The semi-synthetic peptide Lin-SB056-1 in combination with EDTA exerts strong antimicrobial and antibiofilm activity against pseudomonas aeruginosa in conditions mimicking cystic fibrosis sputum

Pseudomonas aeruginosa is a major cause of chronic lung infections in cystic fibrosis (CF) patients. The ability of the bacterium to form biofilms and the presence of a thick and stagnant mucus in the airways of CF patients largely contribute to antibiotic therapy failure and demand for new antimicrobial agents able to act in the CF environment. The present study investigated the anti-P. aeruginosa activity of lin-SB056-1, a recently described semi-synthetic antimicrobial peptide, used alone and in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA). Bactericidal assays were carried out in standard culture conditions and in an artificial sputum medium (ASM) closely resembling the CF environment. Peptideâ\u80\u99s structure and interaction with large unilamellar vesicles in media with different ionic strengths were also investigated through infrared spectroscopy. Lin-SB056-1 demonstrated fast and strong bactericidal activity against both mucoid and non-mucoid strains of P. aeruginosa in planktonic form and, in combination with EDTA, caused significant reduction of the biomass of P. aeruginosa mature biofilms. In ASM, the peptide/EDTA combination exerted a strong bactericidal effect and inhibited the formation of biofilm-like structures of P. aeruginosa. Overall, the results obtained highlight the potential of the lin-SB056-1/EDTA combination for the treatment of P. aeruginosa lung infections in CF patients

Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β- stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH$_{2}$, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH$_{2}$, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β- SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state $^{19}$F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056- lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β- SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by $^{31}$P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands

Heme proteins: the role of solvent on the dynamics of gates and portals

Water plays a pivotal role in the correct functioning of proteins. Hydration is fundamental to their stability and flexibility, to folding process and specific functions, and to protein protein interactions. In this work, the effects of solvation on proteins dynamics have been investigated by employing molecular dynamics simulations and using myoglobin as a model system. The investigation has been focused on solvent waters residing around/inside the protein, with average times of up to tens of nanoseconds, revealing that these slow waters may have significant effects on biological functioning of the protein. Our study pointed out that water is able to interact with proteins in diverse ways, leading to different kinds of perturbations in their intrinsic dynamic behavior. In particular, for myoglobin it was found that a water molecule can (i) "block" entry/escape of ligands to/from a particular docking site, (ii) act as a "wedge" modulating the dynamics of internal cavities, or (iii) join a "flow" of waters taking a ligand into (or "washing" a ligand away from) the protein interior. The information gathered in this work allowed us to provide a fingerprint of protein solvation state, the hydration sites map, which may represent a novel tool for comparing different forms/species of globular proteins

Heme Proteins: The Role of Solvent on the Dynamics of Gates and Portals Revealed by MD Simulations

In the family of respiratory proteins, hemoglobins and myoglobins have been the first to be crystallized in ‘50. Despite the availability of 3D structures, issues regarding the microscopic functioning remain open, such as, for instance, the R to T switching mechanism in hemoglobin or the ligand escape process in myoglobin. Due to the relatively small number of residues, myoglobin is the suitable candidate to investigate the more general structure-function paradigm, being defined as the hydrogen atom of biology. In this work, to complement our recent study on the dynamics of internal cavities of myoglobin[1], the effect of solvation on these intrinsic pathways has been explored. In particular, 60ns-long molecular dynamics simulation of horse heart met-myoglobin was further analyzed and the dynamics of waters residing around/inside the protein with average residence times of up to tens of nanoseconds was monitored. Together with the knowledge obtained previously[1], the analysis of solvent revealed that myoglobin has in fact only few stable hydration sites in which a water molecule can stay for time longer than 2 ns. Strikingly, all of these sites are close to protein/solvent portals observed in previous studies focused on the entry/escape and migration of various ligands in myoglobin[2-4]. 1. Scorciapino, M. A.; Robertazzi, A.; Casu, M.; Ruggerone, P.; Ceccarelli, M. J. Am. Chem. Soc. 2009, 131, 11825-11832. 2. Cohen, J.; Arkhipov, A.; Braun, R.; Schulten, K. Biophys. J. 2006, 91, 1844-1857. 3. Ruscio, J. Z.; Kumar, D.; Shukla, M.; Prisant, M. G.; Murali, T. M.; Onufriev, A. V. Proc. Natl. Acad. Sci. USA 2008, 105, 9204-9209. 4. Ceccarelli, M.; Anedda, R.; Casu, M.; Ruggerone, P. Proteins 2008, 71, 1231-1236

Structural analysis of hemoglobins and myoglobins using MD simulations

In the family of respiratory proteins, hemoglobins and myoglobins have been the first to be crystallized in '50. Despite their precise 3D structures are available at high resolution, some questions regarding the microscopic functioning remain yet open. The R to T switching mechanism in hemoglobins and the ligand escape process in myoglobins remain still under debate. Thanks to the small size, myoglobin is the preferred candidate also for the more general structure-funcion paradigm. In the interior of myoglobin five main docking sites have been identified, especially with Xe NMR, and for long time these Xenon cavities have been classified as packing defects. Recently, it was shown that they might be involved in ligands migration path, even if mechanisms used by myoglobin to connect these cavities is still unknown as well as processes regulating its biologic functions. In this work we made use of standard MD simulations of solvated myoglobin to characterize internal cavities. Our principal results is that we have found several secondary cavities showing volume and occurrence at least comparable to that of Xenon cavities. In order to rationalize and in-depth analyze such a huge amount of data (ca. 30000 cavities/10 ns MD), special cluster-analysis was applied: we classified all cavities with respect to the position, size and occurrence as function of simulation time ascribing them to different clusters. This analysis implicitly highlights possible ligand migration paths for small ligands within the protein matrix allowing to quantitatively compare dynamical behaviour of different myoglobins towards different ligands. Our suggestion that the secondary cavities constitute the preferred path for ligand escape is also supported by explicit metadynamics simulations of ligand escape

Antimicrobial Dendrimeric Peptides: Structure, Activity and New Therapeutic Applications

Microbial resistance to conventional antibiotics is one of the most outstanding medical and scientific challenges of our times. Despite the recognised need for new anti-infective agents, however, very few new drugs have been brought to the market and to the clinic in the last three decades. This review highlights the properties of a new class of antibiotics, namely dendrimeric peptides. These intriguing novel compounds, generally made of multiple peptidic sequences linked to an inner branched core, display an array of antibacterial, antiviral and antifungal activities, usually coupled to low haemolytic activity. In addition, several peptides synthesized in oligobranched form proved to be promising tools for the selective treatment of cancer cells