The growing problem of bacterial resistance to conventional antibiotic compounds and the need for new antibiotics has stimulated interest in the development of antimicrobial peptides (AMPs) as human therapeutics. Development of topically applied agents, such as pexiganan (also known as MSI-78, an analog of the naturally occurring magainin2, extracted from the skin of the African frog Xenopus laevis) has been the focus of pharmaceutical development largely because of the relative safety of topical therapy and the uncertainty surrounding the long-term toxicology of any new class of drug administered systemically. The main hurdle that has hindered the development of antimicrobial peptides is that many of the naturally occurring peptides (such as magainin), although active in vitro, are effective in animal models of infection only at very high doses, often close to the toxic doses of the peptide, reflecting an unacceptable margin of safety. Though MSI-78 did not pass the FDA approval, it is still the best-studied AMP to date for therapeutic purposes. Biophysical studies have shown that this peptide is unstructured in solution, forms an antiparallel dimer of amphipathic helices upon binding to the membrane, and disrupts membrane via toroidal-type pore formation. This article covers functional, biophysical, biochemical and structural studies on pexiganan.

Parkinson’s disease (PD) is the second most common neurodegenerative disease, affecting approximately one-percent of the population over the age of sixty. Although many animal models have been developed to study this disease, each model presents its own advantages and caveats. A unique new model has arisen to study the role of alpha-synuclein (aSyn) in the pathogenesis of PD. This model involves the conversion of recombinant monomeric aSyn protein to a fibrillar form—the aSyn pre-formed fibril (aSyn PFF)—which is then injected into the brain or introduced to the media in culture. Although many groups have successfully adopted and replicated the aSyn PFF model, issues with generating consistent pathology have been reported by investigators. To improve the replicability of this model and diminish these issues, The Michael J. Fox Foundation for Parkinson’s Research (MJFF) has enlisted the help of field leaders who performed key experiments to establish the aSyn PFF model to provide the research community with guidelines and practical tips for improving the robustness and success of this model. Specifically, we identify key pitfalls and suggestions for avoiding these mistakes as they relate to generating the aSyn PFFs from monomeric protein, validating the formation of pathogenic aSyn PFFs, and using the aSyn PFFs in vivo or in vitro to model PD. With this additional information, adoption and use of the aSyn PFF model should present fewer challenges, resulting in a robust and widely available model of PD.

Leap frog: Antimicrobial peptides based on the structure of the magainin peptides from the African clawed frog have shown great promise as therapeutic agents. However their efficacy in vivo is limited by their susceptibility to proteolysis. We show that incorporating the fluorous amino acid hexafluoroleucine into a magainin analogue (see scheme) dramatically improves stability to proteolysis while retaining biological activity.

Protegrins are potent members of the beta-hairpin-forming class of antimicrobial peptides. Key to their antimicrobial activity is their assembly into oligomeric structures upon binding to the bacterial membrane. To examine the relationship between the physicochemical properties of the peptide and its biological activity, we have synthesized variants of protegrin-1 in which key residues in the hydrophobic core, valine-14 and -16, are changed to leucine and to the extensively fluorinated analogue hexafluoroleucine. These substitutions have the effect of making the peptide progressively more hydrophobic while minimally perturbing the secondary structure. The leucine-containing peptide was significantly more active than wild-type protegrin against several common pathogenic bacterial strains, whereas the hexafluoroleucine-substituted peptide, in contrast, showed significantly diminished activity against several bacterial strains. Isothermal titration calorimetry measurements revealed significant changes in the interaction of the peptides binding to small unilamelar vesicles that mimic the lipid composition of the bacterial membrane. The binding isotherms for wild-type and leucine-substituted protegrins indicate that electrostatic interactions dominate the membrane-peptide interaction, whereas the isotherm for the hexafluoroleucine-substituted protegrin suggests a diminished electrostatic component to binding. Notably both of these substitutions appear to alter the stoichiometry of the lipid-peptide interaction, suggesting that these substitutions may stabilize oligomerized forms of protegrin that are postulated to be intermediates in the assembly of the beta-barrel membrane pore structure.

In previous in vivo studies, amyloid fibers formed from a peptide ubiquitous in human seminal fluid (semen-derived enhancer of viral infection (SEVI)) were found to dramatically enhance the infectivity of the HIV virus (3-5 orders of magnitude by some measures). To complement those studies, we performed in vitro assays of PAP(248-286), the most active precursor to SEVI, and other polycationic polymers to investigate the physical mechanisms by which the PAP(248-286) promotes the interaction with lipid bilayers. At acidic (but not at neutral) pH, freshly dissolved PAP(248-286) catalyzes the formation of large lipid flocculates in a variety of membrane compositions, which may be linked to the promotion of convective transport in the vaginal environment rather than transport by a random Brownian motion. Furthermore, PAP(248-286) is itself fusiogenic and weakens the integrity of the membrane in such a way that may promote fusion by the HIV gp41 protein. An alpha-helical conformation of PAP(248-286), lying parallel to the membrane surface, is implicated in promoting bridging interactions between membranes by the screening of the electrostatic repulsion that occurs when two membranes are brought into close contact. This suggests that nonspecific binding of monomeric or small oligomeric forms of SEVI in a helical conformation to lipid membranes may be an additional mechanism by which SEVI enhances the infectivity of the HIV virus.