Structure based discovery of small molecule suppressors targeting bacterial lysozyme inhibitors Arnout Voet a,1 , Lien Callewaert b,1 , Tim Ulens a , Lise Vanderkelen b , Joris M. Vanherreweghe b , Chris W. Michiels b , Marc De Maeyer a,⇑ a Laboratory for Biomolecular Modelling and BioMacS, Katholieke Universiteit Leuven, Celestijnenlaan 200G bus 2403, 3001 Heverlee, Leuven, Belgium b Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium article info Article history: Received 14 January 2011 Available online 20 January 2011 Keywords: Salmonella PliC MliC Pharmacophore Docking Drug design SMPPII abstract The production of lysozyme inhibitors, competitively binding to the lysozyme active site, is a bacterial strategy to prevent the lytic activity of host lysozymes. Therefore, suppression of the lysozyme–inhibitor interaction is an interesting new approach for drug development since restoration of the bacterial lyso- zyme sensitivity will support bacterial clearance from the infected sites. Using molecular modelling tech- niques the interaction of the Salmonella PliC inhibitor with c-type lysozyme was studied and a protein– protein interaction based pharmacophore model was created. This model was used as a query to identify molecules, with potential affinity for the target, and subsequently, these molecules were filtered using molecular docking. The retained molecules were validated as suppressors of lysozyme inhibitory proteins using in vitro experiments revealing four active molecules. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Lysozymes (EC 3.2.1.17) are enzymes from the innate immune system of most animals. The common feature of lysozymes is to exert antibacterial activity by cleaving the b-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in pepti- doglycan, the major bacterial cell wall polymer. In the animal king- dom, three major lysozyme types have been identified, commonly designated as the c-type (chicken type e.g. hen egg white lysozyme (HEWL)), the g-type (goose type e.g. salmon lysozyme) and the i- type (invertebrate type e.g. Tapes japonica lysozyme) lysozyme [1]. The defensive role of animal lysozymes against pathogenic bacteria is widely recognised and experimentally well-established by knock-out and overexpression of the lysozyme genes in trans- genic animals [2–4]. The c-type HEWL was the first enzyme to have its three dimensional structure determined by X-ray crystallogra- phy, revealing that the enzyme is divided into two domains by a deep cleft containing the active site [5]. Meanwhile, the catalytic importance of the conserved residues Glu35 and Asp52 was con- firmed by site directed mutagenesis [6]. The importance of peptidoglycan modifications like N-deacety- lation or O-acetylation as mechanisms to evade c-type lysozyme attack as part of the innate host defences and thereby directly influencing the virulence of pathogens has already been demon- strated for Listeria monocytogenes and Staphylococcus species [7,8]. On the other hand, a very specific, but only recently discovered bacterial resistance mechanism, is the production of proteinaceous lysozyme inhibitors. At present, two families of c-type lysozyme inhibitors have been described, namely the Ivy proteins (Inhibitor of vertebrate lysozyme) and the PliC/MliC family (Periplasmic/ Membrane bound lysozyme inhibitors of c-type lysozyme) [9,10]. In addition, recently both i- and g-type lysozyme inhibitor families (namely Periplasmic lysozyme inhibitors of i- or g-type lysozyme, abbreviated as, respectively PliI and PliG inhibitors), have been iso- lated and identified, indicating that bacteria have evolved inhibi- tors against all major types of lysozyme occurring in animals [11,12]. These lysozyme inhibitors physically interact with their corre- sponding lysozyme specifically restraining its enzymatic activity, and contribute to lysozyme tolerance in bacterial cells [10–13]. In particular for c-type lysozyme inhibitors, more direct evidence for a role in bacteria–host interactions is accumulating. The lysozyme inhibitor Ivy from Escherichia coli is indispensable for survival of this bacterial species in human saliva, which is naturally rich in lysozyme [14]. In the intracellular pathogen Salmonella typhi, on the other hand, expression of the MliC homolog was induced in cells residing in macrophages. MliC even seemed indispensable 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.053 ⇑ Corresponding author. Fax: +32 16 327974. E-mail addresses: arnout.voet@fys.kuleuven.be (A. Voet), lien.callewaert@biw. kuleuven.be (L. Callewaert), lise.vanderkelen@biw.kuleuven.be (L. Vanderkelen), joris.vanherreweghe@biw.kuleuven.be (J.M. Vanherreweghe), chris.michiels@biw. kuleuven.be (C.W. Michiels), marc.demaeyer@fys.kuleuven.be (M. De Maeyer). 1 Equally contributed. Biochemical and Biophysical Research Communications 405 (2011) 527–532 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc