Design and synthesis of 2-oxindole based multi-targeted inhibitors of PDK1/Akt signaling pathway for the treatment of glioblastoma multiforme.

Aggressive behavior and diffuse infiltrative growth are the main features of Glioblastoma multiforme (GBM), together with the high degree of resistance and recurrence. Evidence indicate that GBM-derived stem cells (GSCs), endowed with unlimited proliferative potential, play a critical role in tumor development and maintenance. Among the many signaling pathways involved in maintaining GSC stemness, tumorigenic potential, and anti-apoptotic properties, the PDK1/Akt pathway is a challenging target to develop new potential agents able to affect GBM resistance to chemotherapy. In an effort to find new PDK1/Akt inhibitors, we rationally designed and synthesized a small family of 2-oxindole derivatives. Among them, compound 3 inhibited PDK1 kinase and downstream effectors such as CHK1, GS3Kα and GS3Kß, which contribute to GCS survival. Compound 3 appeared to be a good tool for studying the role of the PDK1/Akt pathway in GCS self-renewal and tumorigenicity, and might represent the starting point for the development of more potent and focused multi-target therapies for GBM.

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.

Enzyme kinetics studies on 29-kDa human liver cathepsin L.

Enzyme kinetics studies reported in the literature showed that human liver Cathepsin L is active only at lysosomal acidic pH values, while biochemical studies in living cells showed that the enzyme works even at neutral pH values (in a condition compatible with the extracellular compartment). Such an apparent ambiguity highlighted the need of analysing in depth the kinetics of ~29-kDa Cathepsin L, which is the form commonly used in experiments. The stability and catalytic activity of this enzyme were investigated at different pH values, reducing and non-reducing environments, presence of copper, iron and zinc ions, and presence of the natural modulator/inhibitor cystatin B. Our experiments showed that ~29-kDa human liver Cathepsin L is stable and catalytically functional even at neutral pH values and under non-reducing conditions, which simulate the extracellular compartment. Under these conditions, Cathepsin L was also proved to interact with cystatin B, being also modulated by physiological concentrations of Cu(++) , Fe(++) and Zn(++) . This paper suppose an advance in the comprehension of the catalytic properties of human liver Cathepsin L, its implications in different physiological processes and its potential use within a drug screening programme in which agents acting extracellularly are being considered.

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.

N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides as potent and selective ligands for A1 adenosine receptors.

A series of N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides were synthesized and tested for their affinity toward A1, A2A , and A3 adenosine receptor subtypes. Biological results demonstrated that the introduction of a fluorine atom at the ortho position of the 9-benzyl group generally enhanced affinity toward A1 subtype and did not significantly affect A2A and A3 affinity. Very interesting is the compound bearing a meta-fluorophenyl substituent on the carbonyl carbon of the amide group, which shows significantly high A1/A2A-A3 selectivity. Compounds of this new series, together with the previously published analogs without the fluorine atom on the 9-benzyl group, constituted the starting dataset for the development of QSAR models. The models obtained were able to rationally describe the affinity trends resulting from biological testing and to enable investigation of the role of different substituents on the 8-azapurine scaffold, as well as the influence of the newly introduced fluorine atom on the benzyl moiety. The said QSAR models can also assist in the design of new compounds selectively active on A1 adenosine receptors. Furthermore, a molecular docking study was carried out to assess hypothetical binding mode of N-[9-(ortho-fluorobenzyl)-2-phenyl-8-azapurin-6-yl]-amides to A1 adenosine receptors.

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.

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.

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.

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.

QSAR study on a novel series of 8-azaadenine analogues proposed as A1 adenosine receptor antagonists.

8-Azaadenines have been recently proposed as a novel promising class of adenosine A1 receptor antagonists. A QSAR study on 45 derivatives, synthesized in our laboratory as antagonists for A1 receptor, is described here. The use of the CODESSA program allowed obtaining a quite simple equation capable of correlating the structural features of these ligands to their activity toward A1 receptor. The model was investigated for reliability and stability by using statistical analysis criteria stricter than usual. Particular care was put in defining the chemical space where the model gave reliable predictions. The model allowed the identification of relevant structural features required for the interaction with the A1 receptor, enabling the prediction of activity of molecules belonging to focused virtual libraries.

N6-1,3-diphenylurea derivatives of 2-phenyl-9-benzyladenines and 8-azaadenines: synthesis and biological evaluation as allosteric modulators of A2A adenosine receptors.

Some 1-[4-(9-benzyl-2-phenyl-9H-purin-6-ylamino)-phenyl]-3-phenyl-urea derivatives and some 1-[4-(9-benzyl-2-phenyl-9H-8-azapurin-6-ylamino)-phenyl]-3-phenyl-urea derivatives were synthesised and evaluated for their interaction with adenosine receptors. It was found that some of these compounds can act as positive enhancers of agonist and antagonist radioligands for the A(2A) adenosine receptors. This evidence was also strengthened by functional data. Other compounds can act as negative modulators. Furthermore these compounds show inhibitory properties for A(1) and A(3) adenosine receptors.

