acid long peptide mimetic 10Panx1 (H-Trp1-Arg2-Gln3-Ala4-Ala5-Phe6-Val7-Asp8-Ser9-Tyr10-OH), has shown potential as a pannexin1 channel inhibitor in both in vitro and in vivo studies. Nonetheless, structural optimization is critical for clinical use. One of the main hurdles to overcome along the optimization process consists of subduing the low biological stability (10Panx1 t1/2 = 2.27 ± 0.11 min). To tackle this issue, identification of important structural features within the decapeptide structure is warranted. For this reason, a structure-activity relationship study was performed to proteolytically stabilize the sequence. Through an Alanine scan, this study demonstrated that the side chains of Gln3 and Asp8 are crucial for 10Panx1's channel inhibitory capacity. Guided by plasma stability experiments, scissile amide bonds were identified and stabilized, while extracellular adenosine triphosphate release experiments, indicative of pannexin1 channel functionality, allowed to enhance the in vitro inhibitory capacity of 10Panx1.

Advantages and Disadvantages of Two In Vitro Assays in Evaluating Aromatase Activity: "A Cell-Based and a Cell-Free Assay".

Objectives: Aromatase is an enzyme that catalyzes the conversion of androgens to estrogens. While inhibition of aromatase is a useful approach for treating breast cancer, it may also have toxicological consequences due to its endocrine disrupting/modulating effect. In this study, sensitivity and performance of two in vitro assays -a cell free and a cell-based- for evaluating aromatase activity were investigated by testing known aromatase inhibitors and partial validation of the methods was performed. Advantages and disadvantages of these methods are also discussed. Materials and Methods: Aromatase activity was evaluated via two in vitro models; direct measurement with a cell-free assay using a fluorescent substrate and recombinant human enzyme and indirect evaluation with a cell-based assay where cell proliferation was determined in estrogen receptor positive human breast cancer cells (MCF-7 BUS) in the absence of estrogen and the presence of testosterone. Results: In the cell-free direct measurement assay, reference compounds ketoconazole and aminoglutethimide have been shown to inhibit the aromatase enzyme with half-maximal inhibitory concentration (IC50) values concordant with literature. In cell-based indirect measurement assay, only ketoconazole dose-dependently inhibited cell proliferation with 3.47 x 10-7 M IC50. Inter-assay and intra-assay reproducibility of both methods was found to be within acceptable deviation levels. Conclusion: Both methods can be successfully applied. However, to evaluate the potential aromatase activity of the novel compounds in vitro, it seems better to perform both the cell-based and the cell-free assays that allows low-moderate biotransformation and eliminate cytotoxicity potential, respectively.

New indole-7-aldehyde derivatives as melatonin analogues; synthesis and screening their antioxidant and anticancer potential.

Over the last decade, there has been substantial interest in the use of melatonin (MLT) and MLT-like compounds in the treatment of several diseases. MLT can scavenge different reactive oxygen species and can also stimulate the synthesis of antioxidant enzymes. Our ongoing study relies on changing the groups in the different modifiable sites of the indole ring to increase the antioxidant activity. In this study a new approach for substitution of indole ring as indole based MLT analogue was proposed. We report the synthesis and characterization of a series of new indole-7-aldehyde hydrazide/hydrazone derivatives as indole-based MLT analogues. Anticancer potential of the compounds were evaluated both by their antioxidant and CYP1 inhibitory activities. In vitro antioxidant capacity of the compounds was investigated both in a cell-based (DCFH assay) and a cell-free (DPPH assay) assay. Potential inhibitory effects of the compounds on CYP1 catalytic activity were investigated via EROD assay. Cytotoxic activity of the compounds was further evaluated by the MTT assay in CHO-K1 cells. MLT analogues having an o-halogenated aromatic moiety exhibited effective antioxidant properties without having any cytotoxic effect. In conclusion, MLT derivatives represent promising scaffolds for discovery of effective antioxidant agents.