Published: June 22, 2011 r2011 American Chemical Society 5237 dx.doi.org/10.1021/jm200701g | J. Med. Chem. 2011, 54, 5237–5244 ARTICLE pubs.acs.org/jmc Refining the Eosinophil Cationic Protein Antibacterial Pharmacophore by Rational Structure Minimization Marc Torrent, †,‡ David Pulido, † Beatriz G. de la Torre, ‡ M. Flor García-Mayoral, § M. Vict oria Nogu  es, † Marta Bruix, § David Andreu,* ,‡ and Ester Boix* ,† † Department of Biochemistry and Molecular Biology, Universitat Aut onoma de Barcelona, E-08193 Cerdanyola del Vall  es, Spain ‡ Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Dr. Aiguader, 88, E-08003 Barcelona, Spain § Rocasolano Institute of Physical Chemistry, CSIC, Serrano 119, 28006 Madrid, Spain b S Supporting Information ’ INTRODUCTION An alarming increase in bacterial resistance to classical anti- biotics has become a serious concern among health professionals and spurred intense efforts toward the development of new anti- microbial leads. In this context, antimicrobial peptides (AMPs) are viewed as promising candidates because of their substantial potency, broad spectrum, and distinct mechanism of action. 1 AMPs target bacterial membranes to which they are driven by mainly electrostatic interactions and which upon binding they disrupt, collapsing transmembrane gradients and eventually causing cell death. 2 Bacterial strategies for resisting AMPs require substantial membrane (phospholipid and protein) remodeling, a demanding task that explains the very low incidence of AMP resistance in bacteria. 3 A number of AMPs are fragments of larger proteins, either naturally derived by proteolysis (e.g., the cathelicidins 4 ) or derived by peptide synthesis approaches. 5 In the latter case, educated deconstruction of complex antimicrobial proteins has allowed the identification of structural features essential for bioactivity. 6,7 Although this pharmacophore dissection process is not straightforward and still requires a substantial amount of trial- and-error, it is worthwhile in that defining such minimal struc- tural motifs provides helpful clues for developing therapeutically useful AMP templates. 8 Eosinophil cationic protein (ECP, RNase 3) is a secretion ribonuclease used as a model for the potential involvement of mammalian RNases in the host defense system. 9,10 Expressed mainly in eosinophils and selectively released at the inflammation area, 11,12 ECP is reportedly involved in immunoregulation and tissue remodeling processes. 13,14 Its broad antibacterial spectrum includes both Gram-negative and -positive strains at a low micro- molar range. 15 Although its mechanism of action is not com- pletely understood, ECP has been described to act through a carpet-like mechanism, causing membrane destabilization and subsequent vesiculation. 16 ECP also has high affinity for bacterial cell wall components, such as lypopolysaccharide and peptido- glycans, 17 and a strong tendency to aggregate Escherichia coli cells. 18 A previous attempt to delineate the antibacterial domain of ECP led to the identification of the N-terminus (residues 145) as the main antibacterial domain of the protein. 19 We have probed deeper into this region and in this paper describe how antimicro- bially equipotent analogues of substantially reduced size (and con- sequently synthetic difficulty and costs) can be successfully derived from ECP by a structural minimization approach. ’ RESULTS AND DISCUSSION Peptide Design and Synthesis. The starting point for a simplified ECP-derived antimicrobial lead candidate was the Received: June 2, 2011 ABSTRACT: Sequence analysis of eosinophil cationic protein (ECP), a ribonuclease of broad antimicrobial activity, allowed identification of residues 145 as the antimicrobial domain. We have further dissected ECP(145) with a view to defining the minimal requirements for antimicrobial activity. Structure- based downsizing has focused on both R-helices of ECP(145) and yielded analogues with substantial potency against Gram- negative and -positive strains. Analogues ECP(836) and ECP(617)-Ahx-(2336) (Ahx, 6-aminohexanoic acid) involve 36% and 40% size reduction relative to (145), respectively, and display a remarkably ECP-like antimicrobial profile. Both retain segments required for self-aggregation and lipolysaccharide binding, as well as the bacterial agglutination ability of parent ECP. Analogue (617)-Ahx-(2336), in particular, is shown by NMR to preserve the helical traits of the native 816 (R1) and 3336 (R2) regions and can be proposed as the minimal structure capable of reproducing the activity of the entire protein.