Prospection of the antiviral effect of bufotenine on the rabies virus infection inhibition

Introduction and Objectives: Rabies, an incurable disease, kills over 70,000 people every year. In vitro experiments showed that nicotinic acetylcholine receptor (nAChR) can participate on the entry of rabies virus into cells. Bufotenine, a tryptophan-derived alkaloid found in skin secretion of Brazilian toads of Rhinella genus and plants of Leguminosae family, can inhibit rabies infection in vitro and increase the survival rate of intracerebrally virus-infected mice from 15 to 40%. To glimpse on possible bufotenine mechanisms of action, this study investigated the action of this alkaloid on cell penetration, interaction with nAChR, through liposomes delivery and andphrenic nerve-diaphragm muscle (PND) preparation, as well as a preliminary proteomic study on bufotenine-treated cells. Material and Methods: Liposomes were obtained from a mixture of phospholipids and cholesterol. We have synthesized both positively and negatively charged liposomes. Size exclusion chromatograph, Ultra-Fast Liquid Chromatography and Electrospray-Ion Trap-Time of Flight were performed in order to identify bufotenine inside the liposomes and verify its ability to penetrate membranes without transmembrane proteins. Male Swiss mice (Mus musculus 30g) were used for twitch-tension studies in an organ bath system, with different concentrations of bufotenine (Butantan Ethics Committee number: CEUA 9532050216). For proteomics experiments, neuroblastoma murine cells (N2A) were treated with different concentrations of bufotenine and lyzed for intracellular protein attainment. MS2 spectra were acquired and protein identification was performed. Results and Discussion: Our experiments showed that bufotenine cannot passively penetrate either liposomes, suggesting that cell receptors, or other surface molecules, are necessary to facilitate bufotenine cellular entry. However, in PND preparations, bufotenine did not caused neuromuscular blockade, indicating that this alkaloid does not bind to the nAchR, and the blockage of such receptor is not the mechanism of action of bufotenine, regarding the inhibition of virus infection. Our preliminary proteomic data indicate that cytoskeleton proteins (vimentin, tubulin, actin and tropomyosin) are differently expressed when compared to untreated cells, suggesting that other physiological systems, such as viral retrograde transport, and not only the direct cell penetration virus inhibition, could be involved