Balancing electrostatic interactions and steric hindrance for the optimization of

University Of Wisconsin La Crosse, La Crosse WI

Investigators

Abstract

DESCRIPTION (provided by applicant): Persistent infections caused by microbes that are resistant to existing antibiotics are a serious threat to public health worldwide. Peptide antibiotics offer one promising solution because they act by perturbing the membrane barrier and thus organisms are less likely to develop resistance to these drugs. The two critical characteristics of existing natural and designed peptide antibiotics are (a) an ability to form ordered structures such as helices, and (b) amphiphilicty, often in the form of some cationic character. The goal of this application is to determine whether the specific placement of charged amino acids can be used in Aib-rich peptides to modulate helical stability, and ultimately antibiotic activity and protease resistance. Aib (1-aminoisobutyric acid) is a naturally occurring 1,1-dialkylated amino acid in bacteria and is well-established in its tendency to promote and stabilize helical conformations due to the steric hindrance at the 1-carbon. Incorporation of the Aib into designed peptide antibiotics has been shown to enhance helicity, increase protease resistance and maintain or even enhance bioactivity. However, while Aib-containing peptides with antimicrobial capabilities and protease resistance have been achieved, these peptides have demonstrated only a moderate degree of helicity in water, likely due in part to the spacing of charged amino acids and in part to the dilute Aib composition. In order to elucidate the roles of steric hindrance and electrostatic interactions on helical stability, a series of six octapeptides will be prepared and their structures characterized using NMR and CD spectroscopies. Isothermal titration calorimetry (ITC) will be used to determine helical stability. The six peptides differ in the positioning (contiguous placement versus one turn apart) and type (alanine, lysine, glutamate) of two guest monoalkylated residues in the sequence. Preliminary evidence indicates that contiguous placement of Ala residues results in a central helical hydrogen-bond that is vulnerable to competition from the solvent due to reduced steric constraints, and that the helical hydrogen-bond breaks in strongly hydrogen-bonding solvents. NMR spectroscopy will be used to determine the three-dimensional structures of the target peptides on an atomic level, and to determine whether particular charge placements exacerbate or alleviate the vulnerable hydrogen-bond. Because the peptide structures will be known from NMR, the CD spectra will provide important information to the field on the magnitude of Aib-rich helical CD signatures, an area that is still the subject of some debate. ITC experiments will reveal the entropic and enthalpic components of hydrogen-bond breakage, and will provide a direct measure of helical stability that is unavailable by other methods for Aib-rich peptides. Ultimately the results of this research will be used to develop design principles for the preparation of more effective antibiotic peptides as eventual therapeutic agents. PUBLIC HEALTH RELEVANCE: Persistent infections caused by microbes that are resistant to existing antibiotics are a serious threat to public health worldwide. Peptide antibiotics offer one promising solution because organisms are less likely to develop resistance to these drugs. This research aims to develop design principles for the preparation of more effective antibiotic peptides as eventual therapeutic agents.