pubs.acs.org/Biochemistry Published on Web 05/11/2009 r 2009 American Chemical Society 5642 Biochemistry 2009, 48, 5642–5657 DOI: 10.1021/bi900272r Antimicrobial Action of Prototypic Amphipathic Cationic Decapeptides and Their Branched Dimers † Pooja C. Dewan, ‡ Aparna Anantharaman, Virander S. Chauhan, and Dinkar Sahal* Malaria Research Laboratory, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India ‡ Present address: Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India Received May 5, 2008; Revised Manuscript Received May 8, 2009 ABSTRACT: Toward delineation of antimicrobial action, a prototypic amphipathic, cationic decapeptide Ac-G-X-R-K-X-H-K-X-W-A-NH 2 was designed and peptides for which X was didehydrophenylalanine (ΔFm), R-aminoisobutyric acid (Um), or phenylalanine (Fm) were synthesized. A growth kinetics experiment indicated that the bacteriostatic effects were nil (Um), mild and transient (Fm), and strong and persistent (ΔFm) respectively. Though at par in binding to lipopolysaccharide, ΔFm and Fm, but not Um, caused outer membrane permeabilization. Inner membrane permeabilization was attenuated and membrane architecture rehabilitated with ΔFm but not Fm. Reverse phase high-performance liquid chromatography revealed that ΔFm was translocated into Escherichia coli, while Um and fragments of Fm were detected in the medium. Among these monomers, only ΔFm was modestly antibiotic [minimum inhibitory concentrations (MICs) of 110 μM(E. coli) and 450 μM(Staphylococcus aureus)]. Interestingly, a linear dimer of ΔFm, viz. (ΔFm) 2 , turned out to be highly potent against E. coli [MIC of 2 μM and minimum bactericidal concentration (MBC) of 2 μM] and modestly potent against S. aureus (MIC of 20 μM and MBC of 20 μM). In contrast, a lysine- based branched dimer of ΔFm, viz. ΔFd, was found to be a potent antimicrobial against both E. coli (MIC of 2.5 μM) and S. aureus (MIC of 5 μM). Studies with analogous branched dimers of Fm and Um have indicated that dimerization represents a scaffold for potentiation of antimicrobial peptides and that the presence of ΔF confers potent activity against both E. coli and S. aureus. De novo design has identified ΔFd as a potent, noncytotoxic, bacterial cell-permeabilizing and -penetrating antimicrobial peptide, more protease resistant than its monomeric counterpart. We report that in comparison to the subdued and sequential “membrane followed by cell interior” mode of action of the monomeric ΔFm, the strong and simultaneous “membrane along with cell interior” targeting by the dimeric ΔFd potentiates and broadens its antibiotic action across the Gram-negative-Gram-positive divide. The arrival of antibiotics was one of the greatest milestones in human and animal health. Today, however, the advent of antibiotic resistance to virtually every known class of natural and synthetic compounds (1, 2) represents a serious problem in clinical medicine. This calls for an urgent need to discover newer designs of antibiotics with novel mechanisms of action. Species across the evolutionary tree produce a large repertoire of anti- biotic peptides (3), sequences of several hundred of which are now in the database http://www.bbcm.univ.trieste.it/∼tossi/antimic. html. However, for reasons of low potency, toxicity, high cost, or short half-life (4), Nature’s peptide antibiotics have not found much use in clinical practice. Different approaches are being used to counter these problems which include preparation of analogues of naturally existing antimicrobial peptides (AMPs) 1 (5), screening of combinatorial libraries (6), and de novo design of AMPs (7). However, a thorough understanding of the mode of action of AMPs is a prerequisite for the rational design of novel AMPs. Various structure-activity relationship studies on AMPs (8) and evasion strategies developed by resistant † This research was funded through a grant (BT/PR3325/BRB/10/283/ 2002) to D.S. from the Department of Biotechnology, Government of India. Research fellowships of the Council of Scientific and Industrial Research, Government of India, to P.C.D. and A.A. are acknowledged. *To whom correspondence should be addressed. Phone: 91-11-2674 1358. Fax: 91-11-2616 2316. E-mail: dinkar@icgeb.res.in. 1 Abbreviations: AMPs, antimicrobial peptides; ATCC, American type Culture Collection; CFU, colony forming units; FACS, fluores- cence-activated cell sorting; FITC, fluorescein isothiocyanate; Fmoc, fluorenyl methoxy carbonyl; LPS, lipopolysaccharide; MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration; PXB, polymyxin B; RPHPLC, reverse phase high-performance liquid chromatography; SEM, scanning electron microscopy; TEM, transmis- sion electron microscopy; Fm, ΔFm, and Um, monomeric peptides with phenylalanine (F), didehydrophenylalanine (ΔF), and R-aminoisobu- tyric acid (U), respectively, in the X positions of the peptide sequence Ac-G-X-R-K-X-H-K-X-W-A-NH 2 ; Fd, ΔFd, and Ud, branched di- meric peptides with phenylalanine (F), didehydrophenylalanine (ΔF), and R-aminoisobutyric acid (U), respectively, at the X positions of the corresponding dimeric peptide sequences; D-Lys-ΔFd, nonhelical di- meric analogue of ΔFd in which lysines 4 and 7 are in the D configura- tion.