Cell-specific pattern of berberine pleiotropic effects on different human cell lines.

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

Astacin gene family of metalloproteinases in planarians: Structural organization and tissue distribution.

Planarian flatworms possess extraordinary regenerative capability and body plasticity, which rely on a composite population of stem cells, the neoblasts. Despite impressive advances have been recently achieved in the knowledge of neoblast biology, few is still known about factors that are released by differentiated tissues and influence the neoblast fate. Extracellular matrix (ECM) is a fundamental component of the stem cell niche and its remodeling affects stem cell fate. Here we provide the characterization of the astacin gene family of metalloproteinases in planarians, good candidate enzymes for generating dynamicity in the ECM. Ten and eighteen astacin isoforms were identified in the planarian species Schmidtea mediterranea and Dugesia japonica, respectively. Besides the already characterized Smedolloid, in Schmidtea mediterranea are present eight astacins with a minimal structure (a signal peptide, an activation domain and a Zn-binding catalytic domain), that are colocalized in large cells organized in a peculiar, not yet morphologically characterized, two-ring-shaped structure located in the middle of the body. A single astacin, characterized by a ShK toxin domain in its C-terminal region, has been found to be produced in gastrodermal cells.

In vitro studies of antifibrotic and cytoprotective effects elicited by proto-berberine alkaloids in human dermal fibroblasts.

BACKGROUND: The pathogenic mechanisms of skin fibrosis are still not completely understood, unlike the profibrotic role played by inflammatory cytokines and transforming growth factor-ß1 (TGF-ß1). Few antifibrotic drugs are available. Nevertheless, folk medicine suggests numerous treatments of fibrotic conditions. Based on information from folk medicine and literature, the hypothesis was made that proto-berberine alkaloids could act as antifibrotic and cytoprotective agents. METHODS: The effects of berberine, dihydroberberine, canadine, stylopine, and coptisine were investigated on an in vitro model of fibrosis purposely set up. The study is based on the use of human dermal fibroblasts (HDF). The ability of the proto-berberine alkaloids investigated to modulate mitochondrial dehydrogenase activity, cell proliferation, collagen production, and inflammatory cytokine (IL-1ß and IL-6) production was tested on HDF cells grown under standard growth conditions, in the presence of 100 µM H(2)O(2), simulating oxidative stress conditions, and in the presence of 34 ng/ml TGF-ß1, simulating fibrotic conditions. Antiradical activity was assayed as well, as it could contribute to cytoprotection. RESULTS: Each alkaloid tested showed peculiar effects on HDF. In particular, all of the alkaloids tested, with the exception of coptisine, inhibited TGF-ß1-induced collagen production. CONCLUSIONS: Due to its irritant effects and the lack of desired properties, coptisine has low exploitation potentialities. The other proto-berberine alkaloids investigated resulted all endowed with activities for which they can be exploited as antifibrotic and cytoprotective agents. Stylopine globally proved to be the most promising compound, being endowed with revitalizing, anti-inflammatory, antifibrotic and wound-healing promoting activities, and showing no toxic effects.

Rare disease registries classification and characterization: a data mining approach.

BACKGROUND: The European Commission and Patients Organizations identify rare disease registries (RDRs) as strategic instruments to develop research and improve knowledge in the field of rare diseases. Interoperability between RDRs is needed for research activities, validation of therapeutic treatments, and public health actions. Sharing and comparing information requires a uniform and standardized way of data collection, so levels of interconnection between RDRs with similar aims and/or nature of data should be identified. The objective of this study is to define a classification and characterization of RDRs in order to identify different profiles and informative needs. METHODS: Exploratory statistical analyses (cluster analysis and random forest) were applied to data derived from the EPIRARE project ('Building Consensus and Synergies for the EU Rare Disease Patient Registration') survey on the activities and needs of RDRs. RESULTS: The cluster analysis identified 3 main typologies of RDRs: public health, clinical and genetic research, and treatment registries. The analysis of the most informative variables, identified by the random forest method, led to the characterization of 3 types of RDRs and the definition of different profiles and informative needs. CONCLUSIONS: These results represent a useful source of information to facilitate the harmonization and interconnection of RDRs in accordance with the different profiles identified. It could help sharing the information between RDRs with similar profiles and, whenever possible, interconnections between registries with different profiles.

Berberine exposure triggers developmental effects on planarian regeneration.

