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 8xMIC 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.

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.

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%; ceftolozanet-azobactam, 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.

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

Murepavadin (POL7080) represents the first member of a novel class of outer membrane protein-targeting antibiotics. It specifically interacts with LptD and inhibits lipopolysaccharide (LPS) transport. Murepavadin is being developed for the treatment of serious infections by Pseudomonas aeruginosa. We determined the plasma protein binding and the pharmacokinetics of murepavadin in plasma and epithelial lining fluid (ELF; pulmonary) in infected animals, and we determined the exposure-response relationship. Treatment of CD-1 neutropenic mice was started 2 h after infection using murepavadin at different dosing frequencies for 24 h, and the number of CFU per lung was determined. The sigmoid maximum-effect model was used to fit the dose-response, and the pharmacodynamic index (PDI) response was used to determine the PDI values, resulting in a static effect and 1-log kill reduction. Using R-2 as an indicator of the best fit, the area under the concentration-time curve for the unbound fraction of the drug (fAUC)/MIC ratio correlated best with efficacy. The mean AUC required to provide a static effect was 36.83 mg h/liter (fAUC = 8.25 mg h/liter), and that to provide a 1-log reduction was 44.0 mg h/liter (fAUC = 9.86 mg h/liter). The mean static fAUC/MIC was determined to be 27.78, and that for a 1-log reduction was 39.85. These data may serve to determine doses in humans that are likely to be efficacious.

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.

This chapter focuses on compounds in late preclinical and clinical development and compounds derived from known and new classes of antibiotics that show promising activity against multidrug-resistant (MDR) gram-negative bacteria. A major emphasis is on compounds that exhibit a new mechanism of action and on anti-pseudomonal antibiotics. In addition, particular attention is devoted to strategies that move away from the older model of identifying broad-spectrum antibiotics in favor of more focused, narrow-spectrum approaches. Antibiotic drug discovery in the past decades has focused efforts on the development of antibiotics against gram-positive bacteria and more specifically against multidrug-resistant Staphylococcus aureus (S. aureus). In contrast, treatment options against some MDR gram-negative pathogens have become very limited, especially due to the emergence of resistance against the last resort antibiotics, colistin and polymyxin B. Compounding the problem of limited financial resources is the fact that gram-negative organisms are inherently difficult to kill. This is due in part to their outer membrane (OM), which is composed of 75% of lipopolysaccharides (LPSs). LPSs are negatively charged, which makes the OM of gram-negative bacteria highly impermeable for many classes of antibiotics, thereby providing the gram-negative bacteria with a formidable shield to prevent entry of antibacterials. Moreover, these organisms are highly efficient at upregulating, mutating, or acquiring genes that code for mechanisms of antibiotic resistance. Most of the antibacterial compounds in preclinical and early development are derived from established classes. Within the established drug classes, novel β lactam antibiotics containing iron-chelating groups are promising.

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.

Novel therapeutic strategies are urgently needed for the treatment of serious diseases caused by viral, bacterial and parasitic infections, because currently used drugs are facing the problem of rapidly emerging resistance. There is also an urgent need for agents that act on novel pathogen-specific targets, in order to expand the repertoire of possible therapies. The high throughput screening of diverse small molecule compound libraries has provided only a limited number of new lead series, and the number of compounds acting on novel targets is even smaller. Natural product screening has traditionally been very successful in the anti-infective area. Several successful drugs on the market as well as other compounds in clinical development are derived from natural products. Amongst these, many are macrocyclic compounds in the 1-2 kDa size range. This review will describe recent advances and novel drug discovery approaches in the anti-infective area, focusing on synthetic and natural macrocyclic compounds for which in vivo proof of concept has been established. The review will also highlight the Protein Epitope Mimetics (PEM) technology as a novel tool in the drug discovery process. Here the structures of naturally occurring antimicrobial and antiviral peptides and proteins are used as starting points to generate novel macrocyclic mimetics, which can be produced and optimized efficiently by combinatorial synthetic methods. Several recent examples highlight the great potential of the PEM approach in the discovery of new anti-infective agents.

ChemInformVolume 34, Issue 4 Natural Products Macrocyclic Hairpin Mimetics of the Cationic Antimicrobial Peptide Protegrin 1: A New Family of Broad-Spectrum Antibiotics. Sasalu C. Shankaramma, Sasalu C. Shankaramma Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorZafiria Athanassiou, Zafiria Athanassiou Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorOliver Zerbe, Oliver Zerbe Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorKerstin Moehle, Kerstin Moehle Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorCarole Mouton, Carole Mouton Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorFrancesca Bernardini, Francesca Bernardini Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorJan W. Vrijbloed, Jan W. Vrijbloed Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorDaniel Obrecht, Daniel Obrecht Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorJohn A. Robinson, John A. Robinson Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this author Sasalu C. Shankaramma, Sasalu C. Shankaramma Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorZafiria Athanassiou, Zafiria Athanassiou Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorOliver Zerbe, Oliver Zerbe Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorKerstin Moehle, Kerstin Moehle Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorCarole Mouton, Carole Mouton Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorFrancesca Bernardini, Francesca Bernardini Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorJan W. Vrijbloed, Jan W. Vrijbloed Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorDaniel Obrecht, Daniel Obrecht Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this authorJohn A. Robinson, John A. Robinson Org.-Chem. Inst., Univ. Zuerich, CH-8057 Zuerich, Switz.Search for more papers by this author First published: 05 March 2003 https://doi.org/10.1002/chin.200304202AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume34, Issue4January 28, 2003 RelatedInformation

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.