Gabriel Siracusano

The Virion Host Shutoff RNase Plays a Key Role in Blocking the Activation of Protein Kinase R in Cells Infected with Herpes Simplex Virus 1

ABSTRACT Earlier studies have shown that active MEK blocks the activation of protein kinase R (PKR), a component of antiviral innate immune responses. In this report we show that the herpes simplex virus 1 virion host shutoff (VHS) RNase protein and MEK (mitogen-activated protein kinase kinase) act cooperatively in blocking the activation of PKR. This conclusion is based on the following. (i) In contrast to viral gene expression in the parental cell line or a cell line expressing a constitutively active MEK, the replication of a VHS mutant is particularly impaired in cells expressing dominant negative MEK. In this cell line PKR is activated by phosphorylation, and the accumulation of several viral proteins is delayed. (ii) In transfected cells, wild-type VHS blocked the activation of PKR, whereas PKR was activated in cells transfected with a mutant VHS or with plasmids encoding the VHS RNase and VP16 and VP22, the two viral proteins that neutralize the RNase activity of VHS. The results suggest that early in infection the VHS RNase degrades RNAs that activate PKR. Coupled with published data, the results suggest that inhibition of activation of PKR or its effect on viral replication is staged early in infection by VHS, postsynthesis of VP16 and VP22 by the γ134.5 protein, and very late in infection by the US11 protein.

Nanoassembly of an amphiphilic cyclodextrin and Zn(II)-phthalocyanine with the potential for photodynamic therapy of cancer

Due to their poor solubility and propensity to aggregate in aqueous media, therapeutic application of several photosensiting agents, such as phthalocyanines, in photodynamic therapy (PDT) of solid tumors is severely hampered. With the aim to propose a novel nanotechnological approach, in this paper biodegradable nanoassemblies based on heptakis (2-oligo(ethyleneoxide)-6-hexadecylthio-)-β-CD (SC16OH) and zinc-phthalocyanine (ZnPc) were developed and tested. Nanoassemblies, prepared by the emulsion–solvent evaporation technique, displayed a hydrodynamic diameter around 200 nm, a negative zeta potential and a satisfactory entrapment efficiency of ZnPc. Steady-state and time resolved fluorescence emission spectroscopy studies showed the entrapment of ZnPc as a monomer in the carrier, with a low tendency to self-aggregate and consequently a fairly good propensity to generate singlet oxygen after photoactivation. The interaction of ZnPc with SC16OH was elucidated by 1H-NMR, which suggested the formation of complexes between drug and both hydrophobic and hydrophilic moieties of the amphiphile. Finally, in vitro potential of the nanoassembly was evaluated in HeLa cells by following cellular uptake and photobiological activity. Overall, results suggest the suitability of the nanoassembly based on SC16OH for delivering ZnPc to cancer cells, thus inducing photodynamic anticancer effects.

Light-Activated Release of Nitric Oxide with Fluorescence Reporting in Living Cells

