Preparation of novel shell-ionotropically crosslinked micelles based on hexadecylamine and tripolyphosphate for cancer drug delivery.

AIMS: Micellar systems have the advantage of being easily prepared, cheap, and readily loadable with bioactive molecular cargo. However, their fundamental pitfall is poor stability, particularly under dilution conditions. We propose to use simple quaternary ammonium surfactants, namely, hexadecylamine (HDA) and hexadecylpyridinium (HDAP), together with tripolyphosphate (TPP) anion, to generate ionotropically stabilized micelles capable of drug delivery into cancer cells. METHODS: optimized mixed HDA/HDAP micelles were prepared and stabilized with TPP. Curcumin was used as a loaded model drug. The prepared nanoparticles were characterized by dynamic light scattering, infrared spectroscopy, transmission electron microscopy, and differential scanning calorimetry. Moreover, their cellular uptake was assessed using flow cytometry and confocal fluorescence microscopy. RESULTS: The prepared nanoparticles were found to be stable under dilution and at high temperatures and to have a size range from 139 nm to 580 nm, depending on pH (4.6-7.4), dilution (up to 100 times), and temperature (25 - 80 °C). They were effective at delivering their load into cancer cells. Additionally, flow cytometry indicated the resulting stabilized micellar nanoparticles to be non-cytotoxic. CONCLUSIONS: The described novel stabilized micelles are simple to prepare and viable for cancer delivery.

Discovery of new PKN2 inhibitory chemotypes via QSAR-guided selection of docking-based pharmacophores.

Serine/threonine-protein kinase N2 (PKN2) plays an important role in cell cycle progression, cell migration, cell adhesion and transcription activation signaling processes. In cancer, however, it plays important roles in tumor cell migration, invasion and apoptosis. PKN2 inhibitors have been shown to be promising in treating cancer. This prompted us to model this interesting target using our QSAR-guided selection of docking-based pharmacophores approach where numerous pharmacophores are extracted from docked ligand poses and allowed to compete within the context of QSAR. The optimal pharmacophore was sterically-refined, validated by receiver operating characteristic (ROC) curve analysis and used as virtual search query to screen the National Cancer Institute (NCI) database for new promising anti-PKN2 leads of novel chemotypes. Three low micromolar hits were identified with IC50 values ranging between 9.9 and 18.6 µM. Pharmacological assays showed promising cytotoxic properties for active hits in MTT and wound healing assays against MCF-7 and PANC-1 cancer cells.

Structure-Based Discovery and Bioactivity Evaluation of Novel Aurora-A Kinase Inhibitors as Anticancer Agents via Docking-Based Comparative Intermolecular Contacts Analysis (dbCICA).

Aurora-A kinase plays a central role in mitosis, where aberrant activation contributes to cancer by promoting cell cycle progression, genomic instability, epithelial-mesenchymal transition, and cancer stemness. Aurora-A kinase inhibitors have shown encouraging results in clinical trials but have not gained Food and Drug Administration (FDA) approval. An innovative computational workflow named Docking-based Comparative Intermolecular Contacts Analysis (dbCICA) was applied-aiming to identify novel Aurora-A kinase inhibitors-using seventy-nine reported Aurora-A kinase inhibitors to specify the best possible docking settings needed to fit into the active-site binding pocket of Aurora-A kinase crystal structure, in a process that only potent ligands contact critical binding-site spots, distinct from those occupied by less-active ligands. Optimal dbCICA models were transformed into two corresponding pharmacophores. The optimal one, in capturing active hits and discarding inactive ones, validated by receiver operating characteristic analysis, was used as a virtual in-silico search query for screening new molecules from the National Cancer Institute database. A fluorescence resonance energy transfer (FRET)-based assay was used to assess the activity of captured molecules and five promising Aurora-A kinase inhibitors were identified. The activity was next validated using a cell culture anti-proliferative assay (MTT) and revealed a most potent lead 85(NCI 14040) molecule after 72 h of incubation, scoring IC50 values of 3.5-11.0 µM against PANC1 (pancreas), PC-3 (prostate), T-47D and MDA-MB-231 (breast)cancer cells, and showing favorable safety profiles (27.5 µM IC50 on fibroblasts). Our results provide new clues for further development of Aurora-A kinase inhibitors as anticancer molecules.

