Newly designed antimicrobial peptides with potent bioactivity and enhanced cell selectivity prevent and reverse rifampin resistance in Gram-negative bacteria.

Advances on chemically modified antimicrobial peptides for generating peptide antibiotics.

The peptidomimetic H-[NLys-tBuAla]6-NH2 (CEP-136), which exhibits low inherent antimicrobial activity against Gram-negative bacteria (MIC = 16-64 μM), was shown to significantly potentiate the antibacterial activity of several clinically important antibiotics against the human pathogens Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Thus, the antibacterial spectrum of rifampicin, clarithromycin, and azithromycin could be extended to include also these Gram-negative bacteria. Additionally, the potentiation effect was demonstrated in a panel of clinically relevant multidrug-resistant isolates including extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing as well as colistin-resistant strains. For some peptidomimetic-antibiotic combinations, the strong synergy corresponded to a more than 50-fold reduction of the minimal inhibitory concentration of the antibiotic. Mechanistic studies indicate that the potentiation arises from a permeabilization effect exerted on the outer membrane lipopolysaccharide layer of the Gram-negative bacteria without significant disruption of the inner membrane. Furthermore, the peptidomimetic enhancer exhibited only a marginal effect on the viability of mammalian HepG2 cells even at concentrations 100-fold higher than that enabling the antibiotic enhancement. Also, a low hemolytic activity combined with limited in vivo acute toxicity of CEP-136 in healthy mice allowed in vivo validation of the potentiation effect on both rifampicin and azithromycin treatment in a murine peritonitis model. Thus, CEP-136 is an interesting hit compound for further development of effective adjuvants for repurposing antibiotics for use against infections by multidrug-resistant Gram-negative bacteria.

Antibiotic Potentiation in Multidrug-Resistant Gram-Negative Pathogenic Bacteria by a Synthetic Peptidomimetic.

New approaches to target antibacterial agents toward Gram-negative bacteria are key, given the rise of antibiotic resistance. Since the discovery of polymyxin B nonapeptide as a potent Gram-negative outer membrane (OM)-permeabilizing synergist in the early 1980s, a vast amount of literature on such synergists has been published. This Review addresses a range of peptide-based and small organic compounds that disrupt the OM to elicit a synergistic effect with antibiotics that are otherwise inactive toward Gram-negative bacteria, with synergy defined as a fractional inhibitory concentration index (FICI) of <0.5. Another requirement for the inclusion of the synergists here covered is their potentiation of a specific set of clinically used antibiotics: erythromycin, rifampicin, novobiocin, or vancomycin. In addition, we have focused on those synergists with reported activity against Gram-negative members of the ESKAPE family of pathogens namely, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and/or Acinetobacter baumannii. In cases where the FICI values were not directly reported in the primary literature but could be calculated from the published data, we have done so, allowing for more direct comparison of potency with other synergists. We also address the hemolytic activity of the various OM-disrupting synergists reported in the literature, an effect that is often downplayed but is of key importance in assessing the selectivity of such compounds for Gram-negative bacteria.

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Synergy by Perturbing the Gram-Negative Outer Membrane: Opening the Door for Gram-Positive Specific Antibiotics

Antimicrobial resistance (AMR) has been increasing unrelentingly worldwide, thus negatively impacting human health. The discovery and development of novel antibiotics is an urgent unmet need of the hour. However, it has become more challenging, requiring increasingly time-consuming efforts with increased commercial risks. Hence, alternative strategies are urgently needed to potentiate the existing antibiotics. In this context, short cationic peptides or peptide-based antimicrobials that mimic the activity of naturally occurring antimicrobial peptides (AMPs) could overcome the disadvantages of AMPs having evolved as potent antibacterial agents. Besides their potent antibacterial efficacy, short peptide conjugates have also gained attention as potent adjuvants to conventional antibiotics. Such peptide antibiotic combinations have become an increasingly cost-effective therapeutic option to tackle AMR. This Review summarizes the recent progress for peptide-based small molecules as promising antimicrobials and as adjuvants for conventional antibiotics to counter multidrug resistant (MDR) pathogens.

