Author Correction: Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chimeric peptidomimetic antibiotics against Gram-negative bacteria.

There is an urgent need for new antibiotics against Gram-negative pathogens that are resistant to carbapenem and third-generation cephalosporins, against which antibiotics of last resort have lost most of their efficacy. Here we describe a class of synthetic antibiotics inspired by scaffolds derived from natural products. These chimeric antibiotics contain a ß-hairpin peptide macrocycle linked to the macrocycle found in the polymyxin and colistin family of natural products. They are bactericidal and have a mechanism of action that involves binding to both lipopolysaccharide and the main component (BamA) of the ß-barrel folding complex (BAM) that is required for the folding and insertion of ß-barrel proteins into the outer membrane of Gram-negative bacteria. Extensively optimized derivatives show potent activity against multidrug-resistant pathogens, including all of the Gram-negative members of the ESKAPE pathogens1. These derivatives also show favourable drug properties and overcome colistin resistance, both in vitro and in vivo. The lead candidate is currently in preclinical toxicology studies that-if successful-will allow progress into clinical studies that have the potential to address life-threatening infections by the Gram-negative pathogens, and thus to resolve a considerable unmet medical need.

Murepavadin antimicrobial activity against and resistance development in cystic fibrosis Pseudomonas aeruginosa isolates.

BACKGROUND: Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. OBJECTIVES: To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. METHODS: MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time-kill assays. Resistant mutants were studied by WGS. RESULTS: The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1-5 h. Murepavadin MICs were 2-9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8×MIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). CONCLUSIONS: Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

Anti-biofilm activity of murepavadin against cystic fibrosis Pseudomonas aeruginosa isolates.

OBJECTIVES: To determine the activity of murepavadin in comparison with tobramycin, colistin and aztreonam, against cystic fibrosis (CF) Pseudomonas aeruginosa isolates growing in biofilms. The biofilm-epidemiological cut-off (ECOFF) values that include intrinsic resistance mechanisms present in biofilms were estimated. METHODS: Fifty-three CF P. aeruginosa isolates from respiratory samples were tested using the Calgary (closed system) device, while 4 [2 clinical (one smooth, one mucoid) and 2 reference strains] were tested using the BioFlux, a microfluidic open model of biofilm testing. Biofilm was stained with SYTO9® and propidium iodide. The minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) were determined. The MBIC-ECOFF and the MBEC-ECOFF were calculated. RESULTS: Colistin, tobramycin and murepavadin presented similar MBIC50/MBIC90 values (4/32, 8/64 and 2/32, respectively). Murepavadin exhibited the lowest MBEC90 (64 mg/L). Aztreonam MBIC and MBEC values were higher than those of the other antibiotics tested. Tobramycin and murepavadin had the lowest MBEC-ECOFF (64 and 128 mg/L, respectively), while those of aztreonam and colistin exceeded 512 mg/L. Using the BioFlux, for the PAO1, PAO mutS and the smooth clinical strain, a significant difference (P < 0.0125) was observed when comparing the fluorescence of treated and untreated biofilms. For the mucoid strain, only the biofilm treated with aztreonam (MBIC and MBEC) and tobramycin (MBEC) showed differences with respect to the untreated biofilm. CONCLUSIONS: Murepavadin demonstrated good activity against P. aeruginosa biofilms both in open and closed systems. The MBIC-ECOFF and the MBEC-ECOFF are proposed as new parameters to estimate the activity of antibiotics on biofilms.

Susceptibility of Pseudomonas aeruginosa Recovered from Cystic Fibrosis Patients to Murepavadin and 13 Comparator Antibiotics.

The objective was to determine the in vitro antimicrobial susceptibility of Pseudomonas aeruginosa isolates cultured from cystic fibrosis (CF) patients and explore associations between strain sequence type and susceptibility. Fourteen antibiotics and antibiotic combinations, including the novel antibacterial peptide murepavadin, were tested for activity against 414 Pseudomonas aeruginosa isolates cultured from respiratory samples of CF patients. The complete genomes of the isolates were sequenced, and minimum spanning trees were constructed based on the sequence types (STs). Percentages of resistance according to CLSI 2019 breakpoints were as follows: cefepime, 14%; ceftazidime, 11%; ceftazidime-avibactam, 7%; ceftolozane-tazobactam, 3%; piperacillin-tazobactam, 12%; meropenem, 18%; imipenem, 32%; aztreonam, 23%; ciprofloxacin, 30%; gentamicin, 30%; tobramycin, 12%; amikacin, 18%; and colistin, 4%. Murepavadin MIC50 and MIC90 were 0.12 mg/liter and 2 mg/liter, respectively. There were no apparent clonal clusters associated with resistance, but higher MICs did appear to occur more often in STs with multiple isolates than in single ST isolates. In general, the CF isolates showed a wide genetic distribution. P. aeruginosa CF isolates exhibited the lowest resistance rates against ceftolozane-tazobactam, ceftazidime-avibactam, and colistin. Murepavadin demonstrated the highest activity on a per-weight basis and may therefore become a valuable addition to the currently available antibiotics for treatment of respiratory infection in people with CF.

