Nicola M. Martucci

Discovery of new inhibitors of Cdc25B dual specificity phosphatases by structure-based virtual screening.

Cell division cycle 25 (Cdc25) proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases and represent attractive drug targets for anticancer therapies. To discover more potent and diverse inhibitors of Cdc25 biological activity, virtual screening was performed by docking 2.1 million compounds into the Cdc25B active site. An initial subset of top-ranked compounds was selected and assayed, and 15 were found to have enzyme inhibition activity at micromolar concentration. Among these, four structurally diverse inhibitors with a different inhibition profile were found to inhibit human MCF-7, PC-3, and K562 cancer cell proliferation and significantly affect the cell cycle progression. A subsequent hierarchical similarity search with the most active reversible Cdc25B inhibitor found led to the identification of an additional set of 19 ligands, three of which were confirmed as Cdc25B inhibitors with IC(50) values of 7.9, 4.2, and 9.9 μM, respectively.

Bioengineered Silicon Diatoms: Adding Photonic Features to a Nanostructured Semiconductive Material for Biomolecular Sensing

Native diatoms made of amorphous silica are first converted into silicon structures via magnesiothermic process, preserving the original shape: electron force microscopy analysis performed on silicon-converted diatoms demonstrates their semiconductor behavior. Wet surface chemical treatments are then performed in order to enhance the photoluminescence emission from the resulting silicon diatoms and, at the same time, to allow the immobilization of biological probes, namely proteins and antibodies, via silanization. We demonstrate that light emission from semiconductive silicon diatoms can be used for antibody-antigen recognition, endorsing this material as optoelectronic transducer.

Nanoparticle-based strategy for personalized B-cell lymphoma therapy

B-cell lymphoma is associated with incomplete response to treatment, and the development of effective strategies targeting this disease remains challenging. A new personalized B-cell lymphoma therapy, based on a site-specific receptor-mediated drug delivery system, was developed in this study. Specifically, natural silica-based nanoparticles (diatomite) were modified to actively target the antiapoptotic factor B-cell lymphoma/leukemia 2 (Bcl2) with small interfering RNA (siRNA). An idiotype-specific peptide (Id-peptide) specifically recognized by the hypervariable region of surface immunoglobulin B-cell receptor was exploited as a homing device to ensure specific targeting of lymphoma cells. Specific nanoparticle uptake, driven by the Id-peptide, was evaluated by flow cytometry and confocal microscopy and was increased by approximately threefold in target cells compared with nonspecific myeloma cells and when a random control peptide was used instead of Id-peptide. The specific internalization efficiency was increased by fourfold when siRNA was also added to the modified nanoparticles. The modified diatomite particles were not cytotoxic and their effectiveness in downregulation of gene expression was explored using siRNA targeting Bcl2 and evaluated by quantitative real-time polymerase chain reaction and Western blot analyses. The resulting gene silencing observed is of significant biological importance and opens new possibilities for the personalized treatment of lymphomas.

Internalization kinetics and cytoplasmic localization of functionalized diatomite nanoparticles in cancer cells by Raman imaging

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses.

Ser/Thr kinases and polyamines in the regulation of non-canonical functions of elongation factor 1A

The link between eukaryotic translation elongation factor 1A (eEF1A) and signal transduction pathways through the regulatory mechanism of phosphorylation has never been considered. In this review, we focus on the different kinases that recognize the Ser and Thr residues of the eEF1A1 and eEF1A2 isoforms and regulate their involvement in different cellular processes like cell survival and apoptosis. In this context, polyamines seem to play a role in the regulation of the translation elongation process by modulating the Ser/Thr kinases involved in the phosphorylation of translation elongation factors.

A new strategy for label-free detection of lymphoma cancer cells.

In this paper, a new strategy for highly selective and sensitive direct detection of lymphoma cells by exploiting the interaction between a peptide and its B-cell receptor, has been evaluated. In particular, an idiotype peptide, able to specifically bind the B-cell receptor of A20 cells in mice engrafted with A20 lymphoma, has been used as molecular probe. The new detection technique has been demonstrated on a planar crystalline silicon chip. Coverage of 85% of silicon surface and detection efficiency of 8.5 × 10(-3) cells/μm(2) were obtained. The recognition strategy promises to extend its application in studying the interaction between ligands and their cell-surface receptors.

