Characterization, modeling, and anticancer activity of L.arginase production from marine Bacillus licheniformis OF2.

BACKGROUND: L-arginase, is a powerful anticancer that hydrolyzes L-arginine to L-ornithine and urea. This enzyme is widely distributed and expressed in organisms like plants, fungi, however very scarce from bacteria. Our study is based on isolating, purifying, and screening the marine bacteria that can produce arginase. RESULTS: The highest arginase producing bacteria will be identified by using microbiological and molecular biology methods as Bacillus licheniformis OF2. Characterization of arginase is the objective of this study. The activity of enzyme was screened, and estimated beside partial sequencing of arginase gene was analyzed. In silico homology modeling was applied to generate the protein's 3D structure, and COACH and COFACTOR were applied to determine the protein's binding sites and biological annotations based on the I-TASSER structure prediction. The purified enzyme was undergone an in vitro anticancer test. CONCLUSIONS: L-arginase demonstrated more strong anti-cancer cells with an IC50 of 21.4 ug/ml in a dose-dependent manner. L-arginase underwent another investigation for its impact on the caspase 7 and BCL2 family of proteins (BCL2, Bax, and Bax/Bcl2). Through cell arrest in the G1/S phase, L-arginase signals the apoptotic cascade, which is supported by a flow cytometry analysis of cell cycle phases.

Phytochemical Profiling and Compound Isolation of Cissus rhombifolia Vahl. Leaves Aqueous Methanolic Extract with the Evaluation of Its Anti-Inflammatory Effect Using Lipopolysaccharide-Induced Inflammation in RAW 264.7 Cells.

The inflammatory disorders represent a serious health issue. Certain Cissus species possess anti-inflammatory effect. Cissus rhombifolia Vahl. leaves' anti-inflammatory activities and phytoconstituents are poorly characterized. In this study, 38 constituents were tentatively characterized in Cissus rhombifolia Vahl. leaves' aqueous methanolic extract (CRLE) using high-performance liquid chromatography combined with mass spectrometry (HPLC/MS) and Proton Nuclear Magnetic Resonance (1 H-NMR). Myricetin, ß-amyrin, and alliospiroside A, were isolated from CRLE using column chromatography. The anti-inflammatory effect of CRLE and its isolated compounds were studied in lipopolysaccharide (LPS)-induced RAW 264.7 cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT assay) was used to assess how CRLE and its isolated compounds affected cell viability. Further, its effects on the production of intracellular NO, and inflammatory cytokines cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) were assessed by the Griess test, and cytokine enzyme-linked immunosorbent assays, respectively. CRLE and its isolated compounds, myricetin, ß-amyrin, and alliospiroside A decreased the NO production. Western blotting was performed to assess the protein expression levels of the inflammatory cytokines inducible nitric oxide synthase (iNOS). Alliospiroside A downregulated IL-6, TNF-α, and COX-2 and inhibited the expression of iNOS. CRLE and its compounds represent effective alternative candidate to treat inflammatory diseases.

Novel 2-oxo-2-phenylethoxy and benzyloxy diaryl urea hybrids as VEGFR-2 inhibitors: Design, synthesis, and anticancer evaluation.

Two series of diaryl urea derivatives, 6a-k and 7a-n, were synthesized. All the newly synthesized compounds were tested against the NCI (US) cancer cell lines via SRB assay. The p-chloro-m-trifluoromethyl phenyl derivatives 6e-g and 7e-g showed the most potent cytotoxic activity with a GI50 value range of 1.2-15.9 µM. Furthermore, the p-fluorobenzyloxy diaryl urea derivative 7g revealed the most potent cytotoxicity against eight cancer cell lines in the MTT assay with IC50 values below 5 µM. Compounds 6a-k and 7a-n were tested for their vascular endothelial growth factor receptor-2 (VEGFR-2) kinase inhibitory activities. The p-chloro-m-trifluoromethyl diaryl urea benzyloxy derivatives 7e-i and the p-methoxydiaryl urea benzyloxy derivatives 7k, 7l, and 7n were found to be the most active compounds as VEGFR-2 inhibitors in the benzyloxy series 7, with an IC50 range of 0.09-4.15 µM. In the 2-oxo-2-phenylethoxy series 6, compounds 6e-g and 6i were reported with IC50 values of 0.94, 0.54, 2.71, and 4.81 µM, respectively. Moreover, compounds 7e and 7g induced apoptosis, causing cell cycle arrest in the G2/M phase. In addition, 7g showed an antimigratory effect in A-375 cells and inhibited the VEGFR-2 expression in an immunohistofluorescence study. Molecular docking simulations on VEGFR-2 as well as ADME properties prediction were also performed.