Efficacy of the Novel Antibiotic POL7001 in Preclinical Models of Pseudomonas aeruginosa Pneumonia.

The clinical development of antibiotics with a new mode of action combined with efficient pulmonary drug delivery is a priority against untreatable Pseudomonas aeruginosa lung infections. POL7001 is a macrocycle antibiotic belonging to the novel class of protein epitope mimetic (PEM) molecules with selective and potent activity against P. aeruginosa We investigated ventilator-associated pneumonia (VAP) and cystic fibrosis (CF) as indications of the clinical potential of POL7001 to treat P. aeruginosa pulmonary infections. MICs of POL7001 and comparators were measured for reference and clinical P. aeruginosa strains. The therapeutic efficacy of POL7001 given by pulmonary administration was evaluated in murine models of P. aeruginosa acute and chronic pneumonia. POL7001 showed potent in vitro activity against a large panel of P. aeruginosa isolates from CF patients, including multidrug-resistant (MDR) isolates with adaptive phenotypes such as mucoid or hypermutable phenotypes. The efficacy of POL7001 was demonstrated in both wild-type and CF mice. In addition to a reduced bacterial burden in the lung, POL7001-treated mice showed progressive body weight recovery and reduced levels of inflammatory markers, indicating an improvement in general condition. Pharmacokinetic studies indicated that POL7001 reached significant concentrations in the lung after pulmonary administration, with low systemic exposure. These results support the further evaluation of POL7001 as a novel therapeutic agent for the treatment of P. aeruginosa pulmonary infections.

Tripeptidyl peptidase-I is essential for the degradation of sulphated cholecystokinin-8 (CCK-8S) by mouse brain lysosomes.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small proteins. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, late infantile neuronal ceroid lipofuscinosis. The pathological consequences of loss of activity are only manifested in neuronal cells suggesting that TPP-I may be involved in the lysosomal degradation of neuropeptides. We have investigated the degradation of the C-terminal octapeptide of sulphated cholecystokinin (CCK-8S) by a lysosomal fraction purified from mouse brain. Degradation products were characterised by reversed phase HPLC and mass spectrometry. Incubation of CCK-8S with brain lysosomes results in the sequential removal of the tripeptides DY(SO(3)H)M and Glycl-Tryptophanyl-Methionine from the N-terminus of CCK-8S. Degradation of CCK-8S in the isolated lysosomal fraction is completely prevented by Ala-Ala-Phe-chloromethyl ketone, an inhibitor of TPP-I. Butabindide, a specific inhibitor of TPP-II, a cell surface peptidase which also cleaves CCK-8S, inhibits TPP-I but kinetic studies indicate that the Ki for inhibition of TPP-I is 1000-fold higher than the Ki for the inhibition of TPP-II. Consequently, higher concentrations of butabindide are required for the inhibition of CCK-8S degradation by TPP-I than by TPP-II. These results indicate that whereas cell surface TPP-II is responsible for regulating extracellular CCK-8S levels, lysosomal TPP-I is largely responsible for the degradation of CCK-8S which enters the cell by receptor-mediated endocytosis.

Exposure to styrene in fiberglass-reinforced plastic manufacture: still a problem.

OBJECTIVE: The aim of the work is to define occupational exposure to styrene in fiberglass manufacture; the phase of stretching styrene resins needs some manual handling and leads workers to be exposed to styrene. METHODS: We surveyed 20 workers in two companies manufacturing fiberglass, checking environmental levels and urinary concentrations of mandelic acid (MA), and phenylglioxylic acid (PGA). Workers completed a questionnaire collecting their medical history. RESULTS: Environmental monitoring showed some styrene concentrations higher than the threshold limit value-time-weighted average. Biological monitoring confirmed these findings and four workers had levels of urinary PGA and MA concentrations higher than the Biological Exposure Indices of the American Conference of Governmental Industrial Hygienists. CONCLUSIONS: This picture confirms that, even though the risk is known and the manufacturing cycle is well-defined, workers nevertheless risk exposure. More research is needed to optimize the work processes and the protection systems to lower this risk.

Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.

Antibiotics with new mechanisms of action are urgently required to combat the growing health threat posed by resistant pathogenic microorganisms. We synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I. Several rounds of optimization gave a lead compound that was active in the nanomolar range against Gram-negative Pseudomonas spp., but was largely inactive against other Gram-negative and Gram-positive bacteria. Biochemical and genetic studies showed that the peptidomimetics had a non-membrane-lytic mechanism of action and identified a homolog of the beta-barrel protein LptD (Imp/OstA), which functions in outer-membrane biogenesis, as a cellular target. The peptidomimetic showed potent antimicrobial activity in a mouse septicemia infection model. Drug-resistant strains of Pseudomonas are a serious health problem, so this family of antibiotics may have important therapeutic applications.

Lysosomal degradation of cholecystokinin-(29-33)-amide in mouse brain is dependent on tripeptidyl peptidase-I: implications for the degradation and storage of peptides in classical late-infantile neuronal ceroid lipofuscinosis.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small peptides. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis (CLN2). This disease is characterized by the accumulation of proteinaceous and autofluorescent material within the lysosomes of neurons, which undergo massive cell death during the course of the disease. The absence of TPP-I may result in the lysosomal accumulation of small peptides and proteins, which eventually compromises lysosomal functions critical to the survival of neurons. To investigate the metabolism of small peptides, we have studied the degradation of cholecystokinin-(29-33)-amide (GWMDF-NH2; cholecystokinin C-terminal pentapeptide) by lysosomal fractions isolated from mouse brain and several other tissues. GWMDF-NH2 is cleaved at only one peptide bond by brain lysosomes, to produce GWM and DF-NH2. Inhibitor studies demonstrate that this reaction is catalysed by TPP-I. In contrast, lysosomal fractions from other mouse tissues additionally cleave a second peptide bond to produce GW and MDF-NH2. Inhibitor studies indicate that this reaction is catalysed by dipeptidyl peptidase-I (DPP-I; cathepsin C). Inhibitors of TPP-I are sufficient to completely block the degradation of GWMDF-NH2 by brain, but inhibitors of both TPP-I and DPP-I are required to completely inhibit the degradation of GWMDF-NH2 by other mouse tissues. Enzyme assays confirm the low activity of DPP-I in brain. An unrelated neuropeptide, neuromedin B, is degraded by a pathway that is partially dependent on TPP-I. These results indicate that TPP-I is required for the partial or complete digestion of certain neuropeptides by brain lysosomes. In the absence of TPP-I, neuropeptides or their degradation products will accumulate in brain lysosomes and may contribute to the pathogenesis of CLN2. Other tissues are spared because they express another peptidase, DPP-I, which has extensive activity on peptides and can compensate for the loss of TPP-I.

Macrocyclic hairpin mimetics of the cationic antimicrobial peptide protegrin I: a new family of broad-spectrum antibiotics.

The problems associated with increasing antibiotic resistance have stimulated great interest in newly discovered families of naturally occurring cationic antimicrobial peptides. These include protegrin, tachyplesin, and RTD-1, which adopt beta-hairpin-like structures. We report here an approach to novel peptidomimetics based on these natural products. The mimetics were designed by transplanting the cationic and hydrophobic residues onto a beta-hairpin-inducing template, either a D-Pro-L-Pro dipeptide or a xanthene derivative. The mimetics have good antimicrobial activity against Gram-positive and Gram-negative bacteria (minimal inhibitory concentration approximately 6-25 microgram mL(-1)). Analogues with improved selectivity for microbial rather than red blood cells (1 % hemolysis at 100 microgram mL(-1)) were identified from a small library prepared by parallel synthesis. Thus, it is possible to separate the antimicrobial and hemolytic activities in this class of mimetics. NMR studies on one mimetic revealed a largely unordered structure in water, but a transition to a regular beta-hairpin backbone conformation in the presence of dodecylphosphocholine micelles. This family of mimetics may provide a starting point for the optimization of antimicrobial agents of potential clinical value in the fight against multiple-drug-resistant microorganisms.