Polymyxin B octapeptide and polymyxin B heptapeptide are potent outer membrane permeability-increasing agents.

Abstract

Polymyxin B octapeptide (PBOP) and polymyxin B heptapeptide (PBHP) were found to be effective permeabilizers of the outer membraneof Escherichia coli and Salmonella typhimurium. PBOP was as effective as polymyxin B nonapeptide (PMBN), the known very potent permeabilizer. As low a PBOPconcentration as 1 /zg/ml sensitized E. coli to rifampicin by a factor of 100. Three /xg ofPBOP per ml was sufficient to sensitize this target to all the other tested hydrophobic antibiotics (erythromycin, fusidic acid, clindamycin, and novobiocin) by a factor of 30. Only a slightly higher (3-fold) concentration of PBHPwas required for a similar sensitizing effect.The outer membrane(OM)of Gram-negative enteric bacteria and Pseudomonasaeruginosa is an effective permeability barrier towards noxious drugs including antibiotics1}. Agents such as divalent chelators and certain polycations are knownto damage and permeabilize the OMand to sensitize the cell to many of those drugs. Chelators remove the stabilizing divalent cations inherent in the OMwhereas polycations bind to the acidic lipopolysaccharide constituent of the OM1}. While the OM permeability-increasing action of chelators is severely blocked by physiologically relevant concentrations of cations2), that of the most potent polycations (such as polymyxin B nonapeptide, PMBN)is not2).Polymyxin nonapeptides (five positive charges, 5 +) are significantly less toxic in animal studies than polymyxins3~6), but able to sensitize, usually at as low concentrations as l~3 //g, the target bacteria to lipophilic antibiotics (such as erythromycin, clindamycin, rifampicin and fusidic acid) by a factor of 303 qq7~ii) AlsQ deaCyipOiymyXmsi2) (6+), the lysine polymer with approx 20 residues8'9) (20+) and the arginine-rich peptide protamine8)9) (17+) are effective sensitizers, while the permeabilizing activity of aminoglycosides (streptomycin; 3+, gentamicin; 5+) is weak or absent in biologically relevant conditions8'13) (numbers in parentheses indicate the net charge). Weakor very weak permeabilizers also include pentalysine13) (5+) and the human granulocyte defensins14) (5 +). Polyamines, including spermine (4+),are not permeabilizers8). Perhaps surprisingly, linear polymyxin-resembling synthetic peptides (linear arginylpolymyxin B decapeptide; 6 + , linear lysylpolymyxin B nonapeptide; 5 + ) also lack the activity1 5).In order to develop an effective and possibly therapeutically useful polycationic permeabilizer, it should be important to evaluate which factors (total number of positive charges, charge density, favorable conformation of the polycation) are critical for the OM-permeabilizing activity. Wewill show in this communication that the OMpermeability-increasing potency of the octapeptide derivative (5+) of

Neopeptide Antibiotics That Function as Opsonins and Membrane-Permeabilizing Agents for Gram-Negative Bacteria

“…Previous studies have shown that PMBN and PMBO increase OM permeability for gram-negative bacteria by binding to the bacterial surface (15,31,32). The test peptides lacked bactericidal activity at concentrations Ն250 g/ml (data not shown).…”

“…The PMB antibiotics (the sequences are given in Table 1) were purchased from Sigma Chemical Co., St. Louis, MO. For certain experiments, PMB (Sigma Chemical Co.) was used to prepare PMBNs and PMBOs by proteolysis of PMB with papain and ficin, respectively, as described elsewhere (9,15). The crude products were purified (Ͼ98%) by HPLC, analyzed, and characterized as described above (see Table 1 for the peptide sequences).…”

Development of new polymyxin derivatives for multi-drug resistant Gram-negative infections

“…Further the cyclic core alone (PMB 4-10; PMB heptapeptide 3d) retains substantial ability to permeabilise the outer membrane. 53 Kanazawa et al 46 further investigated the roles of the individual amino acids of Polymyxin by alanine scanning and confirmed the importance of the diaminobutyrate (Dab) residues in the cyclic core as would be expected from the model of Mares et al 33 These early findings have guided the design of next generation polymyxins.…”

“…Treatment with bromelain leads to the polymyxin octapeptide PMB (3-10) (PMBO; 3c) or with Nagarse (Subtilisin B, Savinase) to the polymyxin heptapeptide PMB (4-10) (PMBH; 3d). 53 A chemical approach using Troc protection followed by cleavage with methane sulfonic acid leading to the protected PMBN has been employed by Okimura et al 54 , albeit in low yield.…”

Characterization of the poxAB Operon Encoding a Class D Carbapenemase in Pseudomonas aeruginosa,

“…Specifically, the bulkiness of the polycations is thought to create transient cracks in the structure of the LPS, thus allowing uptake of other compounds such as hydrophobic antibiotics and of the polycations themselves in a process known as self-promoted uptake. Examples of antibiotics that promote their own entry into the cell by disrupting the normal structure of LPS via their cationic nature include aminoglycosides, polymyxins, cationic detergents and polycationic peptides, among others (256,(276)(277)(278)(279)(280)(281). Permeabilization of the outer membrane with such compounds can additionally sensitize the cells to permeation by other compounds and antibiotics including lysozyme, β-lactams, and a number of hydrophobic antibiotics (erythromycin, clindamycin, novobiocin, fusidic acid and cloxacillin) (274,(282)(283)(284).…”