Interaction of designed cationic antimicrobial peptides with the outer membrane of gram-negative bacteria.

The outer membrane (OM) is a hallmark feature of gram-negative bacteria that provides the species with heightened resistance against antibiotic threats while cationic antimicrobial peptides (CAPs) are natural antibiotics broadly recognized for their ability to disrupt bacterial membranes. It has been well-established that lipopolysaccharides present on the OM are among major targets of CAP activity against gram-negative species. Here we investigate how the relative distribution of charged residues along the primary peptide sequence, in conjunction with its overall hydrophobicity, affects such peptide-OM interactions in the natural CAP Ponericin W1. Using a designed peptide library derived from Ponericin W1, we determined that the consecutive placement of Lys residues at the peptide N- or C-terminus (ex. "PonN": KKKKKKWLGSALIGALLPSVVGLFQ) enhances peptide binding affinity to OM lipopolysaccharides compared to constructs where Lys residues are interspersed throughout the primary sequence (ex. "PonAmp": WLKKALKIGAKLLPSVVKLFKGSGQ). Antimicrobial activity against multidrug resistant strains of Pseudomonas aeruginosa was similarly found to be highest among Lys-clustered sequences. Our findings suggest that while native Ponericin W1 exerts its initial activity at the OM, Lys-clustering may be a promising means to enhance potency towards this interface, thereby augmenting peptide entry and activity at the IM, with apparent advantage against multidrug-resistant species.

Enhanced proteolytic resistance of cationic antimicrobial peptides through lysine side chain analogs and cyclization.

While cationic antimicrobial peptides (CAPs) are compelling candidates for antimicrobial therapy, their clinical development is largely hampered by their rapid and non-specific enzymatic degradation in physiological fluids. We have earlier de novo designed and synthesized a novel category of CAPs typified by the sequence KKKKKK-AAFAAWAAFAA-NH2 (termed "6K-F17") that have remarkable membrane-penetrating power, are highly selective for bacterial rather than host membranes, and are non-cytotoxic. Here we pursue the design and validation of the Lys chain-shortened 6K-F17 analogs 6Dap-F17 (Dap = diaminopropionic acid), 6Dab-F17 (Dab = diaminobutyric acid), and 6Orn-F17 (Orn = ornithine). Intriguingly, although initially designed to specifically resist trypsin vs. their original Lys sites, all three derivatives of 6K-F17 showed markedly improved stability not only against trypsin, but also against the major proteolytic enzymes elastase and proteinase K at a 1:100 enzyme-to-peptide (E:P) ratio. When the least stable analog, 6Dap-F17, was then cyclized ('stapled') - with reduced main chain hydrophobicity to avoid erythrocyte hemolysis - the peptide became robust towards all three enzymes up to 60 min at a 1:100 E:P ratio, and retained strong presence even at an enhanced 1:1 E:P ratio, as determined by HPLC and mass spectrometry. These results suggest that the application of Lys chain-shortening, either alone or in combination with macrocyclization, may enhance metabolic stability of CAPs, and thus their clinical potential.

Uncoupling Amphipathicity and Hydrophobicity: Role of Charge Clustering in Membrane Interactions of Cationic Antimicrobial Peptides.

Peptides with a combination of high positive charge and high hydrophobicity have high antimicrobial activity, as epitomized by peptide venoms, which are designed by nature as disruptors of host membranes yet also display significant efficacy against pathogens. To investigate this phenomenon systematically, here we focus on ponericin W1, a peptide venom isolated from Pachycondyla goeldii ants (WLGSALKIGAKLLPSVVGLFKKKKQ) to examine whether Lys positioning can be broadly applied to optimize the functional range of existing natural sequences. We prepared sets of ponericin W1 analogues, where Lys residues were either distributed in an amphipathic manner throughout the sequence (PonAmp), clustered at the N-terminus (PonN), or clustered at the C-terminus (PonC), along with their counterparts of reduced hydrophobicity through 2-4 Leu-to-Ala replacements. We found that wild-type ponericin W1 and all three variants displayed toxicity against human erythrocytes, but hemolysis was eliminated by the replacement of two or more Leu residues by Ala residues. As well, peptides containing up to 3 Leu-to-Ala replacements retained antimicrobial activity against E. coli bacteria. Biophysical analyses of peptide-membrane interaction patterns by circular dichroism spectroscopy revealed a novel mode of cluster-dependent peptide positioning vis-à-vis the water-membrane interface, where PonAmp and PonC peptides displayed full or partial helical structures, while PonN peptides were unstructured, likely due, in part, to dynamic interchange between aqueous and membrane surface environments. The overall findings suggest that the lower membrane penetration of N-terminal charge-clustered constructs coupled with moderate sequence hydrophobicity may be advantageous for conferring enhanced target selectivity for bacterial versus mammalian membranes.

The long form of pVHL is artifactually modified by serine protease inhibitor AEBSF.

von Hippel-Lindau protein (pVHL) is the tumor suppressor responsible for ubiquitylating the hypoxia-inducible factor (HIF) family of transcription factors for degradation under normoxic conditions. There are two major pVHL isoforms with the shorter isoform (pVHL19 ) lacking the acidic domain present in the N-terminus of the longer isoform (pVHL30 ). Although both isoforms can degrade HIF and suppress tumor formation in experimental systems, previous research suggests that pVHL30 can undergo posttranslational modifications (PTM) and interact with unique proteins. Indeed, pVHL30 has long been observed to migrate as two species on a reducing polyacrylamide gel, indicating the presence of an uncharacterized PTM on the slower-migrating pVHL30 without an identifiable biological consequence. Thus, there has been considerable effort to elucidate the exclusive biological activity of pVHL30 , if any, by first defining the unique features of the slower-migrating species. We show here that the migration of pVHL30 , but not pVHL19 , is retarded by 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), an irreversible serine protease inhibitor commonly found in protease inhibitor cocktails.