The mechanisms of action underlying the pharmacological properties of the natural alkaloid berberine still need investigation. Planarian regeneration is instrumental in deciphering developmental responses following drug exposure. Here we report the effects of berberine on regeneration in the planarian Dugesia japonica. Our findings demonstrate that this compound perturbs the regenerative pattern. By real-time PCR screening for the effects of berberine exposure on gene expression, we identified alterations in the transcriptional profile of genes representative of different tissues, as well as of genes involved in extracellular matrix (ECM) remodeling. Although berberine does not influence cell proliferation/apoptosis, our experiments prove that this compound causes abnormal regeneration of the planarian visual system. Potential berberine-induced cytotoxic effects were noticed in the intestine. Although we were unable to detect abnormalities in other structures, our findings, sustained by RNAi-based investigations, support the possibility that berberine effects are critically linked to anomalous ECM remodeling in treated planarians.

Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli.

Organophosphates are potent poisoning agents that cause severe cholinergic toxicity. Current treatment has been reported to be unsatisfactory and novel antidotes are needed. In this study, we used a single-chain variable fragment (scFv) library to select a recombinant antibody fragment (WZ1-14.2.1) with butyrylcholinesterase-like catalytic activity by using an innovative method integrating genetic selection and the bait-and-switch strategy. Ellman assay demonstrated that WZ1-14.2.1 has Michaelis-Menten kinetics in the hydrolysis of all the three substrates used, acetylthiocholine, propionylthiocholine and butyrylthiocholine. Notably, the catalytic activity was resistant to the following acetylcholinesterase inhibitors: neostigmine, iso-OMPA, chlorpyrifos oxon, dichlorvos, and paraoxon ethyl. Otherwise, the enzymatic activity of WZ1-14.2.1 was inhibited by the selective butyrylcholinesterase inhibitor, ethopropazine, and by the Ser-blocking agent phenylmethanesuphonyl fluoride. A hypothetical 3D structure of the WZ1-14.2.1 catalytic site, compatible with functional results, is proposed on the basis of a molecular modeling analysis.

Combining structure- and ligand-based approaches for studies of interactions between different conformations of the hERG K+ channel pore and known ligands.

Drug-induced insurgence of cardiotoxic effects signaled by the prolongation of the QT interval in the electrocardiogram, has the potential to evolve into a characteristic arrhythmic event named Torsade de Pointes (TdP). Although several different mechanisms can theoretically lead to prolonged QT interval, most of drugs showing this side effect, prolong the cardiac repolarization time through the inhibition of the rapid component of the delayed repolarizing current (IKr) which in humans is carried by a K(+) channel protein encoded by hERG. In this study, four 3D-models, representing different conformational states of hERG K(+) channel, were built by a homology-based technique. A dataset of 59 compounds was collected from the literature and rationally selected according to the availability of IC50 values derived from whole-cell patch clamp performed at 37 °C on HEK cells. Molecular docking was carried out on each one of the four conformations of the channel, hundreds of docking-based molecular descriptors were obtained and used, together with other 2D and 3D molecular descriptors, to develop QSAR models. The statistical parameters describing the accordance between predicted and experimental data and the interpretation of the QSAR models enabled us to assess the reliability of the four 3D-models of the channel pore, thus allowing to look in more depth at binding modes and key features of the interactions occurring between the hERG K(+) channel and ligands endowed of blocking activity.

Planarians as a model to assess in vivo the role of matrix metalloproteinase genes during homeostasis and regeneration.

Matrix metalloproteinases (MMPs) are major executors of extracellular matrix remodeling and, consequently, play key roles in the response of cells to their microenvironment. The experimentally accessible stem cell population and the robust regenerative capabilities of planarians offer an ideal model to study how modulation of the proteolytic system in the extracellular environment affects cell behavior in vivo. Genome-wide identification of Schmidtea mediterranea MMPs reveals that planarians possess four mmp-like genes. Two of them (mmp1 and mmp2) are strongly expressed in a subset of secretory cells and encode putative matrilysins. The other genes (mt-mmpA and mt-mmpB) are widely expressed in postmitotic cells and appear structurally related to membrane-type MMPs. These genes are conserved in the planarian Dugesia japonica. Here we explore the role of the planarian mmp genes by RNA interference (RNAi) during tissue homeostasis and regeneration. Our analyses identify essential functions for two of them. Following inhibition of mmp1 planarians display dramatic disruption of tissues architecture and significant decrease in cell death. These results suggest that mmp1 controls tissue turnover, modulating survival of postmitotic cells. Unexpectedly, the ability to regenerate is unaffected by mmp1(RNAi). Silencing of mt-mmpA alters tissue integrity and delays blastema growth, without affecting proliferation of stem cells. Our data support the possibility that the activity of this protease modulates cell migration and regulates anoikis, with a consequent pivotal role in tissue homeostasis and regeneration. Our data provide evidence of the involvement of specific MMPs in tissue homeostasis and regeneration and demonstrate that the behavior of planarian stem cells is critically dependent on the microenvironment surrounding these cells. Studying MMPs function in the planarian model provides evidence on how individual proteases work in vivo in adult tissues. These results have high potential to generate significant information for development of regenerative and anti cancer therapies.