Combining Stochastic Deformation/Relaxation and Intermolecular Contacts Analysis for Extracting Pharmacophores from Ligand-Receptor Complexes.

We previously combined molecular dynamics (classical or simulated annealing) with ligand-receptor contacts analysis as a means to extract valid pharmacophore model(s) from single ligand-receptor complexes. However, molecular dynamics methods are computationally expensive and time-consuming. Here we describe a novel method for extracting valid pharmacophore model(s) from a single crystallographic structure within a reasonable time scale. The new method is based on ligand-receptor contacts analysis following energy relaxation of a predetermined set of randomly deformed complexes generated from the targeted crystallographic structure. Ligand-receptor contacts maintained across many deformed/relaxed structures are assumed to be critical and used to guide pharmacophore development. This methodology was implemented to develop valid pharmacophore models for PI3K-γ, RENIN, and JAK1. The resulting pharmacophore models were validated by receiver operating characteristic (ROC) analysis against inhibitors extracted from the CHEMBL database. Additionally, we implemented pharmacophores extracted from PI3K-γ to search for new inhibitors from the National Cancer Institute list of compounds. The process culminated in new PI3K-γ/mTOR inhibitory leads of low micromolar IC50s.

Exploring the influence of culture conditions on kefir's anticancer properties.

Cancer is a major health problem in many parts of the world. Conventional anticancer treatments are painful, expensive, and unsafe. Therefore, demand is increasing for cancer treatments preferentially in the form of functional foods or nutritional supplements. Kefir, a traditional fermented milk dairy product, has significant antimutagenic and antitumor properties. This research addresses the hypothesis that kefir's anticancer properties are affected by fermentation conditions. Initially, kefir extracts prepared under standard conditions were screened against 7 cancer cell lines using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Colon cancer and chronic myelogenous leukemia cells were found to be most susceptible to kefir extracts. Subsequently, a factorial design was implemented to assess the effects of 3 fermentation times (24, 48, and 72 h), 3 kefir-to-milk ratios (2, 5, and 10% wt/vol), and 3 fermentation temperatures (4, 25, and 40°C) on kefir's anticancer properties. Remarkably, exploration of the fermentation conditions allowed the anticancer properties of kefir to be enhanced by 5- to 8-fold against susceptible cell lines. Overall, these results demonstrate the possibility of optimizing the anticancer properties of kefir as a functional food in cancer therapy.

Unsupervised pharmacophore modeling combined with QSAR analyses revealed novel low micromolar SIRT2 inhibitors.

Situin 2 (SIRT2) enzyme is a histone deacetylase that has important role in neuronal development. SIRT2 is clinically validated target for neurodegenerative diseases and some cancers. In this study, exhaustive unsupervised pharmacophore modeling was combined with quantitative structure-activity relationship (QSAR) analysis to explore the structural requirements for potent SIRT2 inhibitors using 146 known SIRT2 ligands. A computational workflow that combines genetic function algorithm with k-nearest neighbor or multiple linear regression was implemented to build self-consistent and predictive QSAR models based on combinations of pharmacophores and physicochemical descriptors. Successful pharmacophores were complemented with exclusion spheres to optimize their receiver operating characteristic curve profiles. Optimal QSAR models and their associated pharmacophore hypotheses were experimentally validated by identification and in vitro evaluation of several new promising SIRT2 inhibitory leads retrieved from the National Cancer Institute structural database. The most potent hit illustrated IC50 value of 5.4µM. The chemical structures of active hits were validated by proton nuclear magnetic resonance and mass spectroscopy.

