Perspective on Antibacterial Lead Identification Challenges and the Role of Hypothesis-Driven Strategies.

For the past three decades, the pharmaceutical industry has undertaken many diverse approaches to discover novel antibiotics, with limited success. We have witnessed and personally experienced many mistakes, hurdles, and dead ends that have derailed projects and discouraged scientists and business leaders. Of the many factors that affect the outcomes of screening campaigns, a lack of understanding of the properties that drive efflux and permeability requirements across species has been a major barrier for advancing hits to leads. Hits that possess bacterial spectrum have seldom also possessed druglike properties required for developability and safety. Persistence in solving these two key barriers is necessary for the reinvestment into discovering antibacterial agents. This perspective narrates our experience in antibacterial discovery-our lessons learned about antibacterial challenges as well as best practices for screening strategies. One of the tenets that guides us is that drug discovery is a hypothesis-driven science. Application of this principle, at all steps in the antibacterial discovery process, should improve decision making and possibly the odds of what has become, in recent decades, an increasingly challenging endeavor with dwindling success rates.

Surotomycin demonstrates low in vitro frequency of resistance and rapid bactericidal activity in Clostridium difficile, Enterococcus faecalis, and Enterococcus faecium.

Surotomycin (CB-183,315) is an orally administered, minimally absorbed, selective bactericidal cyclic lipopeptide in phase 3 development for the treatment of Clostridium difficile-associated diarrhea. The aim of this study was to evaluate the emergence of resistance in C. difficile (ATCC 700057 and three recent clinical isolates from the restriction endonuclease analysis groups BI, BK, and K), vancomycin-susceptible (VS) Enterococcus faecalis (ATCC 49452), vancomycin-resistant (VR) E. faecalis (ATCC 700802), VS Enterococcus faecium (ATCC 6569), and VR E. faecium (ATCC 51559) under anaerobic conditions. The rate of spontaneous resistance was below the limit of detection (<10(-8) to <10(-9)) for surotomycin at 16 and 32× the MIC for all isolates tested. Under selective pressure by serial passage, C. difficile grew in a maximum of 4 µg/ml surotomycin (final MICs of 2 to 8 µg/ml [4- to 16-fold higher than those of the naive control]) at day 15, with the exception of the C. difficile BK strain, which grew in 16 to 32 µg/ml (final MICs of 8 to 32 µg/ml [16- to 64-fold higher than those of the naive control]). Enterococci remained relatively unchanged over 15 days, growing in a maximum of 8 µg/ml surotomycin (final MICs of 2 to 16 µg/ml [8- to 64-fold higher than those of the naive control]). Of the isolates tested, no cross-resistance to vancomycin, rifampin, ampicillin, metronidazole, or moxifloxacin was observed. Surotomycin at 20× MIC demonstrated equally rapid bactericidal activity (≥ 3-log-unit reduction in CFU/ml in ≤ 8 h) against naive and reduced-susceptibility isolates of C. difficile, VS Enterococcus (VSE), and VR Enterococcus (VRE), except for C. difficile BK (2.6-log-unit reductions for both). These results suggest that emergence of resistance to surotomycin against C. difficile, E. faecalis, and E. faecium is likely to be rare.

Additional routes to Staphylococcus aureus daptomycin resistance as revealed by comparative genome sequencing, transcriptional profiling, and phenotypic studies.

