Daptomycin is a highly efficient last-resort antibiotic that targets the bacterial cell membrane. Despite its clinical importance, the exact mechanism by which daptomycin kills bacteria is not fully understood. Different experiments have led to different models, including (i) blockage of cell wall synthesis, (ii) membrane pore formation, and (iii) the generation of altered membrane curvature leading to aberrant recruitment of proteins. To determine which model is correct, we carried out a comprehensive mode-of-action study using the model organism Bacillus subtilis and different assays, including proteomics, ionomics, and fluorescence light microscopy. We found that daptomycin causes a gradual decrease in membrane potential but does not form discrete membrane pores. Although we found no evidence for altered membrane curvature, we confirmed that daptomycin inhibits cell wall synthesis. Interestingly, using different fluorescent lipid probes, we showed that binding of daptomycin led to a drastic rearrangement of fluid lipid domains, affecting overall membrane fluidity. Importantly, these changes resulted in the rapid detachment of the membrane-associated lipid II synthase MurG and the phospholipid synthase PlsX. Both proteins preferentially colocalize with fluid membrane microdomains. Delocalization of these proteins presumably is a key reason why daptomycin blocks cell wall synthesis. Finally, clustering of fluid lipids by daptomycin likely causes hydrophobic mismatches between fluid and more rigid membrane areas. This mismatch can facilitate proton leakage and may explain the gradual membrane depolarization observed with daptomycin. Targeting of fluid lipid domains has not been described before for antibiotics and adds another dimension to our understanding of membrane-active antibiotics.antibiotics | daptomycin | membrane potential | cell wall biosynthesis | Bacillus subtilis

dMersacidin, gallidermin, and nisin are lantibiotics, antimicrobial peptides containing lanthionine. They show potent antibacterial activity. All three interfere with cell wall biosynthesis by binding lipid II, but they display different levels of interaction with the cytoplasmic membrane. On one end of the spectrum, mersacidin interferes with cell wall biosynthesis by binding lipid II without integrating into bacterial membranes. On the other end of the spectrum, nisin readily integrates into membranes, where it forms large pores. It destroys the membrane potential and causes leakage of nutrients and ions. Gallidermin, in an intermediate position, also readily integrates into membranes. However, pore formation occurs only in some bacteria and depends on membrane composition. In this study, we investigated the impact of nisin, gallidermin, and mersacidin on cell wall integrity, membrane pore formation, and membrane depolarization in Bacillus subtilis. The impact of the lantibiotics on the cell envelope was correlated to the proteomic response they elicit in B. subtilis. By drawing on a proteomic response library, including other envelope-targeting antibiotics such as bacitracin, vancomycin, gramicidin S, or valinomycin, YtrE could be identified as the most reliable marker protein for interfering with membrane-bound steps of cell wall biosynthesis. NadE and PspA were identified as markers for antibiotics interacting with the cytoplasmic membrane.

Background: NAI-107 is a potent lantibiotic with an unknown mode of action. Results: NAI-107 targets bactoprenol-bound cell envelope precursors, e.g. lipid II, and in addition affects the bacterial membrane. Conclusion: Cell wall biosynthesis is blocked by sequestration of lipid II and functional disorganization of the cell wall machinery. Significance: The dual mechanism of action may explain the potency of NAI-107 and related lantibiotics.

[(Pyridylmethyl)sulfinyl]benzimidazoles 1 (PSBs) are a class of highly potent antisecretory (H+,K+)-ATPase inhibitors which need to be activated by acid to form their active principle, the cyclic sulfenamide 4. Selective inhibitors of the (H+,K+)-ATPase in vivo give rise to the nonselective thiophile 4 solely at low pH, thus avoiding interaction with other thiol groups in the body. The propensity to undergo the acid-catalyzed transformation is dependent on the nucleophilic/electrophilic properties of the functional groups involved in the formation of 2 since this step is both rate-determining and pH-dependent. The aim of this study was to identify compounds with high (H+,K+)-ATPase inhibitory activity in stimulated gastric glands possessing acidic pH, but low reactivity (high chemical stability) at neutral pH as reflected by in vitro (Na+,K+)-ATPase inhibitory activity. The critical influence of substituents flanking the pyridine 4-methoxy substituent present in all derivatives was carefully studied. The introduction of a 3-methoxy group gave inhibitors possessing a combination of high potency, similar to omeprazole and lansoprazole, but increased stability. As a result of these studies, compound 1a (INN pantoprazole) was selected as a candidate drug and is currently undergoing phase III clinical studies.

SUMMARY Inhibition of the gastric proton pump is gaining acceptance as the treatment of choice for severe gastrooesophageal reflux disease, and for treatment of duodenal and gastric ulceration. Three of these drugs are now available (omeprazole, lansoprazole and pantoprazole) and more are being developed. Proton pump inhibitors share the same core structure, but differ in terms of substituents on this core. The substitutions are able to modify some important chemical properties of the compounds. For example, pantoprazole is significantly more acid‐stable than omeprazole or lansoprazole. E3810 is significantly less stable than the other compounds. We present an explanation for this finding that depends on the relative pK values for the pyridine and benzimidazole nitrogens, especially the former. Pantoprazole formulated in an enteric‐coated tablet displays high bioavailability and linear pharmacokinetics whether on single or multiple dose regimens. Although all three proton pump inhibitors provide a similar chemical conversion to sulphenamides, which are highly reactive cysteine reagents, these reagents derivatize different cysteines in the extracytoplasmic or membrane domain of the pump and inhibit the pump at different rates. Whereas the differences in chemical reactivity can be explained by the solution chemistry of the compounds, selective derivatization of different cysteines on the protein argues for an involvement of pump structure in response to the presence of the proton pump inhibitor on its luminal surface. This suggests that the proton pump inhibitors, which were originally designed to take advantage of only the highly acidic space generated in the parietal cell by the production of the sulphenamide, are made even more selective by the protein they target. Pantoprazole is metabolized by a combination of phase I and phase II metabolism, and has also been shown to have a very low potential for drug interaction. Studies of acid secretion in man have shown this compound to be an effective and long lasting inhibitor of acid secretion. The pharmacodynamics explain the cumulative effect of repeated doses and maximal acid secretory capacity with a once daily dosage.