ABSTRACT
The light chain of botulinum neurotoxin A (BoNT/A-LC) is a Zn-dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C-terminus of SNAP25 (residues 141-206) retains full activity for BoNT/A-LC-catalyzed cleavage at P1-P1' (Gln197-Arg198). Using the structure of a complex between the C-terminus of SNAP25 and BoNT/A-LC as a model to design SNAP25-derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala-Asn-Gln-Arg (residues 195-198) at the substrate cleavage site, with the intent to identify possible side-chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1-P1' residues to explore the spatial tolerance within the active-site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A-LC. Among the SNAPIs tested, several known cleavage-resistant, single-point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly-insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A-LC. Two of the Gly-insertion mutants exhibited 10-fold lower IC50 values than the wildtype 66-mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.
Subject(s)
Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Glycine/chemistry , Synaptosomal-Associated Protein 25/chemistry , Binding Sites/physiology , Catalytic Domain , Models, Molecular , Synaptosomal-Associated Protein 25/antagonists & inhibitors , Synaptosomal-Associated Protein 25/genetics , Synaptosomal-Associated Protein 25/metabolismABSTRACT
We report a one-pot synthesis of silver nanoparticle-polymer composites (Ag-PNCs) in water by a novel finding involving the polycondensation of methoxybenzyl chlorides (MeO-BzCl) directly on Ag nanoparticle surfaces at room temperature, leading to highly soluble antimicrobial nanocomposites. The composites, which are soluble in a range of organic solvents, precipitate in the reaction vessel, making their separation simple. Solutions of the composites can be casted directly on substrates or made into freestanding films. The material was found to be stable for nearly 2 years. A range of substrates have been shown to become antibacterial by direct application of this material. The experiments were conducted with Ag-PNC-loaded filter paper strips and glass substrates. The samples were found to be antimicrobial (against Escerichia coli and Aspergillus niger). The simple one-pot approach of this kind to make organic-soluble antibacterial coatings could have wide implications.
Subject(s)
Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/pharmacology , Metal Nanoparticles/chemistry , Nanotechnology/methods , Organic Chemicals/chemistry , Polymers/chemical synthesis , Silver/pharmacology , Aspergillus niger/drug effects , Escherichia coli/drug effects , Metal Nanoparticles/ultrastructure , Microbial Sensitivity Tests , Polymers/chemistry , Polymers/pharmacology , Solubility/drug effects , Spectrophotometry, UltravioletABSTRACT
OBJECTIVES: The aim of this study was to investigate the antimicrobial activities of calcium ions and other cross-linking agents of alginate dressings, as well as their compatibility with commonly used topical antimicrobials. METHODS: The antimicrobial activities of cross-linking agents and antimicrobials (five antibiotics and four antiseptics) were evaluated by the broth dilution method. The interactions between individual cross-linking agents and antimicrobials were evaluated using the chequerboard test against common skin pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. RESULTS: From the MIC determined, antibiotics were the most active, followed by the antiseptics and cross-linking agents. Calcium ions, which are commonly used to cross-link alginate, exhibited very weak antimicrobial activity and higher fractional inhibitory concentration than the other cross-linking agents. The use of calcium and gentamicin resulted in antagonism against S. aureus. In contrast, aluminium, zinc and copper ions exhibited higher antimicrobial activities but insignificant interactions with the antimicrobials. CONCLUSIONS: Commonly used topical antimicrobials that are active against the skin pathogens S. aureus and P. aeruginosa could be potentially incompatible with calcium alginate dressings. Copper, zinc and aluminium ions are more suitable cross-linking agents for alginate as they do not show antagonism with the antimicrobials and could impart antimicrobial property to the resultant dressing.