Development of classification models for identifying "true" P-glycoprotein (P-gp) inhibitors through inhibition, ATPase activation and monolayer efflux assays.

P-glycoprotein (P-gp) is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external) validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as "true" P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

The in vivo effect of chelidonine on the stem cell system of planarians.

The presence of adult pluripotent stem cells and the amazing regenerative capabilities make planarian flatworms an extraordinary experimental model to assess in vivo the effects of substances of both natural and synthetic origin on stem cell dynamics. This study focuses on the effects of chelidonine, an alkaloid obtained from Chelidonium majus. The expression levels of molecular markers specific for stem or differentiated cells were compared in chelidonine-treated and control planarians. The use of these markers demonstrates that chelidonine produces in vivo a significant anti-proliferative effect on planarian stem cells in a dose-dependent fashion. In response to chelidonine treatment mitotic abnormalities were also observed and the number of cells able to proceed to anaphase/telophase appeared significantly reduced with respect to the controls. Our results support the possibility that chelidonine acts on cell cycle progression by inhibition of tubulin polymerization. These studies provide a basis for preclinical evaluation in vivo of the effects of chelidonine on physiologically proliferating stem cells.

Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase.

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)(2)D(3) analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner-Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80-1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3), further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)(2)D(3) and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.

Identification of selective ligands for human fibrin recognition using high-throughput docking.

The ultimate aim of this study is to identify new molecules that are able to recognize polymerized fibrin, which is the main component of a thrombus. These selective ligands can be exposed on the surface of particular nanoparticles used for the targeted delivery of fibrinolytic drugs. The targeted delivery of these drugs is expected to help to keep under control the severe side effects which can occur if the drugs are administered systemically. The study focuses on the application of high-throughput docking methods used to screen a library of thousands of commercial compounds. The aim was to identify molecules that are potentially capable of interacting with the human fibrin γ(312-324) epitope. The best scoring compounds were purchased and tested through fluorimetric assays in order to estimate their affinity toward fibrin. The results show that the protocol proposed here for identifying new compounds of interest may provide a valuable contribution to the discovery of lead molecules for human fibrin recognition.

Structure-activity relationships on purine and 2,3-dihydropurine derivatives as antitubercular agents: a data mining approach.

Nowadays, many people still fall victim to tuberculosis, the disease that has a worldwide spreading. Moreover, the problem of resistance to isoniazid and rifampin, the two most effective antitubercular drugs, is assuming an ever-growing importance. The need for new drugs active against Mycobacterium tuberculosis represents nowadays a quite relevant problem in medicinal chemistry. Several purine and 2,3-dihydropurine derivatives have recently emerged, showing considerable antitubercular properties. In this work, a quantitative structure-activity relationship (QSAR) model was developed, which is able to predict whether new purine and 2,3-dihydropurine derivatives belong to an 'Active' or 'Inactive' class against the above micro-organism. The obtained prediction model is based on a classification tree; it was built with a small number of descriptors, which allowed us to outline structural features important to predict antitubercular activity of such classes of compounds.

Three-dimensional models of the oligomeric human asialoglycoprotein receptor (ASGP-R).

The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R), by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2), which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal oligomeric form, necessary for it to bind multi-antennary carbohydrate ligands. The ultimate target of the study is to contribute to increasing the knowledge of interactions between the human ASGP-R and carbohydrate ligands, at the molecular level, pertinent to applications in the field of hepatic tissue engineering.

Enhancer and competitive allosteric modulation model for G-protein-coupled receptors.

A new mathematical model, referred to as Enhancer and Competitive Allosteric Modulator (ECAM) model, developed with the aim of quantitatively describing the interaction of an allosteric modulator with both enhancer and competitive properties towards G-protein-coupled receptors is described here. Model simulations for equilibrium (displacement-like and saturation-like), and kinetic (association and dissociation) binding experiments were performed. The results showed the ability of the model to interpret a number of possible ligand-receptor binding behaviors. In particular, the binding properties of PD81723, an enhancer and competitive allosteric modulator for the adenosine A(1) receptor, were experimentally evaluated by radioligand binding assays and interpreted by the ECAM model. The results also offer a theoretical background enabling the design and optimization of compounds endowed with allosteric enhancer, competitive, agonist, antagonist, and inverse agonist properties.

Chemical reactivity predictions: use of data mining techniques for analyzing regioselective azidolysis of epoxides.

