Maen Zeino

The ability of molecular docking to unravel the controversy and challenges related to P-glycoprotein—a well-known, yet poorly understood drug transporter

SummaryP-glycoprotein is the most crucial membrane transporter implicated in tumor resistance. Intensive efforts were paid to elucidate the complex mechanism of transport and to identify modulators of this transporter. However, the borderline between substrates and modulators is very thin and identification of the binding sites within P-glycoprotein is complex. Herein, we provide an intensive review of those issues and use molecular docking to assess its ability: first, to differentiate between three groups (substrates, modulators and non-substrates) and second to identify the binding sites. After thorough statistical analysis, we conclude despite the various challenges that molecular docking should not be underestimated as differences between the distinct groups were significant. However, when it comes to defining the binding site, care must be taken, since consensus throughout literature could not be reached.

Synthesis and study of cytotoxic activity of 1,2,4-trioxane- and egonol-derived hybrid molecules against Plasmodium falciparum and multidrug-resistant human leukemia cells

Malaria and cancer cause the death of millions of people every year. To combat these two diseases, it is important that new pharmaceutically active compounds have the ability to overcome multidrug resistance in cancer and Plasmodium falciparum strains. In search of effective anti-cancer and anti-malaria hybrids that possess improved properties compared to their parent compounds, a series of novel 1,2,4-trioxane-based hybrids incorporating egonol and/or ferrocene fragments were synthesized and tested in vitro against P. falciparum strains, CCRF-CEM cells and the multidrug-resistant P-glycoprotein-over-expressing CEM/ADR5000 cells. The most active compounds against P. falciparum strains were artesunic acid homodimers 12 and 13 (IC50 of 0.32 and 0.30 nM, respectively), whereas novel hybrids 7 (1,2,4-trioxane-ferrocene-egonol), 9 (1,2,4-trioxane-ferrocene) and 11 (artesunic acid-egonol) showed a remarkable cytotoxicity toward CCRF-CEM cells (IC50 of 0.07, 0.25 and 0.18 μM, respectively). A cooperative and synergistic effect of the three moieties 1,2,4-trioxane, ferrocene and egonol in hybrid molecule 7 is significant and is obviously stronger than in hybrids 9 (1,2,4-trioxane-ferrocene) and 11 (artesunic acid-egonol), which comprises of only two of the three considered parent compounds. Interestingly, hybrid 9 containing a 1,2,4-trioxane and a ferrocene fragment has shown to be the most effective among the studied hybrids against the tested multidrug-resistant leukemia CEM/ADR5000 cells (IC50 of 0.57 μM) and possesses a degree of cross-resistance of 2.34.

Cytotoxicity of seven naturally occurring phenolic compounds towards multi-factorial drug-resistant cancer cells.

INTRODUCTION In medical oncology, multi-drug resistance (MDR) of cancer cells continues to be a major impediment. We are in quest of novel anti-proliferative agents to overcome drug-resistant tumor cells. METHODS In the present study, we investigated the cytotoxicity of 7 naturally occurring phenolic compounds including two isoflavonoids alpinumisoflavone (1) and laburnetin (2), one biflavonoid amentoflavone (3), three lignans pycnanthulignene A (4), pycnanthulignene B (5), and syringaresinol (7) and one xanthone, euxanthone (6) against 9 drug-sensitive and MDR cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analyzed via flow cytometry. RESULTS The IC50 values for the investigational phenolics ranged from 5.91 µM (towards leukemia CEM/ADR5000 cells) to 65.65 µM (towards drug-resistant breast adenocarcinoma MDA-MB-231-BCRP cells) for 1, 27.63 µM (towards leukemia CCRF-CEM cells) to 107.57 µM (towards MDA-MB-231-pcDNA cells) for 2, from 5.84 µM (towards CEM/ADR5000 cells) to 65.32 µM (towards colon carcinoma HCT116 (p53(-/-)) cells) for 4 and 0.20 µM (towards CCRF-CEM cells) to 195.12 µM (towards leukemia CEM/ADR5000) for doxorubicin. Phenolics 3, 5, 6 and 7 displayed selectivity cytotoxic effects on cancer cells lines. Compounds 1 and 4 induced apoptosis in CCRF-CEM cells, mediated by loss of MMP and increase ROS production. CONCLUSIONS The studied phenolics and mostly isoflavonoid 1 and lignan 4 are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.

Identification of new P-glycoprotein inhibitors derived from cardiotonic steroids

P-glycoprotein (ABCB1, MDR1) is capable of extruding chemotherapeutics outside the cell and its overexpression in certain cancer cells may cause failure of chemotherapy. Many attempts were carried out to identify potent inhibitors of this transporter and numerous compounds were shown to exert inhibitory effects in vitro, but so far none were able to make their way to the clinic due to serious complications. Natural compounds represent a great source of therapeutics, which are believed to be safe and effective. Therefore, we have screened a large library of naturally occurring cardiotonic steroids and their derivatives using high throughput flow cytometry. We were able to identify six compounds capable of modulating P-glycoprotein activity. By using P-glycoprotein ATPase assays, molecular docking in silico studies and resazurin reduction assays, the outcome of this high throughput screening platform has been validated. These novel compounds may serve as candidates to reverse doxorubicin resistance in leukemia cells.