Daptomycin is an extensively used anti-staphylococcal agent due to the rise in methicillin-resistant Staphylococcus aureus, but the mechanism(s) of resistance is poorly understood. Comparative genome sequencing, transcriptomics, ultrastructure, and cell envelope studies were carried out on two relatively higher level (4 and 8 µg/ml(-1)) laboratory-derived daptomycin-resistant strains (strains CB1541 and CB1540 respectively) compared to their parent strain (CB1118; MW2). Several mutations were found in the strains. Both strains had the same mutations in the two-component system genes walK and agrA. In strain CB1540 mutations were also detected in the ribose phosphate pyrophosphokinase (prs) and polyribonucleotide nucleotidyltransferase genes (pnpA), a hypothetical protein gene, and in an intergenic region. In strain CB1541 there were mutations in clpP, an ATP-dependent protease, and two different hypothetical protein genes. The strain CB1540 transcriptome was characterized by upregulation of cap (capsule) operon genes, genes involved in the accumulation of the compatible solute glycine betaine, ure genes of the urease operon, and mscL encoding a mechanosensitive chanel. Downregulated genes included smpB, femAB and femH involved in the formation of the pentaglycine interpeptide bridge, genes involved in protein synthesis and fermentation, and spa encoding protein A. Genes altered in their expression common to both transcriptomes included some involved in glycine betaine accumulation, mscL, ure genes, femH, spa and smpB. However, the CB1541 transcriptome was further characterized by upregulation of various heat shock chaperone and protease genes, consistent with a mutation in clpP, and lytM and sceD. Both strains showed slow growth, and strongly decreased autolytic activity that appeared to be mainly due to decreased autolysin production. In contrast to previous common findings, we did not find any mutations in phospholipid biosynthesis genes, and it appears there are multiple pathways to and factors in daptomycin resistance.

Reduced pulmonary surfactant interaction of daptomycin analogs via tryptophan replacement with alternative amino acids.

Daptomycin was shown to interact in vitro with pulmonary surfactant leading to reduction of its antibacterial activity. We report herein the preparation and anti-staphylococcal activity of a series of daptomycin analogs with reduced pulmonary surfactant interaction by replacing tryptophan with various amino acids.

In vitro and in vivo characterization of CB-183,315, a novel lipopeptide antibiotic for treatment of Clostridium difficile.

CB-183,315 is a novel lipopeptide antibiotic structurally related to daptomycin currently in phase 3 clinical development for Clostridium difficile-associated diarrhea (CDAD). We report here the in vitro mechanism of action, spontaneous resistance incidence, resistance by serial passage, time-kill kinetics, postantibiotic effect, and efficacy of CB-183,315 in a hamster model of lethal infection. In vitro data showed that CB-183,315 dissipated the membrane potential of Staphylococcus aureus without inducing changes in membrane permeability to small molecules. The rate of spontaneous resistance to CB-183,315 at 8× the MIC was below the limit of detection in C. difficile. Under selective pressure by serial passage with CB-183,315 against C. difficile, the susceptibility of the bacteria changed no more than 2-fold during 15 days of serial passages. At 16× the MIC, CB-183,315 produced a ≥3-log reduction of C. difficile in the time-kill assay. The postantibiotic effect of CB-183,315 at 8× the MIC was 0.9 h. At 80× the MIC the postantibiotic effect was more than 6 h. In the hamster model of CDAD, CB-183,315 and vancomycin both demonstrated potent efficacy in resolving initial disease onset, even at very low doses. After the conclusion of dosing, CB-183,315 and vancomycin showed a similar dose- and time-dependent pattern with respect to rates of CDAD recurrence.

Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins.

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.

LC-MS/MS characterization of phospholipid content in daptomycin-susceptible and -resistant isolates of Staphylococcus aureus with mutations in mprF.

Daptomycin (DAP) is a cyclic lipopeptide antibiotic used for the treatment of certain Staphylococcus aureus infections. Although rare, strains have been isolated that are DAP resistant. These strains usually have mutations in mprF, a gene encoding a membrane protein with both lysylphosphatidylglycerol (LPG) synthase and flippase activities. Because ΔmprF strains have increased DAP susceptibility, the mechanism of resistance is not likely due to a loss of mprF function. In this study, we developed an LC-MS assay to examine the effect of different mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in the membrane. Our assay demonstrated that some, but not all, mutations in the flippase and synthase domains result in small but reproducible increases in the proportion of LPG relative to PG. Techniques described herein represent a higher throughput and more sensitive method for measuring relative phospholipids levels. These results offer guidance in the understanding of how mprF confers DAP resistance; namely, mprF-mediated resistance may be through more than one mechanism, including increased overall LPG synthesis and increased LPG present on the outer leaflet of the cytoplasmic membrane.

VraSR two-component regulatory system contributes to mprF-mediated decreased susceptibility to daptomycin in in vivo-selected clinical strains of methicillin-resistant Staphylococcus aureus.

