Scope and limitations of the designer proline-rich antibacterial peptide dimer, A3-APO, alone or in synergy with conventional antibiotics Marco Cassone a , Paraskevi Vogiatzi a , Raffaele La Montagna a , Vanessa De Olivier Inacio b , Predrag Cudic b , John D. Wade c , Laszlo Otvos Jr. a, * a Sbarro Institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA 19122, United States b Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, United States c Howard Florey Institute and Department of Chemistry, University of Melbourne, Victoria 3010, Australia 1. Introduction The antimicrobial drug industry is not keeping in pace with the overwhelming appearance and circulation of pathogens causing known or novel infectious syndromes. Perhaps the most worrisome event is the worldwide spread of antibiotic resistance in hospitals and the community alike [17,28]. Without discounting the efficacy of preventive measures, there is an impelling need to develop new antimicrobial molecules, and to use antimicrobial combinations capable of exerting synergistic activities. Antimicrobial peptides, which are largely diffused in nature, are a promising emerging class of anti-infective drugs. Among those, proline-rich peptides are unique since they have a very specific mechanism of action, allowing structure–activity relationships (SAR) studies [21]. In general, antimicrobial peptides carry little potential to induce resistance [11], however their systemic use is frequently hampered by unacceptable pharmacokinetic parameters and low safety margins [5]. Peptide A3-APO is a flagship representative of a new class of synthetic peptide antimicrobials derived from natural insect products [22]. A3-APO selectively binds to the multihelical lid peptides 29 (2008) 1878–1886 article info Article history: Received 30 June 2008 Received in revised form 22 July 2008 Accepted 22 July 2008 Published on line 5 August 2008 Keywords: A3-APO Synergy Antimicrobial peptide DnaK abstract The proline-rich antimicrobial peptide dimer, A3-APO, was designed based on a statistical analysis of native antibacterial peptide and protein sequences. Analysis of a series of structural analogs failed to identify any single or multiple amino acid modification or architectural changes that would significantly improve its potential as a clinical therapeutic. However, a single chain Chex1-Arg20 version, a natural in vivo metabolite, showed a 2 to 8- fold increase in activity against test Enterobacteriaceae strains. In addition to bacterial species close to Escherichia coli in phylogeny, A3-APO analogs were able to effectively kill Pseudomonas aeruginosa and Staphylococcus saprophyticus. Antibacterial efficacy analysis together with biochemical experiments provided further evidence for a multiple mode of action of A3- APO that includes binding and inhibition of the bacterial heat shock protein DnaK. Through inactivating of resistance enzymes, A3-APO was able to recover the lost activity of con- ventional antibiotics including chloramphenicol, b-lactams, sulfonamides or trimethoprim against multidrug resistant strains with partial or full synergy. However, the synergy appeared to be individual strain and small molecule drug combination-dependent. # 2008 Elsevier Inc. All rights reserved. * Corresponding author at: BioLife Sciences Building, 1900 North 12th Street, Philadelphia, PA 19122, United States. Tel.: +1 215 204 4020; fax: +1 215 204 4021. E-mail address: otvos@temple.edu (L. Otvos Jr.). available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/peptides 0196-9781/$ – see front matter # 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2008.07.016