Embed Size (px)
Citation preview
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
Structure based discovery of small molecule suppressors targeting bacteriallysozyme 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, Belgiumb Laboratory of Food Microbiology and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, 3001 Leuven, Belgium
a r t i c l e i n f o
Article history:Received 14 January 2011Available online 20 January 2011
Keywords:SalmonellaPliCMliCPharmacophoreDockingDrug designSMPPII
0006-291X/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.bbrc.2011.01.053
⇑ Corresponding author. Fax: +32 16 327974.E-mail addresses: [email protected] (A
kuleuven.be (L. Callewaert), [email protected]@biw.kuleuven.be (J.M. Vanherrekuleuven.be (C.W. Michiels), [email protected]
1 Equally contributed.
a b s t r a c t
The production of lysozyme inhibitors, competitively binding to the lysozyme active site, is a bacterialstrategy to prevent the lytic activity of host lysozymes. Therefore, suppression of the lysozyme–inhibitorinteraction 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 identifymolecules, with potential affinity for the target, and subsequently, these molecules were filtered usingmolecular docking. The retained molecules were validated as suppressors of lysozyme inhibitory proteinsusing in vitro experiments revealing four active molecules.
� 2011 Elsevier Inc. All rights reserved.
1. Introduction
Lysozymes (EC 3.2.1.17) are enzymes from the innate immunesystem of most animals. The common feature of lysozymes is toexert antibacterial activity by cleaving the b-(1,4) glycosidic bondbetween 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, commonlydesignated 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 pathogenicbacteria is widely recognised and experimentally well-establishedby knock-out and overexpression of the lysozyme genes in trans-genic animals [2–4]. The c-type HEWL was the first enzyme to haveits three dimensional structure determined by X-ray crystallogra-phy, revealing that the enzyme is divided into two domains by adeep cleft containing the active site [5]. Meanwhile, the catalyticimportance of the conserved residues Glu35 and Asp52 was con-firmed by site directed mutagenesis [6].
ll rights reserved.
. Voet), [email protected] (L. Vanderkelen),weghe), [email protected] (M. De Maeyer).
The importance of peptidoglycan modifications like N-deacety-lation or O-acetylation as mechanisms to evade c-type lysozymeattack as part of the innate host defences and thereby directlyinfluencing 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 discoveredbacterial resistance mechanism, is the production of proteinaceouslysozyme inhibitors. At present, two families of c-type lysozymeinhibitors have been described, namely the Ivy proteins (Inhibitorof 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]. Inparticular for c-type lysozyme inhibitors, more direct evidence fora role in bacteria–host interactions is accumulating. The lysozymeinhibitor Ivy from Escherichia coli is indispensable for survival of thisbacterial species in human saliva, which is naturally rich inlysozyme [14]. In the intracellular pathogen Salmonella typhi, onthe other hand, expression of the MliC homolog was induced incells residing in macrophages. MliC even seemed indispensable
528 A. Voet et al. / Biochemical and Biophysical Research Communications 405 (2011) 527–532
for survival of S. typhi in these phagocytosing cells, where antibac-terial peptides including lysozyme and membrane permeabilizersare produced to eliminate bacteria [15].
Besides this inhibition of the direct bacteriolytic activity of lyso-zyme, lysozyme inhibitors might also indirectly affect bacteria–host interactions. By binding lysozyme, they possibly influencethe release of peptidoglycan fragments, which is known to modu-late the immune response and inflammation [16–18].
Meanwhile, the 3D-structure for a representative of each of thec-type lysozyme inhibitor families (Ivy from E. coli and MliC fromPseudomonas aeruginosa), and its interaction with HEWL has beenunraveled [13,19]. This structure revealed a different HEWL bind-ing mode for MliC compared to Ivy. The crystal structure of theHEWL-Ivy complex shows that a five-residue loop protrudes theHEWL active site, where the Ivy His60 residue forms hydrogenbonds with the lysozyme catalytic residues Glu35 and Asp52, caus-ing an inhibition of lysozyme activity. However, Ivy only protrudesa small area of the active site cleft, as opposed to MliC, which in-serts two conserved loops (residues 86–92 and 97–104) to fillthe active site. The latter interaction is stabilized via a hydrogenbond (MliC Ser89 with HEWL Asp52) and ionic bonds (MliCLys103 with HEWL Asp52 and Glu35).
Overall, these findings strongly suggest a role for lysozymeinhibitors in virulence of pathogens by protecting bacteria againstthe lytic activity of host lysozyme in the infection process. As such,lysozyme inhibitors constitute an attractive novel target for thedevelopment of a new type of antibacterial agent. Since geneticallyknocking-out the inhibitor production results in increased bacte-rial lysozyme sensitivity [10,20], we anticipate that a similar effectwill be obtained if the activity of the inhibitor is disabled pharma-ceutically. Suppressor molecules interfering with the lysozyme–inhibitor interaction can be predicted to increase lysozyme sensi-tivity of the bacteria, and accordingly diminish their pathogenicity.
In the current work, we report a first step towards this novelantibacterial approach, by providing proof of principle for thefeasibility to neutralize the activity of a c-type lysozyme inhibitorby using suppressor molecules. Although these compounds areweakly active at current state, medicinal chemistry efforts candeliver optimized derivatives. Since the PliC/MliC inhibitor familyis more widely distributed than the Ivy family, we have chosen aPliC lysozyme inhibitor (from Salmonella typhimurium, an impor-tant pathogen) as target.
