Binding of Antimicrobial Lactoferricin Peptides to Targets in the Angiogenesis Pathway

Abstract

Peptides derived from lactoferricin B (LfB25; FKCRRWQWRMKKLGAPSITCVRRAF; +8), a 25-residue cationic innate immunity peptide released from bovine lactoferrin, exhibit broad spectrum antimicrobial and anti-angiogenic properties. An increase in drug-resistant bacteria and the role of angiogenesis in promoting tumor growth make LfB peptides attractive candidates for future drug development. An important principle for the design of peptide drugs is to reduce the number of amino acids and the sequence complexity, while maintaining maximal activity and minimal toxicity. LfB25 is proposed to inhibit angiogenesis, the formation of new blood vessels, by competing with fibroblast growth factor (FGF) for binding to negatively charged heparin sulfate proteoglycans on endothelial cell surfaces. Previously we used isothermal titration calorimetry (ITC) to characterize the binding of LfB25 and LfB6 (RRWQWR-NH2; +4; underlined above), the ‘antimicrobial core’ of LfB25, to heparin and sucrose octasulfate (SOS), a heparin analogue. The binding of LfB25 with SOS and heparin was found to fit a two site model, with Kd values on the order of 10−6 and 10−7; whereas the isotherms for LfB6 fit a single binding site model, with Kd values on the order of 10−6.We now report ITC binding assays for two new LfB peptides, LfB25-Ala3,20 and LfB11-Ala2,9 (KARRWQWRAKK-NH2; +7;sequence italicized above). To remove the disulfide bond, the two cysteines were replaced to give LfB25-Ala3,20. To reduce sequence length and complexity, in LfB11-Ala2,9 a cysteine and a methionine were changed to alanine, and the sequence was reduced to 11 residues. The binding of both LfB25-Ala3,20 and LfB11-Ala2,9 to SOS and heparin fit a single-site model, with Kd values on the order of 10−6, similar to those for LfB6. Results from antimicrobial and hemolytic assays will be presented.