Studio dell’attività antivirale e/o anti-infiammatoria di nuove molecole di sintesi e/o di farmaci noti

This thesis collects the work I have done during the three-year of my PhD Course. During my first year I have started a path that has allowed me to acquire different techniques to set up and culture animal cell lines as well as to evaluate the cytotoxic and anti-picornavirus activities of a class of novel synthetic quinoxaline (Part I). Part of the second and all the third year was spent at the NIAID/NIH, Hamiltion MT, USA, in the laboratory of persistent viral diseases under the supervision of dr. Bruce Chesebro investigating the effect of statins for the control of neuroinflammation (Part II). Part I - Antiviral activity of quinoxalines derivates as new leads against Picornavirus Aim: New quinoxaline derivatives were synthesized and tested for cytotoxicity and antiviral activity against human coxsackie virus B5 [CVB-5] and polio virus type-1 [Sb-1], focusing on the mode of action of the more potent compound Ethyl 4-[(2,3-dimethoxyquinoxalin-6- yl)methylthio]benzoate (7a). Methods:Virus multiplication in Vero76 cells were monitored by plaque reduction assay. Time of addition assays were performed to determine the influence of the time of treatment on the antiviral activity. Initial steps of virus infection were dissected into adsorption and penetration steps by switching the incubation temperature from 4°C to 37°C. Results: Four compounds (7a, 7b, 8a, 8b) showed a very potent antiviral activity (EC50s range 0.06-3.8 μM) against CVB-5. Compound 7a resulted the most active and selective derivative (SI >1000). Time of addition assays revealed that compound 7a interfered with the penetration/uncoating stage of the virus cycle. Conclusion: Further experiments to confirm the mechanism of action of the compoundare ongoing. Part II - In vitro studies on the anti-inflammatory activity of statins during neuroinflammation Aim: Due to the known pleiotropic activity of statins, Four different types of statins were investigated for their effect on the production of pro-inflammatory cytokines (IL-6 and CXCL10) in mouse and rat cortical microglia and astrocytes primary cultures. Methods: Primary cell cultures were obtained from 2 days old mice and Sprague-Dawley rats. The purity of the cell cultures was verified by immunofluorescence staining. The production of pro-inflammatory cytokines was stimulated by the mitogen LPS. The amount of cytokines produced in the absence and in the presence of statins was determined by using an extracellular protein kit specific for the cytokines analized. An MTT assay was performed in order to verify the viability of the cell cultures and to exclude a possible stimulation by statins themselves. Results: No LPS/stimulation and no statin/ cytokine inhibition was obtained in rat cells, while in mice derived cells, there was a good LPS stimulation and three out of the four statins inhibited of the production of IL-6 by mouse microglia. Conclusion: Statins seems to have a good anti-inflammatory activity on mouse cells, however, further studies should be performed in order to confirm these results. In rat cells, further studies should be performed in order to find better conditions of stimulation. In vivo testing of the effect of Atorvastatin and Simvastatin in mice affected by scrapie are ongoing.