Novel α-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity

et al. (2013) Novel a-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity. PLoS ONE 8(4): e61614. doi:10.1371/journal.pone.0061614 Novel a-MSH Peptide Analogues with Broad Spectrum Antimicrobial Activity Paolo Grieco 0 Alfonso Carotenuto 0 Luigia Auriemma 0 Antonio Limatola 0 Salvatore Di Maro 0 Francesco Merlino 0 Maria Luisa Mangoni 0 Vincenzo Luca 0 Antonio Di Grazia 0 Stefano Gatti 0 Pietro Campiglia 0 Isabel Gomez-Monterrey 0 Ettore Novellino 0 Anna Catania 0 Gunnar F. Kaufmann, The Scripps Research Institute and Sorrento Therapeutics, Inc., United States of America 0 1 Department of Pharmacy, University of Naples Federico II , Naples , Italy , 2 Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Biochemical Science, 'A. Rossi Fanelli', University of Rome 'La Sapienza' , Rome , Italy , 3 Center for Preclinical Investigation, Fondazione IRCCS Ca'Granda - Ospedale Maggiore Policlinico , Milano , Italy , 4 Department of Pharmaceutical Science, University of Salerno , Fisciano, Salerno , Italy Previous investigations indicate that a-melanocyte-stimulating hormone (a-MSH) and certain synthetic analogues of it exert antimicrobial effects against bacteria and yeasts. However, these molecules have weak activity in standard microbiology conditions and this hampers a realistic clinical use. The aim in the present study was to identify novel peptides with broadspectrum antimicrobial activity in growth medium. To this purpose, the Gly10 residue in the [DNal(29)-7, Phe-12]-MSH(6-13) sequence was replaced with conventional and unconventional amino acids with different degrees of conformational rigidity. Two derivatives in which Gly10 was replaced by the residues Aic and Cha, respectively, had substantial activity against Candida strains, including C. albicans, C. glabrata, and C. krusei and against gram-positive and gram-negative bacteria. Conformational analysis indicated that the helical structure along residues 8-13 is a key factor in antimicrobial activity. Synthetic analogues of a-MSH can be valuable agents to treat infections in humans. The structural preferences associated with antimicrobial activity identified in this research can help further development of synthetic melanocortins with enhanced biological activity. - Funding: This work was supported by Italian Ministry of Education, University and Research (PRIN 2008, 2010MCLBCZ_002) and by Fondazione Fiera Milano, Italy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. a-Melanocyte stimulating hormone (a-MSH), a pro-opiomelanocortin (POMC) derivative, is an ancient tridecapeptide that exerts pleiotropic influences on the host physiology [1]. A major contribution of a-MSH to tissue protection resides in its capacity to prevent cell injury induced by harmful stimuli including endotoxin, reperfusion injury, blood loss, and oxidative stress [1,2]. Of interest, a-MSH involvement in host defense includes antimicrobial activity [3,4]. Indeed, the peptide and its C-terminal sequence Lys-Pro-Val were found to inhibit growth of both the yeast Candida albicans and the gram-positive bacterium Staphylococcus aureus [4]; further, the N-terminal sequence His-Phe-Arg-Trp showed antimicrobial activity against Cryptococcus neoformans [5]. Although the natural a-MSH peptide has a very short half-life that makes it unsuitable for clinical use, synthetic analogues of it could form the basis for novel antimicrobial agents. However, key issues need to be solved before a therapeutic use is realistic. A crucial question concerns potency of the antimicrobial activity. Indeed, a-MSH and the synthetic derivatives described to date were found to exert their activity against infectious agents that were suspended in physiologic solution or in water but not in culture media that allow microorganisms to grow [4,6,7,8,9,10]. Therefore, although data indicate a potential for melanocortin derivatives to combat infections, none of the known molecules is active against microorganisms in growth medium. Because this is a major obstacle to a realistic clinical use, the aim in this research was to design novel melanocortin analogues that could overwhelm this weakness. The lead sequence selected was [DNal(29)-7, Phe12]MSH(613) (DNal), a promising antimicrobial compound that contains the invariant core sequence His-Phe-Arg-Trp (69) common to all melanocortins [7]. Indeed, although DNal did not kill Candida in growth medium, it did kill 100% organisms incubated in distilled water [7]. Conformational analysis of DNal indicated the presence of two b-turns along residues 710 and 10 13 (type I, and distorted type III, respectively) [11]; these two turns, which likely form the pharmacophoric moieties, are linked by a highly flexible glycine residue that can orient them differently [11]. Therefore, in the present research we produced novel antimicrobial peptides based on replacement of Gly10 in the DNal sequence with natural and unnatural amino acids with different degrees of conformational rigidity (Table 1). Activity against various pathogenic agents including gram-positive and gramnegative bacteria and yeasts was assessed using standard culture tests. We report results of a conformational study on the most effective peptides. Peptide Sequence H-His-D(29)Nal-Arg-Trp-Gly-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Aib-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Deg-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-tBuGly-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Ac3c-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Ac4c -Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Ac5c-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Ac6c -Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Aic-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Phe-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Cha-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-bAla-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Acpc-Lys-Phe-Val-NH2 H-His-D(29)Nal-Arg-Trp-Gly-Gly-Lys-Phe-Val-NH2 Materials and Methods Materials N9-fluorenylmethoxycarbonyl (Fmoc)-protected natural amino acids were purchased from GLS Biochem (Shangai-China), NFmoc-protected special amino acids from NeoMPS (StasbourgFrance) and Chem-Impex International, Inc. (Wood Dale-Illinois), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) from Iris Biotech GmbH (Marktredwitz-Germany) and Rink amide resin from Advanced Chemtech (Lousville-KY). For the NFmocprotected amino acids, the following side chain protecting groups were used: Arg(Nc-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5sulfonyl (Pbf)), His(Nim- triphenylmethyl(trityl) (Trt)), Trp(Nin- tertbutyloxycarbonyl (Boc)), Lys(Boc). Peptide synthesis solvents, reagents, as well as CH3CN for HPLC were reagent grade and were acquired from commercial sources and used without further purificati (...truncated)