RESUMEN
Here we examined the membrane binding and pore formation of amphidinol 3 (AM3) and its truncated synthetic derivatives. Importantly, both of the membrane affinity and pore formation activity were well correlated with the reported antifungal activity. Our data clearly demonstrated that the C1-C30 moiety of AM3 plays essential roles both in sterol recognition and stable pore formation. Based on the current findings, we updated the interacting model between AM3 and sterol, in which the moiety encompassing from C21 to C67 accommodates a sterol molecule with forming hydrogen bonds with the sterol hydroxy group and van der Waals contact between AM3 polyol and sterol skeleton. Although the conformation of the C1-C20 moiety of AM3 is hard to specify due to its flexibility, the region likely contributes to stabilization of pore structure.
Asunto(s)
Anfidinoles , Esteroles , Esteroles/farmacología , Esteroles/química , Alquenos/química , Piranos/químicaRESUMEN
Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 µM, as in the case of methyl-ß-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.