Daptomycin and A54145 are homologous lipopeptide antibiotics that permeabilize the cell membranes of Gram-positive bacteria. Membrane permeabilization depends on the presence of both phosphatidylglycerol (PG) and calcium, and it involves the formation of oligomeric transmembrane pores that consist of approximately 6-8 subunits. We here show that each lipopeptide molecule binds two calcium ions in separable, successive steps. The first calcium ion causes the lipopeptide molecule to bind to the target membrane, and likely to form a loosely associated oligomer. Higher calcium concentrations induce binding of a second ion, which produces the more tightly associated and more deeply membrane-inserted final, functional form of the oligomer. Both calcium-dependent steps are accompanied by fluorescence signals that indicate transition of specific amino acid residues into less polar environments, suggestive of insertion into the target membrane. Our findings agree with the earlier observation that two of the four acidic amino acid residues in the daptomycin molecule are essential for antibacterial activity.

α-Azido acids have been used in solid phase peptide synthesis (SPPS) for almost 20 years. Here we report that peptides bearing an N-terminal α-azidoaspartate residue undergo elimination of an azide ion when treated with reagents that are commonly used for removing the Fmoc group during SPPS. We also report an alternative solid-phase route to the synthesis of an analog of daptomycin that uses a reduced number of α-azido amino acids and without elimination of an azide ion.

Eighty-four cancer patients at risk of infection because of neutropenia were randomized to receive nalidixic acid as an alternative to trimethoprim-sulfamethoxazole (TMP-SMX) for infection prophylaxis. Infections were documented significantly earlier and more often among patients who entered the trial with neutrophil counts of less than 0.1 X 10(9)/liter. TMP-SMX recipients experienced fewer microbiologically documented infections and bacteremias and were free of infection for a higher proportion of days with severe neutropenia (less than 0.1 X 10(9)/liter) than nalidixic acid recipients. Gram-negative bacillary and Staphylococcus aureus infections accounted for the major differences. Although the majority of aerobic gram-negative bacilli were eliminated from the feces after 1 week of prophylaxis with either agent, TMP-SMX was proved superior to nalidixic acid in this regard and was associated with acquired drug resistance by gram-negative bacilli less frequently. Both agents selected for colonization and subsequent infection by gram-positive cocci. Our data suggest that prophylaxis is most likely to be effective if administered to patients for at least 1 week before they become severely neutropenic. Nalidixic acid used as a single agent in doses of 4 g daily, however, cannot be recommended as an alternative to TMP-SMX for infection prophylaxis in neutropenic cancer patients.

The lipopeptide antibiotic daptomycin is active against Gram-positive pathogens. It permeabilizes bacterial cell membranes, which involves the formation of membrane-associated oligomers. We here studied a dimer of daptomycin whose two subunits were linked through a bivalent aliphatic acyl chain. Unexpectedly, the dimer had very low activity on vegetative Staphylococcus aureus and Bacillus subtilis cells. However, activity resembled that of monomeric daptomycin on liposomes and on B. subtilis L-forms. These findings underscore the importance of the bacterial cell wall in daptomycin resistance. Keywords lipopeptide antibiotics, antibiotic resistance, cell wall permeability, bacterial L-formsThis document is the unedited Author's version of a Submitted Work that was subsequently accepted for publication in ACS Infectious Diseases,© American Chemical Society after peer review. To access the final edited and published work see http://pubs.acs.org/doi/full/10.1021/acsinfecdis.7b00019.Daptomycin is a calcium-dependent lipopeptide antibiotic that depolarizes the cell membranes of Gram-positive bacteria (1, 2). In previous studies, we have shown that permeabilization involves the formation of daptomycin oligomers on target cell membranes (3,4). Oligomer formation has an entropy cost, and we reasoned that lowering this cost by joining two daptomycin molecules together might produce a derivative with greater antimicrobial activity. Within the daptomycin oligomer, the fatty acyl tails of adjoining subunits are in close proximity (5, 6),suggesting that joining them covalently should not impose any unfavourable steric constraints on oligomer formation; we thus used a bivalent fatty acyl moiety to effect dimerization (see Figure 1).The dimer was then tested for antibacterial activity against several strains of Staphylococcus aureus and Bacillus subtilis. Unexpectedly, the MIC was higher than that of the monomer with all tested strains by one to two orders of magnitude (see Tables 1 and 2).Figure 1: Structure of the semisynthetic daptomycin dimer characterized in this study. Each half of the molecule corresponds to a native daptomycin monomer. Details on the synthetic procedure and on the characterization are given in the Supplementary Materials. Several explanations might conceivably account for the marked reduction in activity. Firstly, the steric constraints introduced by dimerization might disrupt the membrane-permeabilizing activity. To test this possibility, we tested the dimer in a liposome model that recapitulates both the oligomer formation (7) and the membrane permeabilization (8) which are also observed in bacterial cells (1,(3)(4)(5) 9). Membrane binding and oligomerization is accompanied by a large increase in the fluorescence intensity of daptomycin's intrinsic kynurenine residue (10, 11). The fluorescence increase occurs in a virtually indistinguishable manner with the monomer and the dimer ( Figure 2A). Similarly, when phosphatidylglycerol was omitted from the liposomes, both the monomer (3) and the dime...