Discovery of novel potent nuclear factor kappa-B inhibitors (IKK-ß) via extensive ligand-based modeling and virtual screening.

Inhibitor kappa-B kinase-beta (IKK-ß) controls the activation of nuclear transcription factor kappa-B and has been linked to inflammation and cancer. Therefore, inhibitors of this kinase should have potent anti-inflammatory and anticancer properties. Accordingly, we explored the pharmacophoric space of 218 IKK-ß inhibitors to identify high-quality binding models. Subsequently, genetic algorithm-based quantitative structure activity relationship (QSAR) analysis was employed to select the best possible combination of pharmacophoric models and physicochemical descriptors that explain bioactivity variation among training compounds. Three successful pharmacophores emerged in 2 optimal QSAR equations (r12175  = 0.733, r12LOO  = 0.52, F1 = 65.62, r12PRESS against 43 test inhibitors = 0.63 and r22175  = 0.683, r22LOO  = 0.52, F2 = 72.66, r22PRESS against 43 test inhibitors = 0.65). Two pharmacophores were merged in a single binding model. Receiver operating characteristic curve validation proved the excellent qualities of this model. The merged pharmacophore and the associated QSAR equations were applied to screen the National Cancer Institute list of compounds. Ten hits were found to exhibit potent anti-IKK-ß bioactivity, out of which, one illustrates IC50 of 11.0nM.

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CLpro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CLpro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CLpro . HKU4-CoV 3CLpro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CLpro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CLpro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CLpro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CLpro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CLpro . HIGHLIGHTS: MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CLpro is a potential drug target for coronavirus infection. HKU4-CoV 3CLpro is a useful surrogate model for the identification of MERS-CoV 3CLpro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CLpro inhibitors development.

Rosmarinic acid reverses the effects of metronidazole-induced infertility in male albino rats.

Rosmarinic acid (RA) is a natural antioxidant that has many biological activities. In the present study we investigated the potential of RA to reverse the negative effects of the widely used antibiotic and antiprotozoal agent metronidazole (MTZ), which is known to induce reversible male infertility. Two doses of RA (5 and 15mg kg-1) were studied in sexually mature rats with and without MTZ-induced infertility. Rats were intraperitoneally injected with 5mg kg-1 RA or 15mg kg-1 RA (in distilled water) and, 45min later, they were intraperitoneally injected with 40mg kg-1 MTZ (in distilled water). Cauda epididymidal sperm suspensions were used to assess sperm count, motility and morphology. Histological and ultrastructural studies were performed on the testes and cauda epididymidis. In rats in which infertility was not induced, neither dose of RA affected the parameters assessed. However, in sexually mature rats in which infertility was induced by 40mg kg-1 MTZ, RA at both 5 and 15mg kg-1 ameliorated the damaging effects of MTZ on final bodyweight (30 days later), sperm motility and morphology. Only 5mg kg-1 RA, and not 15mg kg-1 RA, improved the harmful effects of MTZ on the sperm count and testis ultrastructure. The findings of the present study have considerable clinical implications and suggest a possible use for RA to reverse the negative effects of MTZ on male fertility, the male reproductive system and spermatogenesis.

Cytotoxic activity of the novel heterocyclic compound G-11 is primarily mediated through intrinsic apoptotic pathway.

Natural and chemically synthesized heterocyclic compounds have been explored for their potential use as anticancer agents. We had synthesized non-natural heterocyclic analogs and evaluated their anti-tumor activity by measuring effect on cell proliferation and induction of apoptosis in different cell lines. Previously, we identified a pyrazole-containing compound (G-11) showing cytotoxic effect towards leukemia and lymphoma cell lines. In this study, we further investigated the mechanistic aspects of anticancer properties of G-11 in HL-60 cell line. We demonstrated that cytotoxic effect of G-11 is mediated by caspase-dependent apoptosis. However, the involvement of mitochondrial dysfunction induced by G-11 was independent of caspases. G-11 triggered generation of ROS, caused disruption of mitochondrial transmembrane potential, increased release of cytochrome c to the cytosol, and altered the expression of Bcl-2 and Bax proteins. These results suggest significant involvement of intrinsic apoptotic pathway. This study comprehensively details the possible mechanisms of action of a novel heterocyclic compound which could find its potential use as an anticancer agent.

