Structure-Function Studies of Polymyxin B Nonapeptide: Implications to Sensitization of Gram-Negative Bacteria # Haim Tsubery, †,‡ Itzhak Ofek, ‡ Sofia Cohen, ‡ and Mati Fridkin* ,† Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel, and Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel Received January 5, 2000 Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived by enzymatic processing from the naturally occurring peptide polymyxin B, is able to increase the permeability of the outer membrane of Gram-negative bacteria toward hydrophobic antibiotics probably by binding to the bacterial lipopolysaccharide (LPS). We have synthesized 11 cyclic analogues of PMBN and evaluated their activities compared to that of PMBN. The synthetic peptides were much less potent than PMBN in their capacity to sensitize Escherichia coli and Klebsiella pneumoniae toward novobiocin and to displace dansyl-PMBN from Escherichia coli LPS. Moreover, unlike PMBN, none of the analogues were able to inhibit the growth of Pseudomonas aeruginosa. The structural-functional features of PMBN were characterized and identified with regard to the ring size, the distance between positive charges and peptide backbone, the chirality of the DPhe-Leu domain, and the nature of the charged groups. Apparently, the structure of PMBN is highly specific for efficient perturbation of the outer membrane of Gram-negative bacteria as well as for LPS binding. The present study further increases our understanding of the complex PMBN-LPS and may, potentially, enable the design of compounds having enhanced permeabilization potency of the Gram-negative outer membrane. Introduction The accelerated emergence of pathogenic bacteria exhibiting a multiple pattern of resistance toward conventional antibiotics is a major threat, especially in severe infections such as septicemia where mortality is high. 1 The development of novel, versatile antibiotic arsenal is thus most urgent. Optimal new antimicrobial agents should withstand enzymatic modification and/ or degradation and be targeted toward a bacterial component which is readily accessible and not likely to undergo mutations. The bacterial endotoxin lipopolysaccharide (LPS) is the major antigen of the outer membrane (OM) of Gram- negative bacteria. It is composed of three major parts, one of which is lipid A, a highly conserved hydrophobic domain of LPS, in Gram-negative bacteria. 2 LPS is essential for survival of the bacteria through establish- ing of an effective permeability barrier. Gram-negative bacteria lacking lipid A have not yet been isolated, and the mutation is thought to be lethal. 2 LPS is a predomi- nant inducer of sepsis. 3 Uncontrolled perturbation of immune cells by LPS followed by cytokine-mediated damage to blood vessels and decrease in vascular resistance frequently lead to collapse of organs and death. 4 Neutralization of the devastating effects of LPS is one of the main targets in combating endotoxicosis. 5 In the past few years various therapeutic strategies and novel agents for the treatment of LPS-mediated septic shock have been evaluated, including anti-LPS antibod- ies, LPS-neutralizing proteins (BPI and LBP), lipid A antagonist (lipid X), polymyxin B (PMB), and polymyxin B-related synthetic peptides. 5-7 Lipid A is a target for cationic proteins and peptides since it contains nega- tively charged phosphorylated disaccharides linked, via ester bonds, to long fatty acid chains which are inter- calated in the bacterial outer membrane. 8 PMB is a naturally occurring cationic cyclic decapep- tide isolated from Bacillus polymyxa. 9,10 PMB is highly bactericidal to Gram-negative bacteria and considered one of the most efficient cell-permeabilizing com- pounds. 11 This capacity is due to its high-affinity binding to lipid A. 12 However, the therapeutic applications of PMB are very limited because of its relatively high toxicity. 13,14 Polymyxin B nonapeptide (PMBN) is a cyclic peptide obtained from PMB by proteolytic removal of its terminal amino acyl residue (Scheme 1). 15 PMBN is an extremely poor antimicrobial compound, 16 but it is still capable of binding, like its parent compound, to LPS, rendering Gram-negative bacteria susceptible to various hydrophobic antibiotics. 17 This ability of PMBN to bind to bacteria with relatively high affinity and to permeabilize their outer membrane is often referred to as “sensitizing activity” and points at a novel therapeu- tic direction. 18 This approach is strengthened by the markedly reduced toxicity of the PMBN derivative as compared with its parental PMB molecule. 11 In addition, an enzyme capable of degrading the cyclic PMBN peptide has not yet been reported. * To whom correspondence should be addressed. Tel: 972-8- 9342505. Fax: 972-8-9344142. E-mail: mati.fridkin@weizmann.ac.il. † The Weizmann Institute of Science. ‡ Tel Aviv University. # Abbreviations: Agb, 2-amino-4-guanidinobutyric acid; CD, circular dichroism; CFU, colon-forming unit; Dab, 2,4-diaminobutyric acid; Dap, 2,3-diaminopropionic acid; DIEA, diisopropylethylamine; MIC, minimal inhibitory concentration; NMM, 4-methylmorpholine; LPS, lipopolysac- charide; PMB, polymyxin B; PMBN, polymyxin B nonapeptide; PyBOP, (benzotriazol-1-yloxy)trispyrolidinophosphonium hexafluorophosphate; TES, triethylsilane; TFA, trifluoroacetic acid; TFE, trifluoroethanol. 3085 J. Med. Chem. 2000, 43, 3085-3092 10.1021/jm0000057 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/13/2000