Design, synthesis and anticancer activity of novel 2-arylbenzimidazole/2-thiopyrimidines and 2-thioquinazolin-4(3H)-ones conjugates as targeted RAF and VEGFR-2 kinases inhibitors.

In the current study, series of 2-arylbenzimidazole-thiopyrimidine and -thioquinazolin-4(3H)-ones conjugates 12a-d, 13a,b and 14a-l have been synthesized. All the synthesized compounds were tested in vitro for their anticancer activities against a panel of cancer cell lines at NCI - US and their growth inhibition (GI) % were determined at 10 µM. Compounds 14c and 14g-i were selected to be screened at the five dose assay and were found to exhibit GI50 values 1.1-30.0 µM. The benzimidazole-quinazolinone derivative 14c, in particular, showed potent anticancer activity against the tested cancer cell lines (GI50 of 1.3-4.2 µM). In addition, compounds 12a,b, 13a, 14a-e, 14g, 14i and 14j were selected to be tested against some cancer cell lines using MTT assay and the benzimidazole-quinazolinone 14g was found to have potent anticancer activities against melanoma (Mel-501 and A-375), breast (MCF-7), colon (HCT-116), prostate (PC-3), lung (A-549) and pancreas (Paca-2) cancer cell lines reporting IC50 values ranging between 0.1 and 6.2 µM. Moreover, the synthesized hybrids were tested in vitro on kinases; BRAF (wt), BRAF (V600E), CRAF and VEGFR-2. The benzimidazole-quinazolinone derivatives 14f,g revealed potent RAF kinases inhibitory activities on BRAF (wt), BRAF (V600E) and CRAF showing IC50 values 0.002-0.1 µM, whereas, the benzimidazole-quinazolinone derivatives 14i and 14k showed moderate VEGFR-2 inhibitory activity (IC50 = 20.60 and 6.14 µM, respectively). Moreover, the representative compounds 14g and 14i caused cell cycle arrest of A-375 melanoma cell line at G2/M phase and were found to induce late apoptosis. CRAF in the DFG-out inactive conformation homology modeling was first reported in this study and molecular docking studies on BRAF, CRAF and VEGFR-2 were also performed to investigate the binding modes of the target compounds and their interactions with the key amino acids; BRAF (Glu500, Cys531 and Asp593), CRAF (Glu393, Cys424 and Asp486) and VEGFR-2 (Glu885, Cys919 and Asp1046).

Novel 1,2,3-Triazole-Coumarin Hybrid Glycosides and Their Tetrazolyl Analogues: Design, Anticancer Evaluation and Molecular Docking Targeting EGFR, VEGFR-2 and CDK-2.

This study represents the design and synthesis of a new set of triazole-coumarin-glycosyl hybrids and their tetrazole hybrid analogues possessing various sugar moieties and modified analogues. All the newly synthesized derivatives were screened for their cytotoxic activities against a panel of human cancer cell lines. The coumarin derivatives 10, 13 and 15 derivatives revealed potent cytotoxic activities against Paca-2, Mel-501, PC-3 and A-375 cancer cell lines. These promising analogues were further examined for their inhibitory assessment against EGFR, VEGFR-2 and CDK-2/cyclin A2 kinases. The coumarin-tetrazole 10 displayed broad superior inhibitory activity against all screened enzymes compared with the reference drugs, erlotinib, sorafenib and roscovitine, respectively. The impact of coumarin-tetrazole 10 upon cell cycle and apoptosis induction was determined to detect its mechanism of action. Additionally, it upregulated the levels of casp-3, casp-7 and cytochrome-c proteins and downregulated the PD-1 level. Finally, molecular docking study was simulated to afford better rationalization and gain insight into the binding affinity between the promising derivatives and their targeted enzymes, which could be used as an optimum lead for further modification in the anticancer field.