Combining molecular dynamics simulation and ligand-receptor contacts analysis as a new approach for pharmacophore modeling: beta-secretase 1 and check point kinase 1 as case studies.

Ligand-based pharmacophore modeling require relatively long lists of active compounds, while a pharmacophore based on a single ligand-receptor crystallographic structure is often promiscuous. These problems prompted us to combine molecular dynamics (MD) simulation with ligand-receptor contacts analysis as means to develop valid pharmacophore model(s). The particular ligand-receptor complex is allowed to perturb over a few nano-seconds using MD simulation. Subsequently, ligand-receptor contact points (≤2.5 Å) are identified. Ligand-receptor contacts maintained above certain threshold during molecular dynamics simulation are considered critical and used to guide pharmacophore development. We termed this method as Molecular-Dynamics Based Ligand-Receptor Contact Analysis. We implemented this new methodology to develop valid pharmacophore models for check point kinase 1 (Chk1) and beta-secretase 1 (BACE1) inhibitors as case studies. The resulting pharmacophore models were validated by receiver operating characteristic curved analysis against inhibitors obtained from CHEMBL database.

The anticancer activity of the substituted pyridone-annelated isoindigo (5'-Cl) involves G0/G1 cell cycle arrest and inactivation of CDKs in the promyelocytic leukemia cell line HL-60.

BACKGROUND/AIMS: The antileukemic potential of isoindigos make them desired candidates for understanding their mechanism of action. We have recently synthesized a novel group of pyridone-annelated isoindigos and identified the derivative 5'-Cl that is cytotoxic to various cancer cell lines. In the present study, we analyzed the effect of this compound on cell cycle of the promyelocytic leukemia cell line HL-60. METHODS: HL-60 cells were treated with 5'-Cl and its effect on cell cycle stages were determined by flow cytometry. Expression of cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) were determined by Western blotting, and activation of CDKs was studied using kinase assays. RESULTS: 5'-Cl remarkably arrested cell cycle in HL-60 cells at the G0/G1 phase in a dose and time-dependent manner. Furthermore, 5'-Cl treatment significantly inhibited expression of D-cyclins, CDK2 and CDK4 and suppressed phosphorylation of the retinoblastoma protein Rb, whereas it increased the level of CKI p21. Molecular modelling experiments show that 5'-Cl may compete with ATP for binding to the catalytic subunit of CDK2 and CDK4 that could lead to inhibition of these enzymes. Indeed, 5'-Cl inhibited the kinase activity of CDK2 and CDK4 both in cell free systems and in treated cells. 5'-Cl also inhibited cell cycle progression in several other tumor cell lines. CONCLUSION: We demonstrate the potent inhibitory effects of 5'-Cl on HL-60 cells could be mediated by arresting cells in the G0/G1 phase.

Enhanced drug encapsulation and extended release profiles of calcium-alginate nanoparticles by using tannic acid as a bridging cross-linking agent.

Calcium alginate nanoparticles (NPs) suffer from sub-optimal stability in bio-relevant media leading to low drug encapsulation efficiency and uncontrolled release profiles. To sort out these drawbacks, a novel approach is proposed herein based on introducing tannic acid into these NPs to act as a bridging cross-linking aid agent. Calcium-alginate NPs were prepared by the ionotropic gelation method and loaded with diltiazem hydrochloride as a model drug. These NPs were characterized in terms of particle size, zeta potential, and morphology, and results were explained in accordance with Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The incorporation of tannic acid led to more than four folds increase in drug encapsulation efficiency (i.e. from 15.3% to 69.5%) and reduced burst drug release from 44% to around 10% within the first 30 min. These findings suggest the possibility of improving the properties of Ca-alginate NPs by incorporating cross-linking aid agents under mild conditions.

