De Novo Design of Selective Antibiotic Peptides by Incorporation of Unnatural Amino Acids

Abstract

The evolution of drug-resistant bacteria is one of the most critical problems facing modern medicine and requires the development of new drugs that exhibit their antibacterial activity via novel mechanisms of action. One potential source of new drugs could be the naturally occurring peptides that exhibit antimicrobial activity via membrane disruption. To develop antimicrobial peptides exhibiting increased potency and selectivity against Gram positive, Gram negative, and Mycobacterium bacteria coupled with reduced hemolytic activity, peptides containing unnatural amino acids have been designed, synthesized, and evaluated. These compounds were designed on the basis of the electrostatic surface potential maps derived from the NMR determined SDS and DPC micelle-bound conformations of (Ala8,13,18)magainin-2 amide. Unnatural amino acids were incorporated into the polypeptide backbone to control the structural and physicochemical properties of the peptides to introduce organism selectivity and potency. The methods and results of this investigation are described below.

Effect of Liposome Surface Charge and Peptide Side Chain Charge Density on Antimicrobial Peptide-Membrane Binding as Determined by Circular Dichroism

“…Adopting different conformations on binding to zwiterionic and anionic membrane models is a requirement for organism selectivity. This is because changing the conformation of an AMP changes its amphipathic nature which in turn changes its physicochemical surface properties [27,43]. The changes in the conformation/physicochemical properties of the AMP will lead to different mechanisms of interaction with zwitter ionic and anionic liposomes and thus to organism selectivity [32,[44][45][46].…”

“…The AMPs used in these investigation were synthesized either manually using tBOC chemistry or with an automated peptide synthesizer using FMOC chemistry [30,31] as previously reported [27,28,32].…”

“…The electrostatic interactions that occur between an AMP and the surface of a phospholipid membrane are believed to be the dominant physicochemical property associated with organism selectivity [21The intent of this investigation is to determine the effect of varying the surface charge and hydrophobicity of liposomes of mixed phospholipid compositions on the binding of four of the AMPs developed in our laboratory in order to shed light on the physicochemical requirements for membrane selectivity. The peptide structure of the AMPs in this study incorporated three dipeptide units consisting of the conformationally restrained unnatural amino acids Tic (Tetrahydroisoquinolinecarboxylic acid)-Oic (Octahydroindolecarboxylic acid) into the peptide's primary sequence, Ac-GF-Tic-Oic-GX-Tic-Oic-GF-Tic-Oic-GX-Tic-XXXX-CONH 2 , where X may be one of the following basic amino acids, Lys, Orn, Dab, Dpr or Arg [27]. The Tic-Oic dipeptide units induce local regions of reduced molecular flexibility onto the peptide backbone and thus result in a reduction of the total conformational flexibility of the peptide during lipid binding.…”

“…This distance is involved in determining the overall surface charge density as well as defining the distance between the membrane surface and the polypeptide backbone. The amino acid sequences of the AMPs used in this investigation are given in Table 1 [27,28].…”

The Design of Bacteria Strain Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids

“…There is a developing preponderance of evidence in the literature supporting the concept that the selectivity and potency of a specific AMP is determined in a large measure by the chemical composition of the target membrane. (Powers & Hancock, 2003;Yeaman & Yount, 2003) In our laboratory we have focused on developing antimicrobial peptides that contain unnatural amino acids to control the conformational and physicochemical properties (Hicks, Bhonsle, Venugopal, Koser, & Magill, 2007) of the resulting peptide. Our original skeletal design of an unnatural AMP incorporated placement of three L-Tic-L-Oic dipeptide units into the polypeptide backbone to induce an ordered structure onto the peptide .…”