De novo design of selective antibiotic peptides by incorporation of unnatural amino acids.

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.

The anthrax protective antigen (PA63) bound conformation of a peptide inhibitor of the binding of lethal factor to PA63: as determined by trNOESY NMR and molecular modeling.

Anthrax protective antigen (PA) is one of the three proteins produced by the gram positive bacteria Bacillus anthracis collectively known as the "anthrax toxin" (Ascenzi, P.; Visca, P.; Ippolito, G.; Spallarossa, A.; Bolognesi, M.; et al. Anthrax toxin: a tripartite lethal combination. FEBS Lett. 2002, 531, 384-388). The role played by PA in anthrax intoxication is to transport the two enzymes lethal factor (LF) and edema factor (EF) into the cell. Collier and co-workers (Mourez, M.; Kane, R. S.; Mogridge, J.; Metallo, S.; Deschatelets, P.; et al. Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 2001, 958). reported the isolation of two peptides via phage display that bind to the PA63 heptamer and inhibit its interaction with LF and EF, and thereby prevent the transport of LF and EF into the cell. One of these peptides, His-Thr-Ser-Thr-Try-Trp-Trp-Leu-Asp-Gly-Ala-Pro (P1), was selected for structural investigation on the basis of its ability to prevent the binding of LF to the PA63 heptamer bundle. Two-dimensional trNOESY experiments coupled with NOE restrained simulated annealing calculations were used to determine the PA63-bound conformation of P1. On binding to PA63, P1 adopts a helical conformation involving residues 3-9 while the C- and N-terminal residues exhibit dynamic fraying.

Nuclear magnetic resonance and molecular modeling analysis of the interaction of the antimalarial drugs artelinic acid and artesunic acid with beta-cyclodextrin.

The artemisinin derivatives artelinic acid and artesunic acid are members of a class of compounds that have shown promise for the treatment of multidrug resistant strains of Plasmodium falciparum. Unfortunately, these compounds exhibit poor solubility and stability in aqueous solution. The research presented herein was conducted to determine whether complexation of artelinic acid or artesunic acid with beta-cyclodextrin would result in complexes with increased aqueous solubility while retaining the potent antimalarial activity of these compounds. Preliminary complexation studies with natural beta-cyclodextrins were conducted as a proof of concept, with a primary focus on understanding the electrostatic interactions that stabilize the resulting complexes. Complex formation was monitored using UV spectroscopy. The structures of the resulting complexes were determined using multidimensional nuclear magnetic resonance spectroscopy (NMR) and molecular modeling. NMR results are most consistent for artelinic acid and beta-cyclodextrin forming complexes in a ratio of 2:1; however, the presence of 1:1, 2:2, and 3:1 complexes in solution cannot be excluded based on the experimental data collected. The NMR data also indicate selective insertion of artelinic acid into the hydrophobic cavity of the beta-cyclodextrin via the primary face. NMR results indicate artesunic acid forms a similar complex with beta-cyclodextrin in a ratio of 1:1; again however, the presence of 1:1, 2:2, and 3:1 complexes in solution cannot be ruled out.

Comparison of the conformation and electrostatic surface properties of magainin peptides bound to sodium dodecyl sulfate and dodecylphosphocholine micelles.

The role played by noncovalent interactions in inducing a stable secondary structure onto the sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelle-bound conformations of (Ala(8,13,18))magainin 2 amide and the DPC micelle bound conformation of magainin 1 were determined. Two-dimensional NMR and molecular modeling investigations indicated that (Ala(8,13,18))magainin 2 amide bound to DPC micelles adopts a alpha-helical secondary structure involving residues 2-16. The four C-terminal residues converge to a lose beta-turn structure. (Ala(8,13,18))magainin 2 amide bound to SDS miscelles adopts a alpha-helical secondary structure involving residues 7-18. The C- and N-terminal residues exhibited a great deal of conformational flexibility. Magainin 1 bound to DPC micelles adopts a alpha-helical secondary structure involving residues 4-19. The C-terminal residues converge to a lose beta-turn structure. The results of this investigation indicate hydrophobic interactions are the major contributors to stabilizing the induced helical structure of the micelle-bound peptides. Electrostatic interactions between the polar head groups of the micelle and the cationic side chains of the peptides define the positions along the peptide backbone where the helical structures begin and end.