Synthesis of new 2-phenyladenines and 2-phenylpteridines and biological evaluation as adenosine receptor ligands.

Synthesis and biological assays of a series of 2-phenylpteridine derivatives are described to compare their affinities to adenosine receptors with those of the corresponding adenines, purposely prepared, and 8-azaadenines previously described. This study demonstrates that the enlargement of the five-membered ring of the adenine nucleus to a six-membered one is a modification that does not allow the molecules to maintain high activity towards adenosine receptors; in fact, pteridine derivatives did not show themselves to be good adenosine receptor ligands. On the contrary, N(6)-cycloalkyl- or N(6)-alkyl-2-phenyladenines showed a very high affinity and selectivity for A(1) adenosine receptors. We demonstrate also that the 9-benzyl substituent is crucial for conferring high affinity for A(3) receptors to molecules having a 2-phenyladenine-like nucleus.

Synthesis, biological activity and molecular modelling of new trisubstituted 8-azaadenines with high affinity for A1 adenosine receptors.

We describe here the synthesis and biological activity of new 8-azaadenines bearing both a phenyl group on C(2) and a 9-benzyl group substituted in the ortho position with a Cl or a F atom or a CF(3) group, to verify the synergistic effect of a combination of these substitution patterns on binding with the A(1) adenosine receptors. In position N(6) aliphatic and cycloaliphatic substituents were chosen which had been shown to bind well with the A(1) receptors. Because of the high lipophilicity of these kinds of molecules, we also introduced a hydroxyalkyl substituent in the same position. The compounds obtained generally showed a very good affinity and selectivity for A(1) receptors. Some of the compounds showed K(i) in the nanomolar range, one even in the subnanomolar range (0.6 M). Molecular docking calculations were performed in order to evaluate the interaction energies between the bovine A(1) receptor model and the selected ligands, and then to correlate these energies with biological activities of the ligands as obtained from the experiments. Molecular docking analysis suggests different binding modes towards A(1) receptors that are plausible for these ligands.

QSAR study on thiazole and thiadiazole analogues as antagonists for the adenosine A1 and A3 receptors.

Thiazole and thiadiazole analogues have been recently proposed as a novel promising class of adenosine A1 and A3 receptor antagonists. When appropriately modified, they show selectivity toward A1 or A3 receptors, which results in a variety of therapeutic potentialities of these ligands. In this work, we carried out a QSAR study on thiazole and thiadiazole analogues as antagonists for adenosine A1 and A3 receptors. To develop reliable models, we focused attention on any possible pitfalls of each step of QSAR process and approached each stage following accurate procedures. Application of datasets by using CODESSA software led to QSAR equations based on three and four descriptors for the adenosine A1 and A3 receptor ligands, respectively. The obtained models allowed us to understand the main structural features that strongly correlate with the target property.

Synthesis and biological evaluation of 2-aminothiazoles and their amide derivatives on human adenosine receptors. Lack of effect of 2-aminothiazoles as allosteric enhancers.

A number of 2-aminothiazoles (2a-e) and their amide derivatives (4-10) were prepared. The 2-aminothiazoles themselves were tested as allosteric enhancers of agonist binding to human adenosine A(1) receptors. In a variety of experimental set-ups the compounds did not show any such effect, in contrast to earlier findings by another research group. Subsequently the 2-aminothiazoles were used as intermediates in the synthesis of a number of amide derivatives of either aromatic (4-6) or aliphatic nature (7-10). Some of the compounds emerged as moderately active antagonists on human adenosine A(1) and/or A(2A) receptors with lower or negligible potency at adenosine A(3) receptors.

Synthesis and biological evaluation of 2,3,5-substituted [1,2,4]thiadiazoles as allosteric modulators of adenosine receptors.

A number of 2,3,5-substituted [1,2,4]thiadiazole analogues of SCH-202676 (N-(2,3-diphenyl[1,2,4]thiadiazole-5(2H)-ylidene)methanamine, 7a) were synthesized and tested as potential allosteric modulators of adenosine receptors. All compounds were capable of displacing the binding of the radiolabeled agonist [(3)H]CCPA to human A(1) adenosine receptors, whereas modest and varying effects were observed on the binding of [(3)H]DPCPX, a radiolabeled antagonist for this receptor subtype. Four compounds, 7a (SCH-202676), 7k (LUF5792), 7l (LUF5794), and 8e (LUF5789), were selected for more detailed characterization. They all proved allosteric inhibitors of agonist binding, with 7k being most potent, whereas their effects on antagonist binding were more ambiguous. Subsequently, experiments were done on human adenosine A(2A) and A(3) receptors. Compounds 7a and 7l displayed peculiar displacement characteristics of both radiolabeled agonist and antagonist binding to A(2A) receptors, whereas 7a showed some activity on A(3) receptors.