Thermodynamic and Spectroscopic Investigations of Novel Antimicrobial Peptides containing Unnatural Amino Acids with Model Membrane Systems

Abstract

With the over prescription and use of antibiotic drugs the evolution of drug resistant bacteria strains has become a world wide health crisis. In response to this crisis, the search for new drugs to kill bacteria via a novel mode of action has become of critical importance to modern medicine. Natural and synthetic antimicrobial peptides (AMPs) have exhibited a broad spectrum of activity against various infectious microorganisms while exhibiting little to no mammalian cell toxicity. It's because of this selectivity that AMPs are considered as a potential new source of therapeutics for drug-resistant bacteria. It is believed that the selectivity for bacteria cell membranes over host cells and the potency between different bacteria strains is derived from the ability of the AMPs to exploit the differences in chemical composition of various organism's membranes. The hypothesis guiding this research states: The physicochemical surface properties of the target cell's membrane interact with the 3D physicochemical surface properties of the approaching AMP inducing a conformational change onto the AMP maximizing the interactions between the two to facilitate AMP-membrane binding and the formation of pores leading to intracellular leakage and cell death. There are two binding interactions that are of interest, the S-state and I-state. The S-state, or surface state, refers to the binding of the peptide to the surface of the membrane, while the I-state involves the insertion of the peptide into the bilayer. By understanding the interactions of various AMPs with different target cell membranes one can develop novel analogs with increased potency and selectivity for a particular strain of bacteria. This knowledge would eventually lead to the design of antimicrobial peptides used as pharmaceuticals to treat bacteria resistant to current antibiotics. In our laboratory we have developed a series of antimicrobial peptides that incorporate unnatural amino acids to impart specific physicochemical properties onto these peptides. Circular Dichroism (CD), isothermal titration calorimetry (ITC) and calcein leakage assays were conducted and it was determined that there are two distinctly different mechanisms of binding occurring between this series of peptides and zwitterionic and anionic membrane models. By understanding the interactions of various AMPs with different target cell membranes one can develop novel analogs with increased potency and selectivity.