Integrated in silico - in vitro strategy for the discovery of potential xanthine oxidase inhibitors from Egyptian propolis and their synergistic effect with allopurinol and febuxostat

Xanthine oxidase (XO) has been well-recognized as a validated target for the treatment of hyperuricemia and gout. Currently, there are two drugs in clinical use that shut down XO overactivity, allopurinol and febuxostat; however, detrimental side effects restrict their applications. Propolis is a unique natural adhesive biomass of structurally variable and biologically active metabolites that exert remarkable health benefits. Moreover, combination drug therapy has become a promising pharmacotherapeutic strategy directed for reformulating existing drugs into new combination entities with potentiating therapeutic impacts. In this study, computer-aided molecular docking and MD simulations accompanied by biochemical testing were used for mining novel pharmacologically active chemical entities from Egyptian propolis to combat hyperuricemia. Further, with a view to decrease the potential toxicity of synthetic drugs and enhance efficacy, propolis hits were subjected to combination analysis with each of allopurinol and febuxostat. More specifically, Glide docking was utilized for a structure-based virtual screening of in-house datasets comprising various Egyptian propolis metabolites. Rosmarinic acid, luteolin, techtochrysin and isoferulic acid were the most promising virtual hits. In vitro XO inhibitory assays demonstrated the ability of these hits to significantly inhibit XO in a dose-dependent manner. Molecular docking and MD simulations revealed a cooperative binding mode between the discovered hits and standard XO inhibitors within the active site. Subsequently, the most promising hits were tested in a fixed-ratio combination setting with allopurinol and febuxostat separately to assess their combined effects on XO catalytic inhibition. The binary combination of each techtochrysin and rosmarinic acid with febuxostat displayed maximal synergy at lower effect levels. In contrast, individually, techtochrysin and rosmarinic acid with allopurinol cooperated synergistically at high dose levels. Taken together, the suggested strategy seems imperative to ensure a steady supply of new therapeutic options sourced from Egyptian propolis to regress the development of hyperuricemia

Olive phenolics as c-Met inhibitors: (-)-Oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models.

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity

The Maxi-K (BK) Channel Antagonist Penitrem A as a Novel Breast Cancer-Targeted Therapeutic

Breast cancer (BC) is a heterogeneous disease with different molecular subtypes. The high conductance calcium-activated potassium channels (BK, Maxi-K channels) play an important role in the survival of some BC phenotypes, via membrane hyperpolarization and regulation of cell cycle. BK channels have been implicated in BC cell proliferation and invasion. Penitrems are indole diterpene alkaloids produced by various terrestrial and marine Penicillium species. Penitrem A (1) is a selective BK channel antagonist with reported antiproliferative and anti-invasive activities against multiple malignancies, including BC. This study reports the high expression of BK channel in different BC subtypes. In silico BK channel binding affinity correlates with the antiproliferative activities of selected penitrem analogs. 1 showed the best binding fitting at multiple BK channel crystal structures, targeting the calcium-sensing aspartic acid moieties at the calcium bowel and calcium binding sites. Further, 1 reduced the levels of BK channel expression and increased expression of TNF-α in different BC cell types. Penitrem A (1) induced G1 cell cycle arrest of BC cells, and induced upregulation of the arrest protein p27. Combination treatment of 1 with targeted anti-HER drugs resulted in synergistic antiproliferative activity, which was associated with reduced EGFR and HER2 receptor activation, as well as reduced active forms of AKT and STAT3. Collectively, the BK channel antagonists represented by penitrem A can be novel sensitizing, chemotherapeutics synergizing, and therapeutic agents for targeted BC therapy

Updated mini-review of the chemistry, pharmacology and validated assays for U.S. FDA approved COVID-19 therapeutic agents

The fish market in Wuhan, China was the epicenter of Covid-19 pandemic, brought about by SARS-Co-V-2.  The latter has radiated worldwide, resulting in significant economic and social disruption “Covid Crisis”.  Although numerous drugs have been indicated for treating the viral infection and/or its symptoms, U.S. FDA has granted approvals for only three therapeutic agents; Remdesivir, Baricitinib and Monoclonal Antibodies (Bamlanivimab/Etesevimab, Casirivimab/Imdevimab).  This review assigns the approved drug entities with respect to their chemical and pharmacological aspects as well as their reported analyses in marketed formulations and biological matrices.  Furthermore, it offers a comparative study between the cited analytical methodologies for each drug separately.  A comprehensive, detailed, yet an instant message of Coronavirus authorized cure is delivered throughout the presented review.</span

Comparative anti-herpes simplex virus type 1 and chemical profiling of <i>Thymus capitatus</i> and <i>Artemisia herba-alba</i> collected from North Africa