The use of docking-based comparative intermolecular contacts analysis to identify optimal docking conditions within glucokinase and to discover of new GK activators.

Glucokinase (GK) is involved in normal glucose homeostasis and therefore it is a valid target for drug design and discovery efforts. GK activators (GKAs) have excellent potential as treatments of hyperglycemia and diabetes. The combined recent interest in GKAs, together with docking limitations and shortages of docking validation methods prompted us to use our new 3D-QSAR analysis, namely, docking-based comparative intermolecular contacts analysis (dbCICA), to validate docking configurations performed on a group of GKAs within GK binding site. dbCICA assesses the consistency of docking by assessing the correlation between ligands' affinities and their contacts with binding site spots. Optimal dbCICA models were validated by receiver operating characteristic curve analysis and comparative molecular field analysis. dbCICA models were also converted into valid pharmacophores that were used as search queries to mine 3D structural databases for new GKAs. The search yielded several potent bioactivators that experimentally increased GK bioactivity up to 7.5-folds at 10 µM.

Evaluation of naproxen and cromolyn activities against cancer cells viability, proliferation, apoptosis, p53 and gene expression of survivin and caspase-3.

We previously reported the inhibitory profiles of naproxen and cromolyn against glycogen synthase kinase-3ß, which partly explain the molecular mechanisms of their anti-cancer properties. In this study, we performed a detailed biochemical evaluation of the two drugs against colorectal adenocarcinoma (Caco2), hepatocellular carcinoma (HepG2), mammary gland carcinoma (MCF7), epitheloid cervix carcinoma (Hela), lung carcinoma (A5W9) and epidermoid larynx carcinoma (Hep2) cell lines. Additionally, we performed cellular viability tests using trypan blue, proliferation MTT assay, apoptosis, p53 and real-time polymerase chain reaction for gene expression of survivin and caspase-3. Not only the two drugs were found to significantly reduce the viability of different cell lines, but they also were shown to have potent dose-dependent reduction of cellular proliferation. They exhibited cytotoxicity IC50 values of 3.69 and 4.16 µM for naproxen and cromolyn, respectively. Viability and proliferation results clearly correlated with apoptosis and p53 experiments in showing that both drugs significantly raised apoptotic percentages. Furthermore, we observed a significant reduction in survivin and elevation of caspase-3 gene expression upon exposure to the two drugs. It can be concluded that both naproxen and cromolyn have significant anti-cancer properties.

Synthesis and antitumor activities of some new N1-(flavon-6-yl)amidrazone derivatives.

A new series of N1-(flavon-6-yl)amidrazones were synthesized by reacting the hydrazonoyl chloride derived from 6-aminoflavone with the appropriate sec-cyclic amines. The antitumor activities of these compounds were evaluated on breast cancer (MCF-7) and leukemic (K562) cell lines. Among the compounds tested, the N-morpholine derivative was the most active against the MCF-7 and K562 cell lines, with IC50 values of 5.18 and 2.89 µM, respectively. Our docking studies showed that the N-morpholino derivative fits and blocks the oncogenic tyrosine kinases bcr/abl and epidermal growth factor receptor (EGFR) in a similar fashion to that of the potent anticancer agent imatinib.

Preparation and in vitro characterization of glibenclamide-loaded alginate hexyl-amide beads: a novel drug delivery system to improve the dissolution rate.

