Biological evaluation of Tyr6 and Ser7 modified drosocin analogues Peter C. de Visser, a,b,  Peter A. V. van Hooft, a,  Anne-Marij de Vries, a Ad de Jong, a Gijsbert A. van der Marel, b Herman S. Overkleeft b and Daan Noort a, * a TNO Defense, Security and Safety, PO Box 45, 2280 AA Rijswijk, The Netherlands b Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands Received 21 December 2004; revised 10 March 2005; accepted 18 March 2005 Available online 19 April 2005 This paper is dedicated to the memory of our colleague Jacques van Boom, who died on July 31, 2004, at the age of 67 Abstract—An array of analogues of the cationic antimicrobial peptide drosocin was synthesized containing substitutions of Tyr6 and Ser7 in order to increase the proteolytic stability. Stabilizing the N-terminus with unnatural amino acids increased the serum stability of analogues by almost a factor 30 over an 8 h period. Ó 2005 Elsevier Ltd. All rights reserved. 1. Introduction Cationic antimicrobial peptides (CAPs) have found wide attraction as lead structures in combating bacterial infections, 1 and considerable progress in elucidating the mode of action of a variety of CAPs has been made in recent years. 2,3 It has been widely accepted that most CAPs, (e.g., polymyxin B) 4 exert their activity through adoption of a defined secondary structure upon contact with Gram-negative bacterial cell membranes. As a re- sult, either bacterial cell lysis or disturbance of mem- brane transport events occurs, with bacterial cell death as a result. The fact that most CAPs appear to be indis- criminate to cell type and are often equally effective in killing mammalian cells normally limits their use to top- ical applications. Drosocin, a CAP isolated from Drosophila melanogas- ter, is a 19-mer oligopeptide containing three PRP re- peats and an O-glycosylation site at Thr11 (Table 1). 5 The antibacterial activity of drosocin, as is the case with other members of the proline-rich CAPs (Table 1), ap- pears not to be based on cell lysis. Rather, it is likely that drosocin activity toward bacterial strains is based on recognition of a specific intracellular target. 6 This obser- vation is underscored by the finding that enantiomeric drosocin, composed of D-amino acids, 7 does not possess any antibacterial activity, whereas most enantiomeric CAPs do. 8 Drosocin, and related proline-rich CAPs, are further distinguished from other CAPs by the lack of toxicity toward human erythrocytes, and their appar- ent bias in activity toward Gram-negative bacteria. 5 These properties combined make drosocin a very attrac- tive lead structure in the search for new and effective antibacterial agents. A major hurdle in the development of drosocin-based antibacterial agents is the inherent instability of droso- cin toward proteolytic activities present in mammalian sera. In fact, drosocin and its congeners are degraded in sera at such a rate that effective treatment of bacterial infections would require large doses of the peptide. 9 Any strategy that leads to drosocin analogues with enhanced proteolytic stability without impairing antibacterial activity should therefore be an important step forward in the development of new antibacterial agents. With this observation in mind, we embarked on a program aimed at the development and evaluation of chemically modified drosocin analogues, the initial results of which are presented here. 2. Results and discussion Our design of drosocin analogues is based on the re- ported finding that the first step in the proteolytic 0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2005.03.074 * Corresponding author. Tel.: +31 15 2843497; fax: +31 15 2843963; e-mail: noortd@pml.tno.nl   Both authors contributed equally to this work. Bioorganic & Medicinal Chemistry Letters 15 (2005) 2902–2905