Daptomycin (DAP) is a new class of cyclic lipopeptide antibiotic highly active against methicillin-resistant Staphylococcus aureus (MRSA) infections. Proposed mechanisms involve disruption of the functional integrity of the bacterial membrane in a Ca-dependent manner. In the present work, we investigated the molecular basis of DAP resistance in a group of isogenic MRSA clinical strains obtained from patients with S. aureus infections after treatment with DAP. Different point mutations were found in the mprF gene in DAP-resistant (DR) strains. Investigation of the mprF L826F mutation in DR strains was accomplished by inactivation and transcomplementation of either full-length wild-type or mutated mprF in DAP-susceptible (DS) strains, revealing that they were mechanistically linked to the DR phenotype. However, our data suggested that mprF was not the only factor determining the resistance to DAP. Differential gene expression analysis showed upregulation of the two-component regulatory system vraSR. Inactivation of vraSR resulted in increased DAP susceptibility, while complementation of vraSR mutant strains restored DAP resistance to levels comparable to those observed in the corresponding DR wild-type strain. Electron microscopy analysis showed a thicker cell wall in DR CB5012 than DS CB5011, an effect that was related to the impact of vraSR and mprF mutations in the cell wall. Moreover, overexpression of vraSR in DS strains resulted in both increased resistance to DAP and decreased resistance to oxacillin, similar to the phenotype observed in DR strains. These results support the suggestion that, in addition to mutations in mprF, vraSR contributes to DAP resistance in the present group of clinical strains.

Evaluating the activity of the RNA polymerase inhibitor myxopyronin B against Staphylococcus aureus.

Myxopyronin B (MyxB) binds to the switch region of RNA polymerase (RNAP) and inhibits transcriptional initiation. To evaluate the potential development of MyxB as a novel class of antibiotic, we characterized the antimicrobial activity of MyxB against Staphylococcus aureus. Spontaneous MyxB resistance in S. aureus occurred at a frequency of 8 × 10(-8) , similar to that of rifampin. The MyxB-resistant mutants were found to be altered in single amino acid residues in the RNAP subunits that form the MyxB-binding site. In the presence of human serum albumin, the MyxB minimum inhibitory concentration against S. aureus increased drastically (≥128-fold) and 99.5% of MyxB was protein bound. Because of the high serum protein binding and resistance rate, we conclude that MyxB is not a viable starting point for antibiotic development.

Regulation of mprF by antisense RNA restores daptomycin susceptibility to daptomycin-resistant isolates of Staphylococcus aureus.

Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin. These results suggest that mprF mutations that reduce susceptibility to cationic antibacterials result in a gain-of-function phenotype.

Genetically engineered lipopeptide antibiotics related to A54145 and daptomycin with improved properties.

Daptomycin is a cyclic lipopeptide antibiotic approved for the treatment of skin and skin structure infections caused by Gram-positive pathogens and for that of bacteremia and right-sided endocarditis caused by Staphylococcus aureus. Daptomycin failed to meet noninferiority criteria for the treatment of community-acquired pneumonia, likely due to sequestration in pulmonary surfactant. Many analogues of daptomycin have been generated by combinatorial biosynthesis, but only two displayed improved activity in the presence of bovine surfactant, and neither was as active as daptomycin in vitro. In the present study, we generated hybrid molecules of the structurally related lipopeptide A54145 in Streptomyces fradiae and tested them for antibacterial activity in the presence of bovine surfactant. Hybrid A54145 nonribosomal peptide synthetase (NRPS) biosynthetic genes were constructed by genetic engineering and were expressed in combination with a deletion of the lptI methyltransferase gene, which is involved in the formation of the 3-methyl-glutamic acid (3mGlu) residue at position 12. Some of the compounds were very active against S. aureus and other Gram-positive pathogens; one compound was also highly active in the presence of bovine surfactant, had low acute toxicity, and showed some efficacy against Streptococcus pneumoniae in a mouse model of pulmonary infection.

Daptomycin exerts bactericidal activity without lysis of Staphylococcus aureus.

The ability of daptomycin to produce bactericidal activity against Staphylococcus aureus while causing negligible cell lysis has been demonstrated using electron microscopy and the membrane integrity probes calcein and ToPro3. The formation of aberrant septa on the cell wall, suggestive of impairment of the cell division machinery, was also observed.

Transcriptional profiling reveals that daptomycin induces the Staphylococcus aureus cell wall stress stimulon and genes responsive to membrane depolarization.

Daptomycin is a lipopeptide antibiotic that has recently been approved for treatment of gram-positive bacterial infections. The mode of action of daptomycin is not yet entirely clear. To further understand the mechanism transcriptomic analysis of changes in gene expression in daptomycin-treated Staphylococcus aureus was carried out. The expression profile indicated that cell wall stress stimulon member genes (B. J. Wilkinson, A. Muthaiyan, and R. K. Jayaswal, Curr. Med. Chem. Anti-Infect. Agents 4:259-276, 2005) were significantly induced by daptomycin and by the cell wall-active antibiotics vancomycin and oxacillin. Comparison of the daptomycin response of a two-component cell wall stress stimulon regulator VraSR mutant, S. aureus KVR, to its parent N315 showed diminished expression of the cell wall stress stimulon in the mutant. Daptomycin has been proposed to cause membrane depolarization, and the transcriptional responses to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nisin were determined. Transcriptional profiles of the responses to these antimicrobial agents showed significantly different patterns compared to those of the cell wall-active antibiotics, including little or no induction of the cell wall stress stimulon. However, there were a significant number of genes induced by both CCCP and daptomycin that were not induced by oxacillin or vancomycin, so the daptomycin transcriptome probably reflected a membrane depolarizing activity of this antimicrobial also. The results indicate that inhibition of peptidoglycan biosynthesis, either directly or indirectly, and membrane depolarization are parts of the mode of action of daptomycin.

Bactericidal action of daptomycin against stationary-phase and nondividing Staphylococcus aureus cells.

Most antibiotics with bactericidal activity require that the bacteria be actively dividing to produce rapid killing. However, in many infections, such as endocarditis, prosthetic joint infections, and infected embedded catheters, the bacteria divide slowly or not at all. Daptomycin is a lipopeptide antibiotic with a distinct mechanism of action that targets the cytoplasmic membrane of gram-positive organisms, including Staphylococcus aureus. Daptomycin is rapidly bactericidal against exponentially growing bacteria (a 3-log reduction in 60 min). The objectives of this study were to determine if daptomycin is bactericidal against nondividing S. aureus and to quantify the extent of the bactericidal activity. In high-inoculum methicillin-sensitive S. aureus cultures in stationary phase (10(10) CFU/ml), daptomycin displayed concentration-dependent bactericidal activity, requiring 32 micro/ml to achieve a 3-log reduction. In a study comparing several antibiotics at 100 microg/ml, daptomycin demonstrated faster bactericidal activity than nafcillin, ciprofloxacin, gentamicin, and vancomycin. In experiments where bacterial cell growth was halted by the metabolic inhibitor carbonyl cyanide m-chlorophenylhydrazone or erythromycin, daptomycin (10 microg/ml) achieved the bactericidal end point (a 3-log reduction) within 2 h. In contrast, ciprofloxacin (10 microg/ml) did not produce bactericidal activity. Daptomycin (2 microg/ml) remained bactericidal against cold-arrested S. aureus, which was protected from the actions of ciprofloxacin and nafcillin. The data presented here suggest that, in contrast to that of other classes of antibiotics, the bactericidal activity of daptomycin does not require cell division or active metabolism, most likely as a consequence of its direct action on the bacterial membrane.

Daptomycin-reversible rifampicin resistance in vancomycin-resistant Enterococcus faecium.

OBJECTIVES: In a previous study, we observed marked synergy between daptomycin and rifampicin against 73% of rifampicin-resistant, vancomycin-resistant Enterococcus faecium (VRE), with approximately 100-fold reductions in rifampicin MICs observed at one-eighth to one-fourth daptomycin MIC. The purpose of this study was to determine whether the synergy between daptomycin and rifampicin could be explained by enhanced entry of rifampicin into the cell or was related to amino acid substitutions in the rifampicin-binding site in the beta subunit (rpo beta) of the RNA polymerase. METHODS: We developed a bioassay for rifampicin to measure cell-bound rifampicin levels as well as metabolic inactivation of rifampicin. In addition, we sequenced the rifampicin-binding site in the rpo beta of VRE strains with and without synergy between daptomycin and rifampicin. RESULTS: Cell-bound rifampicin levels were the same in rifampicin-susceptible VRE as in rifampicin-resistant VRE showing daptomycin synergy and were not affected by the presence of daptomycin. In contrast, rifampicin-resistant VRE without daptomycin synergy had undetectable cell-bound rifampicin. Sequencing the rpo beta rifampicin-binding site revealed that the synergistic strains had the same sequence as rifampicin-susceptible wild-type E. faecium. The daptomycin synergy-resistant strains all had mutations in known rifampicin-binding sites. CONCLUSIONS: Daptomycin is able to reverse rifampicin resistance in some strains of VRE, but the mechanism could not be explained by an effect of daptomycin on entry of rifampicin into or transport out of the cell, by inactivation of rifampicin or by mutation involving the rifampicin-binding site.