Interaction Between the Antibiotic Tetracycline and the Elongation Factor 1α from the Archaeon Sulfolobus solfataricus

The interaction between tetracycline and the archaeal elongation factor 1α from Sulfolobus solfataricus was investigated. The effects produced by this eubacterial antibiotic indicated that this interaction involved the G‐domain of the elongation factor 1α from S. solfataricus, although also the M‐domain was required. In fact, in the presence of the antibiotic, an increase in the fluorescence quantum yield of the aromatic region was observed for elongation factor 1α from S. solfataricus and its truncated form lacking the C‐terminal domain, but not for that lacking also the M‐domain. The increase in quantum yield was restored when the G‐domain of elongation factor 1α from S. solfataricus was fused to the M and the C‐domains of the eubacterial analogue elongation factor Tu. Tetracycline inhibits protein synthesis catalysed by elongation factor 1α from S. solfataricus; this is accompanied by an increase in the GDP/GTP exchange rate and a slight inhibition of the intrinsic GTPase, suggesting that a main effect of the antibiotic was exerted on the GTP‐bound form of the enzyme. Furthermore, the mixed inhibition observed for GTPase confirmed that the interaction, besides the G‐domain, involved also other region(s) of elongation factor 1α from S. solfataricus. These results can be useful for studying potential side effects arising from the interaction between tetracycline and eukaryotic elongation factors.

Raf kinases in signal transduction and interaction with translation machinery

Abstract In recent years, a large amount of evidence has given a central role to translational control in diseases such as cancer, tissue hypertrophy and neurodegeneration. Its deregulation can directly modulate cell cycling, transformation and survival response. The aim of this review is to describe the interaction between Raf activation and the main characters of the translational machinery, such as the elongation factor 1A (eEF1A), which has been recognized in recent years as one of the most interesting putative oncogenes. A particular emphasis is given to an intriguing non-canonical role that eEF1A can play in the relationship between the Ras→Raf-1→MEK1→ERK-1/2 and PI3K→Akt signaling pathways. Recently, our group has described a C-Raf kinase-mediated phosphorylation of eEF1A triggered by a survival pathway induced upon interferon alpha (IFNα) treatment in the human epidermoid cancer cell line (H1355). This phosphorylation seems to be the center of the survival pathway that counteracts the well-known pro-apoptotic function of IFNα. Furthermore, we have identified two new phosphorylation sites on eEF1A (Ser21 and Thr88) that are substrates for Raf kinases in vitro and, likely, in vivo as well. These residues seem to have a significant functional role in the control of cellular processes, such as cell proliferation and survival. In fact, overexpression of eEF1A2 in gemcitabine-treated cancer cells caused the upregulation of phosphoAkt and an increase in cell viability, thereby suggesting that eEF1A2 could exert its oncogenic behavior by participating in the regulation of PI3K pathway.

B-cell receptor-guided delivery of peptide-siRNA complex for B-cell lymphoma therapy

BackgroundDespite the clinical response of conventional anticancer therapy, including chemotherapeutic treatments, radiation therapy and corticosteroids, tumorigenic B-cell lymphomas show an incomplete response to clinical practices that result in a minimal residual disease (MRD) where few residual neoplastic cells undetected in vivo, replenish the cancer cell reservoir. This scenario, which is also shared with other cancer diseases, requires the development of strategies to advance in novel, selective targeting toward the tumorigenic cells that survive to the anticancer agents.MethodsHere, we have taken advantage of the therapeutic properties of an idiotype specific peptide (pA20-36) that bind specifically to murine B-lymphoma cells in the setting of an anti cancer strategy, based on the selected delivery of electrostatic-based complex, peptide-siRNA. To this end, two engineered, arginine rich, peptides that included the pA20-36 targeting sequence were designed to bind fluorescent-labelled siRNA. One peptide presented 9 Arg at the C-terminal of pA20-36 whereas the other included 5 Arg at the N- and C-terminus, respectively.ResultsCompared to the control and random peptide-siRNA complexes, both pA20-36-siRNA complexes were endowed with the selective delivering of fluorescent-labelled siRNA toward the A20 murine B-cell lymphoma, as evaluated by cytofluorimetry and confocal microscopy, whereas fluorescent-labelled siRNA alone was not internalized in the selected cells. Compared to peptide controls, the use of the modified pA20-36 peptides complexed with siRNA anti-GAPDH and anti-Bcl2 showed a down-regulation in the expression levels of the corresponding genes.ConclusionsPeptide-siRNA complex can be suitable tool for both selective peptide-driven cell targeting and gene silencing. In this setting, the improvement of this strategy is expected to provide a safe and non-invasive approach for the delivery of therapeutic molecules.

Biochemical characterisation of the D60A mutant of the elongation factor 1α from the archaeon Sulfolobus solfataricus

The D60A mutant of the elongation factor (EF) 1alpha from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1alpha; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.