Short Peptides and Their Mimetics as Potent Antibacterial Agents and Antibiotic Adjuvants.

The development of new antibacterial agents and therapeutic approaches is of high importance to address the global problem of antibiotic resistance. Although antimicrobial peptides are known to synergize with certain antibiotics, their clinical application is limited by their systemic toxicity, protease instability, and high production cost. To overcome these problems, nine dilipid ultrashort tetrabasic peptidomimetics (dUSTBPs) were prepared consisting of three basic amino acids separated by a molecular scaffold, bis(3-aminopropyl)glycine, and were ligated to two fatty acids. Several nonhemolytic dUSTBPs were shown to enhance the activity of several antibiotics against pathogenic Gram-negative bacteria. More importantly, dUSTBP 8, consisting of three l-arginine units and a dilipid of 8 carbons long, potentiated novobiocin and rifampicin consistently against multidrug-resistant (MDR) clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae. Preliminary studies suggested that dUSTBPs were likely to potentiate antibiotics through outer membrane permeabilization and/or disruption of active efflux and that dUSTBP 8 exhibited enhanced resistance to trypsin in comparison to the previously described di-C9-KKKK-NH2 antibiotic potentiator. The antibacterial activity of rifampicin and novobiocin was enhanced by dUSTBP 8 comparable to other known outer membrane permeabilizing potentiators including the gold standard polymyxin B nonapeptide. Our results indicate that ultrashort tetrabasic peptidomimetics are potent adjuvants that repurpose novobiocin and rifampicin as potent agents against priority MDR Gram-negative pathogens.

Dilipid Ultrashort Tetrabasic Peptidomimetics Potentiate Novobiocin and Rifampicin Against Multidrug-Resistant Gram-Negative Bacteria

Outer membrane (OM) drug impermeability typically associated with a molecular weight above 600 Da and high hydrophobicity prevents accumulation of many antibiotics in Gram-negative bacteria (GNB). Previous studies have shown that ultrashort tetrabasic lipopeptides (UTBLPs) containing multiple lysine residues potentiate Gram-positive bacteria (GPB)-selective antibiotics in GNB by enhancing OM permeability. However, there is no available information on how N-substitution at the ζ-position of lysine in UTBLPs affects antibiotic potentiation in GNB. To study these effects, we prepared a series of branched and linear UTBLPs that differ in the degree of N-ζ-methylation and studied their potentiating effects with GPB-selective antibiotics including rifampicin, novobiocin, niclosamide, and chloramphenicol against wild-type and multidrug-resistant GNB isolates. Our results show that increasing N-ζ-methylation reduces or abolishes the potentiating effects of UTBLPs with rifampicin, novobiocin, and niclosamide against GNB. No trend was observed with chloramphenicol that is largely affected by efflux. We were unable to observe a correlation between the strength of the antibiotic potentiating effect to the increase in fluorescence in the 1-N-phenylnaphthylamine (NPN) OM permeability assay suggesting that other factors besides OM permeability of NPN play a role in antibiotic potentiation. In conclusion, our study has elucidated crucial structure–activity relationships for the optimization of polybasic antibiotic potentiators in GNB.