OBJECTIVE: This investigation aimed to synthesize amphiphilic hexyl amidic derivative of alginate to be used in the preparation of glibenclamide-loaded release system of improved dissolution rate. MATERIALS AND METHODS: Hexyl amine was associated to the activated carboxylic acid moieties of alginate to synthesize alginate hexyl amide polymer (AHAP). This polymer in comparison to alginate was used in different concentrations for preparing beads containing glibenclamide by an ionic gelation using Ca(++) as gelling ion. The prepared beads were characterized by DSC, FTIR and scanning electron microscope. The swelling behavior, drug loading capacity and release behavior were studied. RESULTS AND DISCUSSION: The results showed that the prepared AHAP beads were smaller in size and more spherical. The surface was highly corrugated with much and wider pore size. The beads showed a high drug loading capacity and efficacy that was affected by the polymer concentration. The drug release rate from AHAP beads reached 100% after 4, 8 and 12 hours in comparison to 75.3%, 73.2% and 69.2% from alginate beads at 3%, 2% and 1% polymer concentrations, respectively. CONCLUSION: It can thus be concluded that the amphiphilic AHAP-based bead is a simple and efficient delivery system of promising industrial significance for the improvement of the dissolution rate.

Synthesis and characterization of new derivatives of alginic acid and evaluation of their iron(III)-crosslinked beads as potential controlled release matrices.

CONTEXT: The excellent gelling and safety profiles of alginic acid combined, however, with drawbacks of its ionotropically crosslinked beads (i.e. their quick release of loaded drugs) prompted us to chemically modify alginic acid. OBJECTIVE: Alginic acid was chemically conjugated with four amines of varying hydrophilic-hydrophobic properties (i.e. tris(hydroxymethyl)methyl-, allyl-, benzyl- or pentyl-amines) in an attempt to enhance the drug release profiles from respective metal crosslinked beads. MATERIALS AND METHODS: Chemical conjugation procedures were performed using dicyclohexylcarbodiimide as a coupling agent and the resulting new derivatives were characterized using proton nuclear magnetic resonance ((1)H NMR), infrared (IR) spectroscopy and differential scanning calorimetry (DSC). These modified polymers were used to prepare iron (III)-crosslinked beads loaded with folic acid as model drug, which were tested in vitro to assess their folic acid release profiles. RESULTS AND DISCUSSION: Interestingly, the resulting beads accessed enteric release kinetics, with tris(hydroxymethyl)methyl-amide alginic conjugate producing most pronounced enteric profile. CONCLUSION: The results suggest the possibility of achieving controlled drug release from alginate-based beads via facile chemical modification of alginic acid.

Synthesis and antitumor activity of model cyclopentene-[g]annelated isoindigos.

A set of cyclopenten-[g]annelated isoindigos (5a-g) has been prepared and tested for their in vitro antiproliferative activities against MCF-7 and HL60 cells. Among, the N-1-methyl-5'-nitro derivative (5g) displayed the highest activity against HL60 cells (IC50 = 67 nM) and acted as the most potent Flt3 inhibitor. Compounds 5d-g exhibited moderate activity against MCF-7 (IC50 = 50-80 µM).

Elaborate ligand-based modeling coupled with multiple linear regression and k nearest neighbor QSAR analyses unveiled new nanomolar mTOR inhibitors.

The mammalian target of rapamycin (mTOR) has an important role in cell growth, proliferation, and survival. mTOR is frequently hyperactivated in cancer, and therefore, it is a clinically validated target for cancer therapy. In this study, we combined exhaustive pharmacophore modeling and quantitative structure-activity relationship (QSAR) analysis to explore the structural requirements for potent mTOR inhibitors employing 210 known mTOR ligands. Genetic function algorithm (GFA) coupled with k nearest neighbor (kNN) and multiple linear regression (MLR) analyses were employed to build self-consistent and predictive QSAR models based on optimal combinations of pharmacophores and physicochemical descriptors. Successful pharmacophores were complemented with exclusion spheres to optimize their receiver operating characteristic curve (ROC) profiles. Optimal QSAR models and their associated pharmacophore hypotheses were validated by identification and experimental evaluation of several new promising mTOR inhibitory leads retrieved from the National Cancer Institute (NCI) structural database. The most potent hit illustrated an IC50 value of 48 nM.