Herpes simplex virus (HSV) can infect millions of people worldwide causing mild to life-threating infections. The current study demonstrates the first comparative anti-HSV type 1 activity and phytochemical investigation of Artemisia herba-alba and Thymus capitatus collected from Egypt and Libya. Liquid chromatography/mass spectrometry (LC/MS) analysis allowed the identification of 56 and 38 compounds in the Egyptian and Libyan Artemisia herba-alba ethanolic extracts, respectively, in addition to 46 and 50 compounds in the Egyptian and Libyan Thymus capitatus ethanolic extracts, respectively. Gas chromatography/mass spectrometry (GC/MS) analysis of their corresponding essential oils revealed the presence of 15, 17, 17 and 8 compounds in Egyptian and Libyan Artemisia herba-alba and Thymus capitatus, respectively. The major chemical classes of the identified compounds were phenolic acids, flavonoids and oxygenated monoterpenes. Evaluation of the anti-HSV1 activities of the studied extracts showed that the Egyptian Thymus capitatus ethanolic extracts were the most potent extract with more than 200-fold reduction in the viral PFU.</p

(-)-Oleocanthal treatment inhibits HGF-induced G1/S cell cycle progression and mitogenic signaling in human MDA-MB-231 breast cancer cells.

<p>(<b>A</b>) Flow cytometry analysis for cell cycle progression in control and (-)-oleocanthal-treated MDA-MB-231 cells. Cells in the various treatment groups were synchronized in G1 phase. Briefly, MDA-MB-231 cells were plated at a density of 1×10⁶ cells/100 mm plates in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. Cells were then washed twice with PBS and starved in control or treatment serum-free medium containing 0.5% FBS for 48 h to synchronize the cells in G1 phase. Afterwards, cells were fed various doses of (-)-oleocanthal in serum-free defined media containing 40 ng/ml HGF as the mitogen for 24 h. <i>Left panel</i> shows histograms generated using CellQuest software (PI staining). <i>Right panel</i> shows percentage of cells in each phase of cell cycle. Vertical bars show the average of 3 independent experiments. (<b>B</b>) Western blot analysis showing (-)-oleocanthal treatment effects on G1/S cell cycle regulatory proteins. Cells in the various treatment groups were synchronized in G1 phase in the same way described above. (-)-Oleocanthal treatment caused a marked downregulation of cyclin D1 and CDK6, while it caused upregulation of p21 and p27. (<b>C</b>) Western blot analysis showing (-)-oleocanthal treatment effects on c-Met downstream mitogenic signaling proteins Akt and MAPK. MDA-MB-231 cells were plated at a density of 1×10⁶ cells/100 mm culture plates in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. Cells were then washed twice with PBS and starved in control or treatment medium containing 0.5% FBS for 72 h and stimulated with 100 ng/ml human recombinant HGF for 10 min before cell lysis. SU11274 was used as a positive control. Afterwards, whole cell lysates were prepared for subsequent separation by polyacrylamide gel electrophoresis followed by Western blot analysis. Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs beside their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane ± SEM, *<i>P</i><0.05 as compared with vehicle-treated controls.</p

(-)-Oleocanthal treatment inhibits HGF-induced Met activation, stabilizes the epithelial phenotype, and reduces mesenchymal phenotype in breast cancer cells.

<p>(<b>A</b>) Western blot analysis. (-)-Oleocanthal caused a dose-dependent inhibition of HGF-induced phosphorylation of c-Met in MDA-MB-231, MCF-7, and BT-474 breast tumor cells with no effect on total Met levels. Cells were plated at 1×10⁶ cells/100 mm culture plates in RPMI-1640 media supplemented with 10% FBS and allowed to adhere overnight. Cells were then washed twice with PBS and starved in control or treatment medium containing 0.5% FBS for 72 h and stimulated with 100 ng/ml human recombinant HGF for 10 min before cell lysis. SU11274 was used as a positive control. (<b>B</b>) Western blot analysis. (-)-Oleocanthal resulted in a marked increase in the level of epithelial markers E-cadherin and Zo-1 in MDA-MB-231, MCF-7, and BT-474 cells and a decrease of mesenchymal marker vimentin expression (in MDA-MB-231 cells) compared to the vehicle-treated control groups. (-)-Oleocanthal treatment resulted in downregulation of β-catenin in MCF-7 and BT-474 cells. Cells were plated at 1×10⁶ cells/100 mm culture plates, allowed to attach overnight and then washed with PBS and incubated in the respective control or treatment in serum-free defined media containing 40 ng/ml HGF as the mitogen for 72 h. Whole cell lysates were prepared for subsequent separation by polyacrylamide gel electrophoresis followed by Western blot analysis. Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs below their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane ± SEM, *<i>P</i><0.05 as compared with vehicle-treated controls.</p

131 -Oxophorbine protopheophorbide A from Ziziphus lotus as a novel mesenchymal-epithelial transition factor receptor inhibitory lead for the control of breast tumor growth in vitro and in vivo.