The rapid delivery of biologically relevant species with precise spatiotemporal control is a hot topic in life science. Light is the most elegant on/off trigger to fulfill these criteria. In fact, the fast response of the photochemical reactions and their instantaneous initiation/stopping depending on the presence or absence of the illumination, represent a powerful tool for the introduction of given amounts of “photocaged” chemical entities in biological systems in a noninvasive way. In addition, the use of photons as external input offers the additional advantage of not affecting important physiological parameters, such as pH, temperature and ionic strength, which is a critical condition for bio-applications. Quantification of the delivery upon uncaging of the chemical entity is another important issue to be faced. One possibility to address this task is to use a fluorescent reporter. This elegant strategy relies on the simultaneous photorelease of the desired caged bioactive species and a fluorescent component from the same nonfluorescent caged precursor. In this way, the uncaging process can be quantified by monitoring the fluorescence emission of the reporter and, furthermore, the spatial distribution of the released species in a biological environment can be followed in real time by fluorescence microscopy. Nitric oxide (NO) is one of the most appealing and well-studied molecules in biomedical sciences. This inorganic radical is a biological mediator in many physiological processes, including neurotransmission, hormone secretion and vasodilatation. Furthermore, NO has proven to reduce radical-mediated oxidative processes, exert cytotoxic effects against infective microorganisms, and inhibit tumor growth. This multifaceted role has stimulated a massive interest in developing NO-releasing systems with the prospect to tackle important diseases. However, the biological effects of this radical have been shown to be highly site, concentration and dosage dependent, creating a complex role for the molecule in opposing beneficial and deleterious events. This dichotomy has made NO-photoreleasing compounds much more desirable than those based on spontaneous thermolysis as demonstrated by the increasing number of papers on NO photodonors with improved photophysical and photochemical characteristics appearing in recent years. Conversely, NO uncaging quantification based on fluorescence reporting has attracted very little attention. As a part of our ongoing interest in developing functional light-controlled NO delivering systems, we recently reported the design and synthesis of molecular conjugate 1 (Scheme 1). It integrates two chromogenic centers within the same covalent skeleton: an anthracene moiety (unit a) and a nitroaniline derivative that we have recently discovered to be a suitable NO photodonor (unit b). This compound was devised in such a way to amplify the NO photorelease from unit b through an effective photoinduced energy transfer a*! b. As a result, the typical emission of the anthracene fluorophore is completely suppressed, making conjugate 1 intrinsically nonfluorescent. Herein, we report novel and intriguing properties of compound 1. In particular, we demonstrate that NO photo-uncaging: 1) leads to the formation of a strongly fluorescent coproduct that can be used as an optical reporter for NO delivery; 2) induces a remarkable level of mortality in cancer cell. The absorption spectrum of 1 shows the distinctive spectral features of the anthracene chromophore below 400 nm and an intense absorption at longer wavelengths extending beyond 450 nm, due to the contribution of the nitroaniline moiety (a in Figure 1 a). These spectral features allow the NO photodonor unit to be selectively excited at lexc>400 nm. Changes in the electronic absorption spectrum observed upon irradiation of a solution of 1 at lexc = 420 nm are strictly related to the photorelease of NO from the unit b (Figure 1 a). A typical NO-release profile, obtained by the direct amperometric detection of this species alternating cycles of light/dark is shown in the inset of Figure 1 a, for the sake of clarity. The photobleaching observed is in line with the photochemical pathway leading to NO release previously proposed in the case of the single NO photodonor unit b. This mechanism involves a nitro-to-nitrite photo-rearrangement followed by the simultaneous release of NO and a phenoxy radical. Eventually, this intermediate generates a phenol derivative as the main stable photoproduct that, in our case, does not absorb in the visible region. Phenol derivatives as the main stable photoproducts have also been reported in the case of other nitroaromatics, for which a NO release mechanism similar to the chromophoric center b applies. Electrospray ionization (ESI) mass spectrometric analysis of the crude reaction mixture, performed immediately after the photolysis experiments (~20 % transformation), validates this proposal ; a main peak with an m/z value of 425.2 corresponding to [M + 1] of the conjugate 2 (see [a] Dr. E. Vittorino, Prof. S. Sortino Laboratory of Photochemistry, Department of Drug Sciences University of Catania, Viale Andrea Doria 6, 95125 Catania (Italy) Fax: (+ 39) 095-580-138 E-mail : ssortino@unict.it [b] Dr. M. T. Sciortino, Dr. G. Siracusano Dipartimento di Scienze della Vita, Sezione di Scienze MicrobiologicheGenetiche e Molecolari, Universit di Messina Salita Sperone, 98166 Messina (Italy)

Nanoassemblies based on non-ionic amphiphilic cyclodextrin hosting Zn(II)-phthalocyanine and docetaxel: Design, physicochemical properties and intracellular effects.

The combination of conventional anticancer therapy with other treatment modalities such as photodynamic therapy (PDT) is paving the way to novel more effective treatment of solid tumors via light exposure. With this idea in mind, in this paper, nanoparticles (NPs) based on Heptakis (2-oligo(ethyleneoxide)-6-hexadecylthio-)-β-CD (SC16OH) for dual delivery of Zinc-Phthalocyanine (ZnPc) and Docetaxel (DTX) were developed pointing to their potential application as nanomedicine for the combined photodynamic and chemo-therapy of solid tumors. NPs prepared by the emulsion-solvent evaporation technique displayed a hydrodynamic diameter of ≅ 200nm, a negative zeta potential (≅ -27mV) and a satisfactory entrapment efficiency of both drugs at a specific mass ratio. On these bases, NPs containing DTX and ZnPc with theoretical loading of 5% and 0.2% respectively (ZnPc/DTX5-NPs) were selected for further investigations. The allocation of ZnPc and DTX into the colloid was investigated by complementary spectroscopic techniques. In particular, fluorescence emission studies showed the entrapment of ZnPc as a monomer in the carrier, with a low tendency to self-aggregate and consequently a fairly high propensity to photogenerate singlet oxygen. The interaction of SC16OH with DTX, co-entrapped with ZnPc, was elucidated by (1)H NMR and 2D ROESY, which suggested the presence of the chemotherapeutic in the hydrophobic portion of SC16OH. ZnPc/DTX5-NPs were fairly stable in different biological relevant media within 24h. Finally, in vitro potential of the nanoassembly was evaluated in HeLa cancer cells by cell viability exploring both effects of DTX and ZnPc. Overall, results suggest the suitability of NPs based on SC16OH for delivering a combination of DTX with ZnPc to cancer cells, thus inducing photodynamic and antimitotic effects.