New 1,2,3-Triazole-Coumarin-Glycoside Hybrids and Their 1,2,4-Triazolyl Thioglycoside Analogs Targeting Mitochondria Apoptotic Pathway: Synthesis, Anticancer Activity and Docking Simulation.

Toxicity and resistance to newly synthesized anticancer drugs represent a challenging phenomenon of intensified concern arising from variation in drug targets and consequently the prevalence of the latter concern requires further research. The current research reports the design, synthesis, and anticancer activity of new 1,2,3-triazole-coumarin-glycosyl hybrids and their 1,2,4-triazole thioglycosides as well as acyclic analogs. The cytotoxic activity of the synthesized products was studied against a panel of human cancer cell lines. Compounds 8, 10, 16 and 21 resulted in higher activities against different human cancer cells. The impact of the hybrid derivative 10 upon different apoptotic protein markers, including cytochrome c, Bcl-2, Bax, and caspase-7 along with its effect on the cell cycle was investigated. It revealed a mitochondria-apoptotic effect on MCF-7 cells and had the ability to upregulate pro-apoptotic Bax protein and downregulate anti-apoptotic Bcl-2 protein and thus implies the apoptotic fate of the cells. Furthermore, the inhibitory activities against EGFR, VEGFR-2 and CDK-2/cyclin A2 kinases for 8, 10 and 21 were studied to detect the mechanism of their high potency. The coumarin-triazole-glycosyl hybrids 8 and 10 illustrated excellent broad inhibitory activity (IC50= 0.22 ± 0.01, 0.93 ± 0.42 and 0.24 ± 0.20 µM, respectively, for compound 8), (IC50 = 0.12 ± 0.50, 0.79 ± 0.14 and 0.15± 0. 60 µM, respectively, for compound 10), in comparison with the reference drugs, erlotinib, sorafenib and roscovitine (IC50 = 0.18 ± 0.05, 1.58 ± 0.11 and 0.46 ± 0.30 µM, respectively). In addition, the docking study was simulated to afford better rationalization and put insight into the binding affinity between the promising derivatives and their targeted enzymes and that might be used as an optimum lead for further modification in the anticancer field.

Synthesis, Characterization, In Vitro Anticancer Potentiality, and Antimicrobial Activities of Novel Peptide-Glycyrrhetinic-Acid-Based Derivatives.

Glycyrrhetinic acid (GA) is one of many interesting pentacyclic triterpenoids showing significant anticancer activity by triggering apoptosis in tumor cell lines. This study deals with the design and synthesis of new glycyrrhetinic acid (GA)-amino acid peptides and peptide ester derivatives. The structures of the new derivatives were established through various spectral and microanalytical data. The novel compounds were screened for their in vitro cytotoxic activity. The evaluation results showed that the new peptides produced promising cytotoxic activity against the human breast MCF-7 cancer cell line while comparing to doxorubicin. On the other hand, only compounds 3, 5, and 7 produced potent activity against human colon HCT-116 cancer cell line. The human liver cancer (HepG-2) cell line represented a higher sensitivity to peptide 7 (IC50; 3.30 µg/mL), while it appeared insensitive to the rest of the tested peptides. Furthermore, compounds 1, 3, and 5 exhibited a promising safety profile against human normal skin fibroblasts cell line BJ-1. In order to investigate the mode of action, compound 5 was selected as a representative example to study its in vitro effect against the apoptotic parameters and Bax/BCL-2/p53/caspase-7/caspase-3/tubulin, and DNA fragmentation to investigate beta (TUBb). Additionally, all the new analogues were subjected to antimicrobial assay against a panel of Gram-positive and Gram-negative bacteria and the yeast candida Albicans. All the tested GA analogues 1-8 exhibited more antibacterial effect against Micrococcus Luteus than gentamicin, but they exhibited moderate antimicrobial activity against the tested bacterial and yeast strains. Molecular docking studies were also simulated for compound 5 to give better rationalization and put insight to the features of its structure.

Towards breast cancer targeting: Synthesis of tetrahydroindolocarbazoles, antibreast cancer evaluation, uPA inhibition, molecular genetic and molecular modelling studies.

