Estudo do possível efeito de alcalóides obtidos a partir da secreção cutânea de Rhinella jimi e R. icterica na penetração do vírus da raiva em células de mamífero mediado pelo receptor nicotínico de acetilcolina

Abstract

Rabies is an acute infectious disease caused by a virus that affects the central nervous system. The virus replicates at the inoculation site and it is believed that it uses the nicotinic acetylcholine receptors, located in the neuromuscular junctions to reach the nerve endings. The genus Bufo, recently split into Bufo in the Old World and Rhinella in the New World, contain a large number of alkaloids and steroids in their skin secretion. The aim of this study was to assay molecules extracted from the skin of amphibians as possible interfering agents in the process of infection of the rabies virus in mammalian cells. Bufo (Rhinella sp.) skin secretions were collected through mechanical stimulation. A liquid-liquid partition (H2O-CH2Cl2) was performed and the two resulting solutions were purified by RP-HPLC, in a C18 column. Structural characterization was performed by mass spectrometry. Cytotoxic tests of the isolated compounds were performed over BHK-21 cells. Briefly, 96-well microtiter plates containing the cells were incubated for 24h in the media containing different dilutions of the purified molecules. For the virologic test, fixed strain PV (Pasteur Virus) was used on fluorescence inhibition test and fluorescent foci inhibition test, with both simultaneous and time course treatment of the cells with the virus and the fractions. Sixteen fractions were obtained by RP- PLC. The cytotoxic tests revealed that 9 fractions were toxic to BHK-21cells. On the virologic test, fraction 2 showed a lasting effect, independent from the simultaneous and time course treatments in both tests. Mass spectrometric analyses showed that this fraction contains a steroid named hellebrigenin. This fraction also showed no cytotoxic effect on MTT test. Fraction 14 was able to reduce rabies virus infection in both tests, apparently showing competition effects. Mass spectrometric analyses showed that this fraction contains two indole alkaloids, N`,N`-dimethyl 5-hydroxytryptamione (bufotenine) and N`,N`,N`-trimethyl 5-hydroxytryptamine (5-HTQ). This fraction showed cytotoxic effect on MTT test (66% viable cells). The two fractions also showed effects against CVS (Chalenge Virus Standard), another sample of rabies virus. The two individual components of fraction 14 were tested for biological activity in order to evaluate which retains the biological effect and only bufotenine showed antiviral effects. NMR analyses confirmed the structure of this molecule. Bufotenine was extracted from seeds of Anadenanthera colubrina in acetone solution and purified by RP-HPLC. The CC50 on BHK-21 cells was calculated after MTT test, and Hoechst/PI test indicated that the cytotoxic effect of bufotenine is possibly related with apoptosis. The IC50 of PV virus was calculated after foci inhibition test with dose-response bufotenine concentrations. When tested in N2A cell line, bufotenine was also effective against different samples of rabies virus isolated from infected animals.