Early activation of MyD88-mediated autophagy sustains HSV-1 replication in human monocytic THP-1 cells

Autophagy is a cellular degradation pathway that exerts numerous functions in vital biological processes. Among these, it contributes to both innate and adaptive immunity. On the other hand, pathogens have evolved strategies to manipulate autophagy for their own advantage. By monitoring autophagic markers, we showed that HSV-1 transiently induced autophagosome formation during early times of the infection of monocytic THP-1 cells and human monocytes. Autophagy is induced in THP-1 cells by a mechanism independent of viral gene expression or viral DNA accumulation. We found that the MyD88 signaling pathway is required for HSV-1-mediated autophagy, and it is linked to the toll-like receptor 2 (TLR2). Interestingly, autophagy inhibition by pharmacological modulators or siRNA knockdown impaired viral replication in both THP-1 cells and human monocytes, suggest that the virus exploits the autophagic machinery to its own benefit in these cells. Taken together, these findings indicate that the early autophagic response induced by HSV-1 exerts a proviral role, improving viral production in a semi-permissive model such as THP-1 cells and human monocytes.

ERK1-Based Pathway as a New Selective Mechanism To Modulate CCR5 with Natural Antibodies

Natural human Abs, recognizing an epitope within the first extramembrane loop of CCR5 (the main HIV coreceptor), induce a long-lasting internalization (48 h) of the protein, whereas all known CCR5 modulating molecules show a short-term kinetics (60–90 min). Despite extensive studies on the regulation of CCR5 signaling cascades, which are the effect of concomitant CCR5 internalization by exogenous stimuli such as Abs, downstream signaling continues to be poorly understood. In this article, we report a hitherto unrecognized mechanism of CCR5 modulation mediated by G protein–dependent ERK1 activity. We further demonstrate that ERK1 is localized mainly in the cytoplasmic compartment and that it interacts directly with the CCR5 protein, thus provoking possible CCR5 degradation with a subsequent de novo synthesis, and that re-expression of CCR5 on the cell membrane required several days. In contrast, the RANTES treatment induces a recovery of the receptor on the cell membrane in short-term kinetics without the involvement of de novo protein synthesis. The said new pathway could be relevant not only to better understand the molecular basis of all pathologic conditions in which CCR5 is involved but also to generate new tools to block viral infections, such as the use of recombinant Abs.

Decorated 6,6′,7,7′-tetrahydro-1H,1′H-2,3′-biindole scaffold as promising candidate for recognition of the CDK2 allosteric site

Progression through the S phase of the cell cycle is controlled by cyclin-dependent kinase 2 (CDK2), the activity of which depends on its binding to regulatory partners (cyclins E and A). Deregulation of the activity of CDK2 has been associated with several types of sickness, such as infectious, neurodegenerative, and proliferative diseases. Based on these data, CDK2 has become an attractive target for the development of new anticancer drugs. Indoledione derivatives have recently received special attention in virtue of their pronounced biological effects, such as antiproliferative, antioxidant and antimicrobial properties. In the present work we have investigated the antiproliferative effect of an indolone-based derivative, namely DPIT, whose synthesis we have recently reported, with the aim to clarify its mechanism of action. Furthermore, docking studies have been performed on the eight stereoisomers of DPIT to investigate their capacity to interact as a ligand with ortho- or allosteric sites of CDK2. The encouraging results showed DPIT as a promising candidate for a new type 3 class of inhibitors of CDK2 that recognize the allosteric site.

The Abrogation of Phosphorylation Plays a Relevant Role in the CCR5 Signalosome Formation with Natural Antibodies to CCR5

The exposure to CCR5 (CC chemokine receptor 5) specific natural antibodies in vitro produces a Class B β-arrestin2-dependent CCR5 retention with the aid of ERK1, due to the formation of a CCR5 signalosome, which remains stable for at least 48 h. Considering that β-arrestins and MAPKs are receptive to environmental signals, their signal complexes could be one of the key junction for GPCRs internalization related signal transduction. Here, we demonstrate that, in T cells, the phosphorylation status of either CCR5 receptor or ERK1 protein is necessary to drive the internalized receptor into the early endosomes, forming the CCR5 signalosome. In particular, our data show that β-arrestin2/ERK1 complex is a relevant transducer in the CCR5 signaling pathway. Understanding the mechanism of CCR5 regulation is essential for many inflammatory disorders, tumorigenesis and viral infection such as HIV.