Azidolysis of epoxides followed by reduction of the intermediate azido alcohols constitutes a valuable synthetic tool for the construction of beta-amino alcohols, an important chemical functionality occurring in many biologically active compounds of natural origin. However, depending on conditions under which the azidolysis is carried out, two regioisomeric products can be formed, as a consequence of the nucleophilic attack on both the oxirane carbon atoms. In this work, predictive models for quantitative structure-reactivity relationships were developed by means of multiple linear regression, k-nearest neighbor, locally weighted regression, and Gaussian Process regression algorithms. The specific nature of the problem at hand required the creation of appropriate new descriptors, able to properly reflect the most relevant features of molecular moieties directly involved in the opening process. The models so obtained are able to predict the regioselectivity of the azidolysis of epoxides promoted by sodium azide, in the presence of lithium perchlorate, on the basis of steric hindrance, and charge distribution of the substituents directly attached to the oxirane ring.

Quantitative structure-activity relationship models for predicting biological properties, developed by combining structure- and ligand-based approaches: an application to the human ether-a-go-go-related gene potassium channel inhibition.

A strategy for developing accurate quantitative structure-activity relationship models enabling predictions of biological properties, when suitable knowledge concerning both ligands and biological target is available, was tested on a data set where molecules are characterized by high structural diversity. Such a strategy was applied to human ether-a-go-go-related gene K(+) channel inhibition and consists of a combination of ligand- and structure-based approaches, which can be carried out whenever the three-dimensional structure of the target macromolecule is known or may be modeled with good accuracy. Molecular conformations of ligands were obtained by means of molecular docking, performed in a previously built theoretical model of the channel pore, so that descriptors depending upon the three-dimensional molecular structure were properly computed. A modification of the directed sphere-exclusion algorithm was developed and exploited to properly splitting the whole dataset into Training/Test set pairs. Molecular descriptors, computed by means of the codessa program, were used for the search of reliable quantitative structure-activity relationship models that were subsequently identified through a rigorous validation analysis. Finally, pIC(50) values of a prediction set, external to the initial dataset, were predicted and the results confirmed the high predictive power of the model within a quite wide chemical space.

Predictive models, based on classification algorithms, for compounds potentially active as mitochondrial ATP-sensitive potassium channel openers.

Heart mitochondrial ATP-sensitive potassium channel (mito-K(ATP) channels) are deeply implicated in the self-defense mechanism of ischemic preconditioning. Therefore, exogenous molecules activating these channels are considered as a promising pharmacological tool to reduce the myocardial injury deriving from ischemia/reperfusion events. In our laboratory, a series of 4-spiro-substituted benzopyran derivatives were earlier synthesized and some of them exhibited anti-ischemic properties. In this study, the above compounds are exploited in order to develop QSAR models, based on classification approaches, capable of discriminating between the ones acting as cardioprotective agents and those that are unable to elicit such a property. Molecules belonging to the whole dataset were subjected to CODESSA and E-Dragon calculations in order to compute a large number of molecular descriptors enabling the construction of classification models. Based on the two program packages used, two different experiments were carried out, with the aim of identify batteries of models to be exploited for designing new cardioprotective agents from libraries of new chemical entities. Both model batteries satisfy the rigorous criteria adopted for the validation, either when tested on the training and test set, according to the most straightforward protocol, and when tested on an additional prediction set. They were proven to ensure successful applications in the field of cardioprotective agent design.

Development of QSAR models for predicting hepatocarcinogenic toxicity of chemicals.

A dataset comprising 55 chemicals with hepatocarcinogenic potency indices was collected from the Carcinogenic Potency Database with the aim of developing QSAR models enabling prediction of the above unwanted property for New Chemical Entities. The dataset was rationally split into training and test sets by means of a sphere-exclusion type algorithm. Among the many algorithms explored to search regression models, only a Support Vector Machine (SVM) method led to a QSAR model, which was proved to pass rigorous validation criteria, in accordance with the OECD guidelines. The proposed model is capable to explain the hepatocarcinogenic toxicity and could be exploited for predicting this property for chemicals at the early stage of their development, so optimizing resources and reducing animal testing.

Synthesis, biological assays and QSAR studies of N-(9-benzyl-2-phenyl-8-azapurin-6-yl)-amides as ligands for A1 adenosine receptors.

2-Phenyl-9-benzyl-8-azapurines, bearing at the 6 position an amido group interposed between the 8-azapurine moiety and an alkyl or a substituted phenyl group, have been synthesised and assayed as ligands for adenosine receptors. All the compounds show high affinity for the A(1) adenosine receptor, and many of them also show a good selectivity for A(1) with respect to A(2A) and A(3) adenosine receptors. Based on the quite rich library containing such compounds and relevant biological data, QSAR models, able to rationalise the results and to give a quantitative estimate of the observed trends were also developed. The obtained models can assist in the design of new compounds selectively active on A(1) adenosine receptor.