International audienceThe failure of chemotherapy especially in triple negative breast cancer (TNBC) patients has been correlated with the overexpression of the mesenchymal-epithelial transition factor (c-Met) receptor. Thus, the hepatocyte growth factor (HGF)/c-Met signaling axis has gained considerable attention as a valid molecular target for breast cancer therapy. This study reports for the first time the discovery of the 131 -oxophorbines pheophorbide A and protopheophorbide A along with chlorophyllide A from Ziziphus lotus, an edible typical Tunisian plant, as the potent antiproliferative compounds against the human breast cancer cells MDA-MB-231 and MCF-7. Compared to other compounds, protopheophorbide A exerted the highest light-independent antiproliferative effect against the metastatic TNBC MDA-MB-231 cells (IC50  = 6.5 μM). In silico, this compound targeted the kinase domain of multiple c-Met crystal structures. It potently inhibited the kinase domain phosphorylation of wild and mutant c-Met in Z-LYTE kinase assay. Protopheophorbide A inhibited HGF-induced downstream c-Met-dependent cell proliferation, survival, adhesion and migration through RAF/MEK/ERK and PI3K/PTEN/AKT signaling pathways modulation, ROS generation and activation of JNK and p38 pathways. Interestingly, this compound impaired the ability of the MDA-MB-231 cells to adhere at different extracellular matrix proteins by reducing the HGF-induced expression of integrins αv, β3, α2, and β1. Moreover, protopheophorbide A exhibited anti-migratory properties (IC50  = 2.2 μM) through impacting the expression levels of E-cadherin, vimentin, β-catenin, FAK, Brk, Rac, and Src proteins. Importantly, treatment with protopheophorbide A significantly inhibited the MDA-MB-231 tumor growth in vivo. Our results suggest that protopheophorbide A could be a novel c-Met inhibitory lead with promise to control c-Met/HGF-dependent breast malignancies

(-)-Oleocanthal treatment induces apoptosis at 25 µM in MDA-MB-231 breast cancer cells.

<p>(<b>A</b>) Flow cytometry analysis. Cells were plated at a density of 5×10⁶ cells/100 mm culture plates, allowed to attach overnight. Afterwards, cells were incubated in the respective control or (-)-oleocanthal-treated RPMI-1640 medium containing 40 ng/ml HGF for 24 h. At the end of the experiment, cells in each treatment group were trypsinized, washed then resuspended in ice-cold 1X Annexin V Binding Buffer. Afterwards, the cells were treated as described in the Materials and Methods. In the dot plot of double variable flow cytometry, LL quadrant (FITC −/PI -) shows living cells; UR quadrant (FITC +/PI +) stands for late apoptotic cells; and LR quadrant (FITC +/PI -) represents early apoptotic cells. (<b>B</b>) Western blot analysis of cleaved caspase 3 and cleaved PARP. (-)-Oleocanthal treatment at 25 µM for 72 h markedly increased levels of cleaved caspase-3 and cleaved PARP. (<b>C</b>) Western blot analysis. (-)-Oleocanthal at 25 µM for 72 h downregulates c-Met levels without affecting EGFR levels, and right panel shows that transfection of MDA-MB-231 cells with c-Met-targeted siRNA totally abolished c-Met protein expression. (<b>D</b>) Western blot analysis of cleaved caspase 8, RIP, cleaved caspase 9 and cytochrome c. Treatment with 25 µM (-)-oleocanthal increased the cleavage of caspase-8 and RIP, but not caspase-9 or cytochrome c. Right panel shows that c-Met-targeted siRNA yielded a pattern of apoptosis that is similar to that following treatment with (-)-oleocanthal at 25 µM for 72 h by causing an increase in caspase-8 and RIP cleavage, with no effect on caspase-9 and cytochrome c levels. (<b>E</b>) Effect of Z-VAD-FMK on (-)-oleocanthal-induced apoptosis. MDA-MB-231 cells were treated with 25 µM (-)-oleocanthal in the presence or absence of caspase inhibitor Z-VAD-FMK (50 µM). After 24-h incubation, cells were analyzed to examine cell death by measuring caspase 3 and caspase 8 cleavage detected by Western blotting. In all the above experiments, whole cell lysates were prepared for subsequent separation by polyacrylamide gel electrophoresis followed by Western blot analysis. Scanning densitometric analysis was performed on all blots done in triplicate and the integrated optical density of each band was normalized with corresponding β-tubulin, as shown in bar graphs beside their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane ± SEM, *<i>P</i><0.05 as compared with vehicle-treated controls.</p