A series of some new tetrahydroindolocarbazole derivatives has been synthesized. The structure of the synthesized compounds has been confirmed by different spectroscopic techniques such as IR, NMR, elemental analysis and mass spectrometry. The target compounds were evaluated for their antitumor activity against breast cancer cell line MCF-7, their GI% and their LC50 have been determined. Six of the synthesized compounds exhibited GI% values against MCF-7 cell lines exceeding 70% ranging from 71.9 to 85.0% in addition that compound 11 expressed GI% values of 99.9% and considered the most active derivatives among the synthesized ones. Compound 11 showed a remarkable decrease of u PA level to 3.5 ng/ml compared to DOX. Compound 5, 11 and 15 showed significant decrease in expression of MTAP and CDKN2A, in addition to a remarkable decrease in DNA damage comet assay method. Molecular modeling studies were performed to interpretate the behavior of active ligands as uPA inhibitors.

In vitro anticancer potentiality and molecular modelling study of novel amino acid derivatives based on N1,N3-bis-(1-hydrazinyl-1-oxopropan-2-yl) isophthalamide.

A series of N1,N3-bis (1-oxopropan-2-yl) isophthalamide-based derivatives 4-16 were prepared and their structures were confirmed by different spectral tools. The cytotoxic potentiality of novel compounds 4-16 was assessed by the MTT assay method on colon, lung and breast tumour cell lines. Compound 5 gave the most significant specificity anticancer activity with safety response on normal cell lines. In vitro enzyme assay and several apoptotic parameters were examined to elucidate the mode of action of compound 5. Molecular docking studies also were simulated to put insight and give better understanding to its structural features.

Toward Rational Design of Novel Anti-Cancer Drugs Based on Targeting, Solubility, and Bioavailability Exemplified by 1,3,4-Thiadiazole Derivatives Synthesized Under Solvent-Free Conditions.

The 1,3,4-thiadiazole derivatives (9a-i) were synthesized under solvent free conditions and their chemical composition was confirmed using different spectral tools (IR, Mass, and NMR spectrometry). All the synthesized compounds were screened for their anti-cancer potentiality over human breast carcinoma (MCF-7) and human lung carcinoma (A-549). Most of the tested compounds showed remarkable anti-breast cancer activity. However, compound 4 showed the most anti-lung cancer activity. Then, compounds with cytotoxic activity ≥ 80% over breast and lung cells were subjected to investigate their specificity on human normal skin cell line (BJ-1). Compounds 9b and 9g were chosen owing to their high breast anti-cancer efficacy and their safety, in order to study the possible anti-cancer mode of action. Otherwise, drug delivery provides a means to overcome the low solubility, un-targeted release, and limited bioavailability of the prepared 1,3,4-thiadiazole drug-like substances. Compounds 9b and 9g were chosen to be encapsulated in Na-alginate microspheres. The release profile and mechanism of both compounds were investigated, and the results revealed that the release profiles of both microspheres showed a sustained release, and the release mechanism was controlled by Fickian diffusion. Accordingly, these compounds are promising for their use in chemotherapy for cancer treatment, and their hydrophilicity was improved by polymer encapsulation to become more effective in their pharmaceutical application.

Design, synthesis and molecular modeling of new 4-phenylcoumarin derivatives as tubulin polymerization inhibitors targeting MCF-7 breast cancer cells.