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Effects of Lysine N-ζ-Methylation in Ultrashort Tetrabasic Lipopeptides (UTBLPs) on the Potentiation of Rifampicin, Novobiocin, and Niclosamide in Gram-Negative Bacteria

Polymyxins are considered a last-line treatment against infections caused by multidrug-resistant (MDR) Gram-negative bacteria. In addition to their use as a potent antibiotic, polymyxins have also been utilized as outer membrane (OM) permeabilizers, capable of augmenting the activity of a partner antibiotic. Several polymyxin derivatives have been developed accordingly, with the objective of mitigating associated nephrotoxicity. The conversion of polymyxins to guanidinylated derivatives, whereby the L-γ-diaminobutyric acid (Dab) amines are substituted with guanidines, are described herein. The resulting guanidinylated colistin and polymyxin B (PMB) exhibited reduced antibacterial activity but preserved OM permeabilizing properties that allowed potentiation of several antibiotic classes. Rifampicin, erythromycin, ceftazidime and aztreonam were particularly potentiated against clinically relevant MDR Gram-negative bacteria. The potentiating effects of guanidinylated polymyxins with ceftazidime or aztreonam were further enhanced by adding the β-lactamase inhibitor avibactam.

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Guanidinylated Polymyxins as Outer Membrane Permeabilizers Capable of Potentiating Rifampicin, Erythromycin, Ceftazidime and Aztreonam against Gram-Negative Bacteria

According to the World Health Organization, antibiotic resistance is a global health threat. Of particular importance are infections caused by multidrug-resistant Gram-negative bacteria including Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa for which limited treatment options exist. Multiple and simultaneously occurring resistance mechanisms including outer membrane impermeability, overexpression of efflux pumps, antibiotic-modifying enzymes, and modification of genes and antibiotic targets have made antibiotic drug development more difficult against these pathogens. One strategy to cope with these challenges is the use of outer membrane permeabilizers that increase the intracellular concentration of antibiotics when used in combination. In some circumstances, this approach can rescue antibiotics from resistance or repurpose currently marketed antibiotics. Tobramycin-based hybrid antibiotic adjuvants that combine two outer membrane-active components have been previously shown to potentiate antibiotics by facilitating transit through the outer membrane, resulting in increased antibiotic accumulation within the cell. Herein, we extended the concept of tobramycin-based hybrid antibiotic adjuvants to tobramycin-based chimeras by engineering up to three different membrane-active antibiotic warheads such as tobramycin, 1-(1-naphthylmethyl)-piperazine, ciprofloxacin, and cyclam into a central 1,3,5-triazine scaffold. Chimera 4 (TOB-TOB-CIP) consistently synergized with ciprofloxacin, levofloxacin, and moxifloxacin against wild-type and fluoroquinolone-resistant P. aeruginosa. Moreover, the susceptibility breakpoints of ceftazidime, aztreonam, and imipenem were reached using the triple combination of chimera 4 with ceftazidime/avibactam, aztreonam/avibactam, and imipenem/relebactam, respectively, against β-lactamase-harboring P. aeruginosa. Our findings demonstrate that tobramycin-based chimeras form a novel class of antibiotic potentiators capable of restoring the activity of antibiotics against P. aeruginosa.

Chimeric Tobramycin-Based Adjuvant TOB-TOB-CIP Potentiates Fluoroquinolone and β-Lactam Antibiotics against Multidrug-Resistant Pseudomonas aeruginosa.

Danyel Ramirez

Papers

Dilipid Ultrashort Tetrabasic Peptidomimetics Potentiate Novobiocin and Rifampicin Against Multidrug-Resistant Gram-Negative Bacteria

Effects of Lysine N-ζ-Methylation in Ultrashort Tetrabasic Lipopeptides (UTBLPs) on the Potentiation of Rifampicin, Novobiocin, and Niclosamide in Gram-Negative Bacteria

Guanidinylated Polymyxins as Outer Membrane Permeabilizers Capable of Potentiating Rifampicin, Erythromycin, Ceftazidime and Aztreonam against Gram-Negative Bacteria

Chimeric Tobramycin-Based Adjuvant TOB-TOB-CIP Potentiates Fluoroquinolone and β-Lactam Antibiotics against Multidrug-Resistant Pseudomonas aeruginosa.

Amphiphilic tribasic galactosamines potentiate rifampicin in Gram-negative bacteria at low Mg++/Ca++concentrations.