Mode of Antibacterial Action of Tomatidine C3-Diastereoisomers

Tomatidine (TO) is a natural narrow-spectrum antibiotic acting on the small colony variant (SCV) with a minimal inhibitory concentration (MIC) of 0.06 µg/mL while it shows no activity against prototypical strains (MIC > 128 µg/mL). To expand the spectrum of activity of TO, the 3β-hydroxyl group was substituted with an ethane-1,2-diamine, resulting in two diastereoisomers, (C3-β) and (C3-α). These molecules are equally potent against prototypical and strains (MIC 8 and 32 µg/mL, respectively), whereas is more potent against SCV (MIC 0.5 µg/mL) and hyperpermeable strains (MIC 1 µg/mL). The differences in their modes of action were investigated. We used membrane vesicles to confirm the inhibition of the bacterial ATP synthase, the documented target of TO, and measured effects on bacterial cell membranes. Both molecules inhibited ATP synthase, with K values of 1.1 µM and 3.5 µM for and , respectively, and the bactericidal effect of was linked to ATP synthase inhibition. Furthermore, had no major effect on the membrane fluidity and gradually reduced membrane potential. In contrast, caused structural damages to membranes and completely disrupted the membrane potential (>90%). We were successful in broadening the spectrum of activity of TO. C3-β-diastereoisomers may have more specific antibacterial action than C3-α.