Cytotoxicity of cardiotonic steroids in sensitive and multidrug-resistant leukemia cells and the link with Na(+)/K(+)-ATPase.

Cardiotonic steroids have long been in clinical use for treatment of heart failure and are now emerging as promising agents in various diseases, especially cancer. Their main target is Na(+)/K(+)-ATPase, a membrane protein involved in cellular ion homeostasis. Na(+)/K(+)-ATPase has been implicated in cancer biology by affecting several cellular events and signaling pathways in both sensitive and drug-resistant cancer cells. Hence, we investigated the cytotoxic activities of 66 cardiotonic steroids and cardiotonic steroid derivatives in sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells. Data were then subjected to quantitative structure-activity relationship analysis (QSAR) and molecular docking into Na(+)/K(+)-ATPase, which both indicated a possible differential expression of the pump in the mentioned cell lines. This finding was confirmed by western blotting, intracellular potassium labeling and next generation sequencing which showed that Na(+)/K(+)-ATPase was less expressed in multidrug-resistant than in sensitive cells.

Cytotoxicity and mode of action of a naturally occurring naphthoquinone, 2-acetyl-7-methoxynaphtho[2,3-b]furan-4,9-quinone towards multi-factorial drug-resistant cancer cells

INTRODUCTION Malignacies are still a major public concern worldwide and despite the intensive search of new chemotherapeutic agents, treatment still remains a challenging issue. The present study was designed to evaluate the cytotoxicity of 2-acetyl-7-methoxynaphtho[2,3-b]furan-4,9-quinone (AMNQ) isolated from the bark of Milletia versicolor towards a panel of drug-sensitive and multidrug-resistant (MDR) cancer cell lines. METHODS The resazurin reduction assay was used to evaluate the cytotoxicity of AMNQ against 9 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species were all analyzed by flow cytometry. RESULTS Following resazurin assay, the naphthoquinone AMNQ displayed IC50 values ranging from 0.79 µM (against HepG2 hepatocarcinoma cells) to 3.26 µM (against MDA-MB231/BCRP breast cancer cells) on 9 tested cancer cell lines, whilst doxorubicin showed IC50 values ranging from 0.40 µM (against CCRF-CEM leukemia cells) to 91.37 µM (against CEM/ADR5000 leukemia cells). IC50 values below 1 µM were recorded with AMNQ towards CCRF-CEM cells (0.57 µM), U87MG.ΔEGFR gliobastoma multiforme cells (0.96 µM cells) and HepG2 cells (0.76 µM). Compared to its corresponding sensitive cell lines U87MG, sensitivity was observed in epidermal growth factor receptor-transfected U87MG.ΔEGFR cells to AMNQ. MMP was found to be the main mode of action of induction of apoptosis by AMNQ. CONCLUSIONS The results of this work demonstrate the cytotoxicity of AMNQ towards various types of cancer cell lines, including MDR phenotypes. AMNQ is a potential antiproliferative natural compound that deserves more investigations to develop novel cytotoxic drugs against sensitive and MDR cancers.

Cytotoxic compounds from the fruits of Uapaca togoensis towards multifactorial drug-resistant cancer cells.

Cancer cells may rapidly acquire multidrug resistance, mainly due to the presence of adenosine triphosphate-binding cassette transporters, epidermal growth factor receptor, or mutations in the p53 tumor suppressor gene. This work was designed to assess the cytotoxicity of the methanol crude extracts and compounds from the fruits of Uapaca togoensis, namely, β-amyryl acetate (1), 11-oxo-α-amyryl acetate (2), lupeol (3), pomolic acid (4), futokadsurin B (5), arborinin (6), and 3-O-β-D-glucopyranosyl sitosterol (7) against nine drug sensitive and multidrug-resistant cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of the fruits of U. togoensis and compounds, whilst the caspase-Glo assay was used to detect the activation of caspase enzymes by the fruits of U. togoensis and compound 6. Cell cycle, mitochondrial membrane potential, and levels of reactive oxygen species were all analyzed via flow cytometry. The acridone alkoid 6 and the crude extract from the fruits of U. togoensis were active on all of the nine tested cancer lines with IC50 values below 32 µM and 30 µg/mL, respectively. Compounds 2 and 5 showed selective activities and IC50 values below 99 µM or 42 µM, respectively, which were obtained towards 3/9 and 6/9 tested cancer cell lines. Compound 6 displayed IC50 values below 10 µM towards seven of the nine tested cancer cell lines. The IC50 values ranged from 3.55 µM (against CEM/ADR5000 cells) to 31.77 µM (against CCRF-CEM cells) for alkaloid 6 and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. The crude extract of the fruits of U. togoensis induced apoptosis in the CCRF-CEM leukemia cells, which was mediated by the disruption of the mitochondrial membrane potential. Compound 6 also strongly induced apoptosis in CCRF-CEM cells and cell cycle arrest in the G0/G1 and S phases. The crude extract from the fruits of this plant as well as aborinin are potential antiproliferative natural products that deserve further investigation to develop novel cytotoxic drugs to fight sensitive and otherwise drug-resistant phenotypes.

