Journal of Peptide Science J. Pept. Sci. 2006; 12: 491–496 Published online 20 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/psc.755 Design of a minimized cyclic tetrapeptide that neutralizes bacterial endotoxins PUIG MORA, a‡ CARLOS MAS-MORUNO, b‡ SILVIA TAMBORERO, a LUIS J. CRUZ, b ENRIQUE P ´ EREZ-PAY ´ A a,c and FERNANDO ALBERICIO b,d * a Department of Medicinal Chemistry, Centro de Investigaciones Principe Felipe, Avda Autopista del Saler, 16 46013-Val ` encia, Spain b Institute of Biomedical Research, Barcelona Science Park, University of Barcelona, Josep Samitier 1, 08028-Barcelona, Spain c Instituto de Biomedicina de Valencia, CSIC, 46010-Valencia, Spain d Department of Organic Chemistry, University of Barcelona, Mart´ ı i Franqu ´ es 1, 08028-Barcelona, Spain Received 28 December 2005; Accepted 27 January 2006 Abstract: Septic shock is a leading cause of mortality in intensive care patients, and no specific drugs are as yet available for its treatment. Therefore, new leads are required in order to increase the number of active molecules that may develop into efficacious and safe LPS-neutralizing molecules during pre-clinical stages. We used peptides, derived from the binding regions of known LPS-binding proteins, as scaffolds to introduce modifications at the amino acid level. Structure–activity relationship studies have shown that these modifications generate highly active peptides. Thus, from a bioactive peptide with an initial 16 amino acid residues, a tetrapeptide sequence was determined. After inserting this sequence in a Cys cyclic peptide, it showed the same biological activity as the parent peptide. This sequence could provide the basis for the design of small molecules with LPS-binding properties. Copyright  2006 European Peptide Society and John Wiley & Sons, Ltd. Keywords: endotoxin; septic shock; sepsis; LALF; peptide mapping; lipid A; disulfide bridge formation; molecular recognition; solid phase; peptides INTRODUCTION Sepsis, a systemic inflammatory response to infection, can lead to multiple organ failure known as septic shock, the first cause of mortality in intensive care units [1]. Recognition of the bacterial LPS by immune system cells is detected on the basis of the pathology [2]. LPS is a pathogen-associated molecular pattern (PAMP) present in the outer leaflet of Gram-negative bacteria [3]. Continuous exposure to LPS in mammalian bloodstream induces the deregulation of inflammatory cytokine release, thereby leading to the pathological condition. The cascade of events is initiated by the recognition and binding of LPS to circulating LPS- binding proteins. Among other proteins, the LPS- binding protein (LBP) binds to LPS and transfers it to the CD14 receptor [4,5]. Although LBP and CD14 are at the top of the cell responsive pathway to LPS, a cell membrane receptor must interact with the complex in order to transduce the signal into the cell. TLR2 and TLR4 receptors, members of the toll-like receptors family (TLR), participate in the transduction of the LPS Abbreviations: BPI, bactericidal/permeability-increasing protein; DPLA, 1-4 ′ -diphosphoryl lipid A; EDT, 1,2-ethanedithiol; LALF, Limulus anti- LPS factor, LAL, Limulus amebocyte lysate; LPS, lipopolysaccharide; LBP, lipopolysaccharide-binding protein; TBME; tert-butyl methyl ether; TIPS, triisopropylsilane * Correspondence to: Fernando Albericio, Institute of Biomedical Research, Barcelona Science Park, University of Barcelona, 08028- Barcelona, Spain; e-mail: albericio@pcb.ub.es. ‡ These two authors contributed equally to the present study. signal to the cell nucleus; an event that initiates the transcription of cytokine genes [6]. Research efforts have been directed towards the characterization of all the members involved in cas- cade recognition events and the full elucidation of the LPS-signalling pathway in order to define pharmaco- logical targets. However, although inhibitors of TNF-α factor and other inflammatory mediators have been targeted for inhibition, to date this approach has not increased the survival of patients with septic shock [7,8]. Therefore, increased interest has been devoted to the inhibition of early events of the process. Com- pounds that could neutralize LPS or its toxic part, the lipid A moiety, may provide a potential source of useful lead compounds of pharmacological relevance [8]. Recent developments in identifying novel strategies to overcome endotoxic shock involve LPS-neutralizing peptides. Of these, special interest has been focused on Limulus anti-LPS factor (LALF), a small (101 amino acids) basic protein that binds and neutralizes LPS with high affinity [9]. From the analysis of the crystal struc- ture of recombinant LALF (rLALF) [9], it was proposed that an amphipathic loop that spans residues 31 to 52 is the true LPS-binding domain [9]. In particular, the minimal LPS-binding domain is a 14-amino acid cyclic peptide (residues 36–47 – named LALF-14c), which binds LPS with an activity comparable to the high- affinity endotoxin-binding peptide polymyxin B (PMB), whereas its linear counterpart has a lower activity [10]. Herein, we report on the minimization of the LPS-binding domain of LALF-14c. We focused on Copyright  2006 European Peptide Society and John Wiley & Sons, Ltd.