pubs.acs.org/jmc Published on Web 08/05/2009 r 2009 American Chemical Society J. Med. Chem. 2009, 52, 7421–7431 7421 DOI: 10.1021/jm900622d Discovery of Trp-His and His-Arg Analogues as New Structural Classes of Short Antimicrobial Peptides Rohit K. Sharma, † Ravi P. Reddy, † Werner Tegge, ‡ and Rahul Jain* ,† † Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, SAS Nagar 160 062, Punjab, India, and ‡ Department of Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany Received May 4, 2009 Naturally occurring antimicrobial peptides contain a large number of amino acid residues, which limits their clinical applicability. In search of short antimicrobial peptides, which represent a possible alternative for lead structures to fight antibiotic resistant microbial infections, a series of synthetic peptide analogues based on Trp-His and His-Arg structural frameworks have been prepared and found to be active against several Gram-negative and Gram-positive bacterial strains as well as against a fungal strain with MIC values of the most potent structures in the range of 5-20 μg/mL ((IC 50 in the range of 1-5 μg/mL). The synthesized peptides showed no cytotoxic effect in an MTT assay up to the highest test concentration of 200 μg/mL. A combination of small size, presence of unnatural amino acids, high antimicrobial activity, and absence of cytotoxicity reveals the synthesized Trp-His and His-Arg analogues as promising candidates for novel antimicrobial therapeutics. Introduction Microbial infections continue to be a major cause of morbidity and mortality worldwide. Moreover, the problems have been continuously exacerbated by widespread antibiotic resistance, emergence of new pathogens in addition to the resurgence of old ones, and the lack of effective new ther- apeutics. The scientific efforts of the last 50 years centered around about a dozen antimicrobial core chemotype scaf- folds. 1,2 The major ones among these, which have become available on the market, are the oxazolidinone synthetic core (e.g., linezolid), the lipopeptides (e.g., daptomycin), and the ketolides (e.g., telithromycin), which are modified macro- lides. 3,4 In the recent years, many reports in the literature have suggested that naturally occurring antimicrobial peptides (AMPs a ), which constitute a major component of the innate self-defense system, have the potential to represent such a class of antibiotics. 5-7 These AMPs are not only lethal to a broad spectrum of pathogens but also have a unique low tendency for resistance development. Although the exact mode of action of AMPs is still not completely understood, it has been well established that AMPs interact with the cell membrane of susceptible microorganisms, where either their accumulation in the membrane causes increased permeability and loss of barrier function or they cross the membrane to access cytoplasmic targets. 8,9 The selective action of generally posi- tively charged AMPs is demonstrated by preferential interac- tion with the anionic phospholipids of the bacterial cell membrane rather than with the neutral mammalian cell membrane, which is made of zwitterionic phospholipids and cholesterol. 10-12 However, in spite of the rapid action of naturally occurring AMPs against a broad spectrum of microorganisms and the fact that the development of resistance by the microorganisms against them is slow because it requires substantial changes in the lipid composition of cell membranes of the microorgan- isms, there are some serious drawbacks that limit their practical use. One main disadvantage with naturally occurring AMPs is their large size, which poses several challenges regarding synthesis, metabolic stability, immunogenicity, bioavailability, route of administration, and production costs. One approach that could be adopted to resolve these problems is to design and develop smaller synthetic peptidomimetics having unnatural residues without compromising the mini- mum requirement for being antimicrobial in nature. Thus, the focus of the present work has been to discover small synthetic antimicrobial peptidomimetics that can ultimately led to promising candidates for novel antimicrobial therapeutics. In our earlier research endeavor that has been recently reported, 13 we tried to ascertain the minimum pharmaco- phore by employing the HipHop module 14 of the software package CATALYST (Accelrys, San Diego, CA). For this purpose, the experimental data reported by Svendsen and co- workers was used wherein the synthesis and evaluation of smaller cationic AMPs composed of two to six natural and synthetic amino acids has been reported. 15,16 In absence of any substantial report regarding the presence of a particular receptor or protein target for cationic AMPs, the study was restricted to include only positively ionizable (PI) and hydro- phobic (HYD) features for pharmacophore generation, as *To whom correspondence should be addressed. Phone: þ91-172- 2214682. Fax: þ91-172-2214692. E-mail: rahuljain@niper.ac.in. a Abbreviations: AMP, antimicrobial peptide; CDI, 1,1 0 -carbonyldii- midazole; DCC, N,N 0 -dicyclohexylcarbodiimide; DIC, N,N 0 -diisopro- pylcarbodiimide; HPLC, high-pressure liquid chromatography; HONB, endo-N-hydroxy-5-norbornene-2,3-dicarboximide; HYD, hydrobho- bic; IC 50 , inhibitory concentration that affords 50% inhibition of microbial growth; MIC, minimum inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MRSE, methicillin resistant S. epidermidis; MTT, 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet- razolium bromide; PI, positively ionizable; YPD, yeast extract/peptone/ dextrose.