Cytotoxicity of two naturally occurring flavonoids (dorsmanin F and poinsettifolin B) towards multi-factorial drug-resistant cancer cells

INTRODUCTION The expression of diverse resistance mechanisms in cancer cells is one of the major barriers to successful cancer chemotherapy. METHODS In the present study, we assessed the cytotoxicity of two naturally occurring flavonoids dorsmanin F (1, a flavanone) and poinsettifolin B (2, a chalcone) against 9 drug-sensitive and multidrug-resistant (MDR) cancer cell lines. The resazurin reduction assay was used to evaluate the cytotoxicity of these compounds, whilst caspase-Glo assay was used to detect caspase activation. Cell cycle, mitochondrial membrane potential (MMP) and levels of reactive oxygen species (ROS) were all analysed via flow cytometry. RESULTS Compounds 1 and 2 displayed cytotoxic effects with IC50 values below 34 µM in all the 9 tested cancer cell lines. The IC50 values for flavanone 1 and chalcone 2 ranged from 5.34 µM and 1.94 µM (towards leukaemia CCRF-CEM cells) to 33.30 µM and 28.92 µM (towards MDA-MB-231-BCRP cells), respectively, and from 0.20 µM (against CCRF-CEM cells) to 195.12 µM (against CEM/ADR5000 cells) for doxorubicin. The compounds induced apoptosis in CCRF-CEM leukaemia cells, mediated by MMP disruption and increased ROS production. CONCLUSIONS Dorsmain F and poinsettifolin B are potential cytotoxic natural products that deserve more investigations to develop novel antineoplastic drugs against multifactorial drug-resistant cancers.

Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B.

Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H(+)-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation.

Molecular docking studies of myxobacterial disorazoles and tubulysins to tubulin

Aims: To investigate the distribution of food allergens in pediatric allergic rhinitis. Methods: 106 children diagnosed with allergic rhinitis were reviewed and divided into two age groups, including preschooler (2-5 years old) group, 48 cases; and school children group (6-13 years), 58 cases. Allergen-specific IgE for the food allergens and inhalant allergens in patient’s serum was determined using the AllergyScreen system (Mediwiss Analytic GmbH, Germany). Results: Out of 106 AR children, 88 (83.02%) were tested positive for food allergen-specific serum IgE (sIgG), 97 (91.51%) positive for inhalant allergen-specific sIgE. Among these children, 79 (74.53%) were positive for both food and inhalant allergens, 9 (8.49%) were positive only for food allergens, and 18 (16.98%) were negative for food allergens but positive for inhalant allergens. Out of 88 children positive for food allergens, 34 (32.08%) were positive for single food allergen, 54 (50.94%) were positive for multiple food allergens. The top 6 food allergens, in the order of their positivity rates, were milk (52.83%), eggs (30.19%), beef (26.42%), cashew (26.42%), lamb (12.26%) and crabs (10.38%). The food allergen-positive rate in the preschooler group was higher than that in the school children group (P<0.05). Conclusion: Majority of the children with allergic rhinitis are also positive for food allergens, suggesting that food allergens might be one of the main causes for pediatric allergic rhinitis.The present study was undertaken to ascertain the affinity of the amino acid D-Tryptophan (D-Trp) towards the particular receptors on the melanophores of a major carp Labeo rohita (Ham.). D-Trp, which is sweet in taste, in the dose range of 4.9 x 10 -16 M to 4.9 x 10 -4 M has induced a dose-dependent aggregation (MSI 3.72 ± 0.15 to 1.09 ± 0.02) in the isolated scale melanophores of the dorso-lateral region of this fish. The precise binding of the ligand D-Trp with its receptors is partially dependent, in eliciting its actions, on the release of neurotransmitters. The melanophores do not get desensitized under 90 min incubation within the agonist D-Trp. The general α adrenoceptors as well as the α 2 adrenoceptors are involved in the tastant (D-Trp) induced aggregation of the melanophores. D-Trp demonstrates that its aggregatory effects are mediated with the intervention of the β adrenoceptors. Inhibition of the D-Trp induced aggregatory effect has also been observed when the melanophores were pretreated with the antibiotic neomycin. The aggregating nature of D-Trp suggests that it could be binding with the receptors of aggregation causing ligands.