Genetic changes that correlate with reduced susceptibility to daptomycin in Staphylococcus aureus.

Daptomycin is a lipopeptide antibiotic with potent activity against gram-positive bacteria. Complete-genome comparisons of laboratory-derived Staphylococcus aureus with decreased susceptibility to daptomycin and their susceptible parent were used to identify genes that contribute to reduced susceptibility to daptomycin. Selective pressure of growth in sublethal concentrations of daptomycin resulted in the accumulation of mutations over time correlating with incremental decreases in susceptibility. Single point mutations resulting in amino acid substitutions occurred in three distinct proteins: MprF, a lysylphosphatidylglycerol synthetase; YycG, a histidine kinase; and RpoB and RpoC, the beta and beta' subunits of RNA polymerase. Sequence analysis of mprF, yycF, yycG, rpoB, and rpoC in clinical isolates that showed treatment-emergent increases in daptomycin MICs revealed point mutations in mprF and a nucleotide insertion in yycG, suggesting a role for these genes in decreased susceptibility to daptomycin in the hospital setting.

Inhibition of daptomycin by pulmonary surfactant: in vitro modeling and clinical impact.

The lipopeptide daptomycin has been approved for use in skin and skin-structure infections but has failed to meet statistical noninferiority criteria in a clinical trial for severe community-acquired pneumonia. Daptomycin exhibited an unusual pattern of activity in pulmonary animal models: efficacy in Staphylococcus aureus hematogenous pneumonia and inhalation anthrax but no activity against Streptococcus pneumoniae in simple bronchial-alveolar pneumonia. Daptomycin was shown to interact in vitro with pulmonary surfactant, resulting in inhibition of antibacterial activity. This effect was specific to daptomycin and consistent with its known mechanism of action. This represents the first example of organ-specific inhibition of an antibiotic.

Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus.

The objective of this study was to further elucidate the role of membrane potential in the mechanism of action of daptomycin, a novel lipopeptide antibiotic. Membrane depolarization was measured by both fluorimetric and flow cytometric assays. Adding daptomycin (5 micro g/ml) to Staphylococcus aureus gradually dissipated membrane potential. In both assays, cell viability was reduced by >99% and membrane potential was reduced by >90% within 30 min of adding daptomycin. Cell viability decreased in parallel with changes in membrane potential, demonstrating a temporal correlation between bactericidal activity and membrane depolarization. Decreases in viability and potential also showed a dose-dependent correlation. Depolarization is indicative of ion movement across the cytoplasmic membrane. Fluorescent probes were used to demonstrate Ca(2+)-dependent, daptomycin-triggered potassium release from S. aureus. Potassium release was also correlated with bactericidal activity. This study demonstrates a clear correlation between dissipation of membrane potential and the bactericidal activity of daptomycin. A multistep model for daptomycin's mechanism of action is proposed.

In vitro bactericidal activities of daptomycin against Staphylococcus aureus and Enterococcus faecalis are not mediated by inhibition of lipoteichoic acid biosynthesis.

Previous studies have suggested that lipoteichoic acid biosynthesis inhibition is the mechanism of action of daptomycin. In this investigation, daptomycin inhibited all macromolecular synthesis in Staphylococcus aureus, Enterococcus faecalis, and Enterococcus hirae without kinetic or dose specificity for lipoteichoic acid. Daptomycin remained bactericidal in the absence of ongoing lipoteichoic acid synthesis. Inhibition of lipoteichoic acid synthesis is apparently not the mechanism of action of daptomycin in these pathogens.