Increasing antibiotic resistance in Gram-negative bacteria, particularly in Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, presents a global medical challenge. No new antibiotics will be available for these ‘superbugs’ in the near future due to the dry antibiotic discovery pipeline. Colistin and polymyxin B are increasingly used as the last-line therapeutic options for treatment of infections caused by multidrug-resistant Gram-negative bacteria. This article surveys the significant progress over the last decade in understanding polymyxin chemistry, mechanisms of antibacterial activity and resistance, structure–activity relationships and pharmacokinetics/pharmacodynamics. In the ‘Bad Bugs, No Drugs’ era, we must pursue structure–activity relationship-based approaches to develop novel polymyxin-like lipopeptides targeting polymyxin-resistant Gram-negative ‘superbugs’. Before new antibiotics become available, we must optimize the clinical use of polymyxins through the application of pharmacokinetic/pharmacodynamic principles, thereby minimizing the development of resistance.

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The antimicrobial lipopeptides polymyxin B and E (colistin) are being used as a ‘last-line’ therapy for infections caused by multidrug-resistant Gram-negative pathogens. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative ‘superbugs’. This report details the structure–activity relationships (SAR) based design, in toto synthesis, and preclinical evaluation of a series of novel polymyxin lipopeptides with better antibacterial activity against polymyxin-resistant Gram-negative bacteria.

Polymyxin B and colistin were examined for their ability to inhibit the type II NADH-quinone oxidoreductases (NDH-2) of three species of Gram-negative bacteria. Polymyxin B and colistin inhibited the NDH-2 activity in preparations from all of the isolates in a concentration-dependent manner. The mechanism of NDH-2 inhibition by polymyxin B was investigated in detail with E. coli inner membrane preparations and conformed to a mixed inhibition model with respect to ubiquinone-1 and a non-competitive inhibition model with respect to NADH. These suggest inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.

Polymyxin B and colistin are currently used as a ‘last-line’ treatment for multidrug-resistant Gram-negative bacteria. However very little is known about the pharmacological differences between polymyxin B1, polymyxin B2, colistin A, colistin B, the major cyclic lipopeptides components present in polymyxin B and colistin products. Here, we report on the in vitro and in vivo antimicrobial activity and toxicity of these major lipopeptide components. All four lipopeptides had comparable MICs (<0.125–4 mg/L) against a panel of clinical Gram-negative isolates. They also had comparable in vivo antimicrobial activity (Δlog10 CFU/mL >-3) and nephrotoxicity (mild to moderate histological damage) in mouse models. However, polymyxin B1 and colistin A showed significantly higher (> 3-fold) in vitro apoptotic effect on human kidney proximal tubular HK-2 cells than polymyxin B2 and colistin B, respectively. Compared to the commercial polymyxin and colistin products, the individual lipopeptide components had slightly more in vivo antimicrobial activity. Our results highlight the need to re-assess pharmacopoeial standards for polymyxins B and colistin and to standardize the composition of the different commercial products of polymyxin antibiotics.

The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative ‘superbugs’ has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains.

Recently, we carried out a statistical analysis of a 'tryptic' peptide tandem mass spectrometry database in order to identify sequence-dependent patterns for the gas-phase fragmentation behavior of protonated peptide ions, and to improve the models for peptide fragmentation currently incorporated into peptide sequencing and database search algorithms [Kapp, E. A., Schutz, F., Reid, G. E., Eddes, J. S., Moritz, R. L., O'Hair, R. A. J., Speed, T. P. and Simpson, R. J. Anal. Chem. 2003, 75, 6251-6264.]. Here, we have reexamined this database in order to determine the effect of a common post-translational or process induced modification, methionine oxidation, on the appearance and relative abundances of the product ions formed by low energy collision induced dissociation of peptide ions containing this modification. The results from this study indicate that the structurally diagnostic neutral loss of methane sulfenic acid (CH3SOH, 64Da) from the side chain of methionine sulfoxide residues is the dominant fragmentation process for methionine sulfoxide containing peptide ions under conditions of low proton mobility, i.e., when ionizing proton(s) are sequestered at strongly basic amino acids such as arginine, lysine or histidine. The product ion abundances resulting from this neutral loss were found to be approximately 2-fold greater than those resulting from the cleavage C-terminal to aspartic acid, which has previously been shown to be enhanced under the same conditions. In close agreement with these statistical trends, experimental and theoretical studies, employing synthetic "tryptic" peptides and model methionine sulfoxide containing peptide ions, have determined that the mechanism for enhanced methionine sulfoxide side chain cleavage proceeds primarily via a 'charge remote' process. However, the mechanism for dissociation of the side chain for these ions was observed to change as a function of proton mobility. Finally, the transition state barrier for the charge remote side chain cleavage mechanism is predicted to be energetically more favorable than that for charge remote cleavage C-terminal to aspartic acid.

The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.