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 ...

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 Escherichia 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 that the inhibition of vital respiratory enzymes in the bacterial inner membrane represents one of the secondary modes of action for polymyxins.

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.