DOI: 10.1002/cbic.201300243 Iterative Antimicrobial Candidate Selection from Informed d-/l-Peptide Dimer Libraries Roman J. Lichtenecker, [a, b] Bernhard Ellinger, [a, c] Hong-Mei Han, [d] Kirtikumar B. Jadhav, [a, e] Sascha Baumann, [a, f] Oliwia Makarewicz, [g] Markus Grabenbauer, [d] and Hans- Dieter Arndt* [a, e] Introduction Antimicrobial peptides (AMPs) endow bacteria, as well as many insects, plants, and vertebrates, with a first line of defense and are considered part of the innate immune system. [1] AMPs target preferentially the cell membranes of bacteria, resulting in a fast mode of action, broad spectrum activity, and slow build-up of resistance. Emerging resistance to commonly used antibiotics stimulated therapeutic studies with AMPs [2, 3] and even led to clinical candidates. [2, 3, 4] Furthermore, a variety of non-natural AMP mimetics were studied as possible antimicro- bials, [5] including peptoids, [6] b-peptides, [7, 8, 9] arylamides, [10] or aggregating d-/l-cyclopeptides. [11, 12] On the other hand, syn- thesizing naturally occurring AMPs with single d-configured residues reduces their often pronounced sensitivity to proteol- ysis [13, 14] but was reported to diminish potency by destabilizing their active conformations. [5, 15] Interestingly, peptide natural products featuring the rare syndiotactic alteration of backbone stereochemistry (d-/l-pep- tides) appear to have enriched antimicrobial activity. Examples include the well-known gramicidin A, [16, 17] but also ramopla- nin, [18] feglymycin, [19] and the large, ribosomally synthesized peptide polytheonamide. [20, 21] Gramicidin A (gA), a potent anti- biotic produced by Bacillus brevis during sporulation, [16, 17] forms small pores in cell membranes after noncovalent dimeri- zation, rendering them permeable by small inorganic cations and water. [22–24] As a result, gA is strongly hemolytic and cannot be systemically applied, but some synthetic variants with improved properties have recently been reported. [25] On the conformational level, gA is promiscuous and adopts differ- ent b-helical folds in organic solvents and lipid bilayers, prefer- ring unique double-helical dimers with left- or right-handed topology. [23, 26, 27] Several synthetic models of d-/l-peptides strengthened this trend [24, 28–31] and were recently used for shaping membrane-embedded nanoenvironments. [32] Covalently linking two d-/l-peptide strands supports the formation of b 5.6 -double helices [24, 28–31] that are topologically distinct from l-peptide a 3.6 - or d-/l-peptide b 6.3 -single helices (Figure 1). Such membrane-associated d-/l-peptides could pre- Growing resistance to antibiotics, as well as newly emerging pathogens, stimulate the investigation of antimicrobial pep- tides (AMPs) as therapeutic agents. Here, we report a new li- brary design concept based on a stochastic distribution of nat- ural AMP amino acid sequences onto half-length synthetic peptides. For these compounds, a non-natural motif of alter- nating d- and l-backbone stereochemistry of the peptide chain predisposed for b-helix formation was explored. Synthet- ic d-/l-peptides with permuted half-length sequences were delineated from a full-length starter sequence and covalently recombined to create two-dimensional compound arrays for antibacterial screening. Using the natural AMP magainin as a seed sequence, we identified and iteratively optimized hit compounds showing high antimicrobial activity against Gram- positive and Gram-negative bacteria with low hemolytic activi- ty. Cryo-electron microscopy characterized the membrane-as- sociated mechanism of action of the new d-/l-peptide antibi- otics. [a] Dr. R. J. Lichtenecker, Dr. B. Ellinger, K. B. Jadhav, Dr. S. Baumann, Prof. Dr. H.-D. Arndt Department of Chemical Biology Max Planck Institute of Molecular Physiology Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) [b] Dr. R. J. Lichtenecker Current address: Institute of Organic Chemistry, University of Vienna Währingerstrasse 38, 1090 Wien (Austria) [c] Dr. B. Ellinger Current address: European Screening Port GmbH Schnackenburgallee 114, 22525 Hamburg (Germany) [d] Dr. H.-M. Han, Dr. M. Grabenbauer Department of Systemic Cell Biology Max Planck Institute of Molecular Physiology Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) [e] K. B. Jadhav, Prof. Dr. H.-D. Arndt Institute of Organic and Macromolecular Chemistry Friedrich Schiller University Humboldtstrasse 10, 07743 Jena (Germany) E-mail : hd.arndt@uni-jena.de [f] Dr. S. Baumann Current address: Helmholtz-Institut für Pharmazeutische Forschung Saarland Campus, Geb. C2.3, Universität des Saarlandes 66123 Saarbrücken (Germany) [g] Dr. O. Makarewicz Center for Infectious Diseases, Jena University Hospital Erlanger Allee 101, 07747 Jena (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201300243.  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemBioChem 2013, 14, 2492 – 2499 2492 CHEMBIOCHEM FULL PAPERS