A new set of 4-phenylcoumarin derivatives was designed and synthesized aiming to introduce new tubulin polymerization inhibitors as anti-breast cancer candidates. All the target compounds were evaluated for their cytotoxic effects against MCF-7 cell line, where compounds 2f, 3a, 3b, 3f, 7a and 7b, showed higher cytotoxic effect (IC50 = 4.3-21.2 µg/mL) than the reference drug doxorubicin (IC50 = 26.1 µg/mL), additionally, compounds 1 and 6b exhibited the same potency as doxorubicin (IC50 = 25.2 and 28.0 µg/mL, respectively). The thiazolidinone derivatives 3a, 3b and 3f with potent and selective anticancer effects towards MCF-7 cells (IC50 = 11.1, 16.7 and 21.2 µg/mL) were further assessed for tubulin polymerization inhibition effects which showed that the three compounds were potent tubulin polymerization suppressors with IC50 values of 9.37, 2.89 and 6.13 µM, respectively, compared to the reference drug colchicine (IC50 = 6.93 µM). The mechanistic effects on cell cycle progression and induction of apoptosis in MCF-7 cells were determined for compound 3a due to its potent and selective cytotoxic effects in addition to its promising tubulin polymerization inhibition potency. The results revealed that compound 3a induced cell cycle cessation at G2/M phase and accumulation of cells in pre-G1 phase and prevented its mitotic cycle, in addition to its activation of caspase-7 mediating apoptosis of MCF-7 cells. Molecular modeling studies for compounds 3a, 3b and 3f were carried out on tubulin crystallography, the results indicated that the compounds showed binding mode similar to the co-crystalized ligand; colchicine. Moreover, pharmacophore constructed models and docking studies revealed that thiazolidinone, acetamide and coumarin moieties are crucial for the activity. Molecular dynamics (MD) studies were carried out for the three compounds over 100 ps. MD results of compound 3a showed that it reached the stable state after 30 ps which was in agreement with the calculated potential and kinetic energy of compound 3a.

Targeting apoptotic anticancer response with natural glucosinolates from cell suspension culture of Lepidium sativum.

BACKGROUND: Finding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential. METHODS: In this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography-mass spectrometric analysis (GC-MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines. RESULTS: The findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an IC50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells. CONCLUSIONS: The anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest.

Nα-1, 3-Benzenedicarbonyl-Bis-(Amino Acid) and Dipeptide Candidates: Synthesis, Cytotoxic, Antimicrobial and Molecular Docking Investigation.

PURPOSE: The objective of our work was to prepare a potent and safe antimicrobial and anticancer agents, through synthesis of several peptides and examine their biological activities, namely as, cytotoxically potent and antimicrobial and antifungal agents. INTRODUCTION: Multidrug-resistant microbial strains have arisen against all antibiotics in clinical use. Infections caused by these bacteria threaten global public health and are associated with high mortality rates. METHODS: The main backbone structure for the novel synthesized linear peptide is Nα-1, 3-benzenedicarbonyl-bis-(Amino acids)-X, (3-11). A computational docking study against DNA gyrase was performed to formulate a mode of action of the small compounds as antimicrobial agents. RESULTS: The peptide-bearing methionine-ester (4) exhibited potent antimicrobial activity compared to the other synthesized compounds, while, peptide (8), which had methionine-hydrazide fragment was the most potent as antifungal agent against Aspergillus niger with 100% inhibition percent. Compounds (6 and 7) showed the highest potency against breast human tumor cell line "MCF-7" with 95.1% and 79.8% of cell inhibition, respectively. The nine compounds possessed weak to moderate antiproliferative effect over colon tumor cell line. The docking results suggest good fitting through different hydrogen bond interactions with the protein residues. In silico ADMET study also evaluated and suggested that these compounds had promising oral bioavailability features. CONCLUSION: The tested compounds need further modification to have significant antimicrobial and antitumor efficacy compared to the reference drugs.

Production and characterisation of exopolysaccharide from Streptomyces carpaticus isolated from marine sediments in Egypt and its effect on breast and colon cell lines.

Twenty streptomycete strains were isolated from marine sediment samples collected from Nabq area, Sharm El-Sheikh, Red Sea Coast, Egypt. Four of them produce exopolysaccharides (EPS) showing marked in vitro antitumor activities. Morphological and cultural characteristics of the most significant strain (No. 3) were shown. Moreover, the sequence of this strain showed similarity with Streptomyces carpaticus. The results reveal that EPS produced by Streptomyces carpaticus No. 3 had high cytotoxicity reaching 51.7% and 59.1% against human tumor cells of breast and colon lines respectively. A chemical analysis of EPS indicated that the composing monosaccharides were galactouronic acid, glucose, xylose, galactose, mannose, and fructose with relative ratio of 3:1:1:2:2:1 respectively, with an average molecular weight (Mw) 1.180 × 105 g/mol and of a number average molecular weight (Mn) 1.052 × 105 g/mol. Also the EPS contained uronic acid (0.5072%) and monosaccharide sulphates (21.753%).