2. Materials and methods
2.1. Homology modelling of the protein complex
To model the 3D structure of the complex of HEWL with PliC ofS. typhimurium, the sequence of the PliC protein (NCBI ID:NP_460216) was aligned with the sequence of P. aeruginosa MliCas available in the crystal structure (PDB ID: 3F6Z) (see Fig. 1)[19]. The optimized alignment was used for homology modellingwithin MOE (Chemical Computing Group, Montreal, Canada). A to-tal of 10 PliC homology complex models were created based on theMliC protein, with the lysozyme structure retained during themodel construction. The quality of the models was assessed withinMOE and the best model (based on ProCheck [21] analysis) was se-lected for future drug discovery efforts.
2.2. Computer aided discovery of hit molecules
The virtual discovery of protein–protein interaction inhibitorsin this work is aimed at the salvage of lysozyme activity in thepresence of PliC of S. typhimurium. Virtual screening experimentswere set up according to a funnel principle discarding in multiple
consecutive steps the least probable inhibitors. A library of±200.000 commercially available molecules was converted into a3D molecular database using the Omega2 algorithm [22]. Theseconformations were filtered using a pharmacophore query usingthe MOE software. This pharmacophore query was based on theinteraction of HEWL with PliC. For this, the molecular model ofthe protein interaction was thoroughly analyzed. Previously Yumet al. reported a small binding cleft in the structure of MliC inwhich a lysozyme loop protrudes [19]. Analysis of the homologymodel and the sequences (MliC and PliC) indicate that the geome-try of this pocket and the loop amino acid interactions with thepocket residues are conserved. Especially the interaction of theconserved lysozyme Thr47 with the conserved PliC Tyr78 andThr91 is interesting and was converted into a pharmacophore fea-ture (see Fig. 2A). To analyze the molecular recognition propertiesof the binding site and to identify hot spot interaction regions,molecular interaction fields were computed using the GRID algo-rithm in MOE [23], supplementing the pharmacophore query withtwo hotspot hydrophobic–aromatic interactions (see Fig. 2D). Fur-thermore, the molecular interaction field analysis of the PliC pock-et shows that the feature overlapping with the Thr47 also couldaccommodate a carbonyl function (see Fig. 2C). Therefore we di-vided our pharmacophore query in two separate queries. Usingthese pharmacophore queries, molecules were selected that agreedwith one of the two queries. The selected structures were subse-quently docked into the PliC pocket. The latest available versionof the GOLD algorithm was used with standard parameters, theGoldScore scoring function and with, for the receptor, rotatable po-lar hydrogens and three simulations per compound [24]. Thedocked poses were rescored with ChemScore [25]. Afterwards,the docked poses for all compounds were evaluated using thepharmacophore queries (using the MOE software). Only com-pounds binding according to our pharmacophore model were re-tained for further selection. A collection of 28 molecules wasmanually picked based on docked score, binding mode and diver-gence. The selected molecules were ordered from SPECS (Delft,The Netherlands) (for a full overview of all ordered molecules seeSupplementary data).
2.3. Production and purification of lysozymes and inhibitors
While HEWL (Hen Egg White Lysozyme, Fluka, 66,000 U/mgprotein) is commercially available, the salmon lysozyme and T.japonica lysozyme were recombinantly produced and purified asrecently described by, respectively Vanderkelen et al. and VanHerreweghe et al. [11,12].
The PliC lysozyme inhibitor from S. typhimurium was recombi-nantly expressed in E. coli BL21(DE3). The corresponding gene(ordered locus name: STM1249; NCBI ID: NP_460216) includingits signal peptide was amplified without stop codon using PhusionDNA-polymerase (Finnzymes, Espoo, Finland) with the primerspliC-Fwd (50-TCAGTCTAGAAGGATACTAATGATGAAACG-30) and pliC-Rev (50-ACTGCTCGAGGTTAGCTAAGGAACAGTTAC-30). After diges-tion with XbaI and XhoI (recognition sites in primers are italicized),the resulting fragment was ligated into pET28b(+) which providesa C-terminal His6-tag followed by a stop codon, and transformed toE. coli BL21(DE3). The recombinant protein PliC-His6 was expressedby inoculating a stationary phase culture, grown well aerated for21 h at 37 �C, in LB broth with kanamycin (Sigma–Aldrich, Bornem,Belgium; 50 lg/ml), 1/100 in fresh medium, addition of 1 mM IPTG(Acros Organics, Geel, Belgium) after 4 h of growth, and furtherovernight growth (37 �C with shaking 200 rpm). PliC-His6 was sub-sequently purified from a periplasmic extract (Deckers et al., 2004)[20] of this culture using a HisTrap™ HP column (GE Healthcare)on an ÄKTA-FPLC system.
Fig. 1. The model of the S. typhimurium PliC in complex with lysozyme was constructed by homology modelling, using MOE with the P. aeruginosa MliC protein (PDB 3F6Z) asa template. Frame A depicts the superposition of the MliC template (blue) with the modeled PliC (yellow). The complex was modeled in the environment of the c-typelysozyme as available in this crystal structure. In frame B the complex of the PliC (yellow) in complex with HEWL (red) is depicted in cartoon mode. Frame C shows theRamachandran plot of the best PliC model. Underneath the alignment of the MliC template with the PliC sequence is given as used during the homology modelling. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Frame A: the modeled structure of the HEWL (red) – PliC complex (yellow) was analyzed to construct a pharmacophore query. Frame B: the key interaction of HEWLwith PliC is the (HEWL) Thr47 hydroxyl interacting with PliC residues Tyr78 and Thr91. Frame C: molecular interaction field analysis indicates that this position is equallybeneficial for interaction with a carbonyl function. Thus two different pharmacophore queries were created. Query 1 describes the interactions by a hydroxyl function whilequery 2 represents the interactions of the carbonyl group. Frame D: F2 describes the hydrogen bridge formation of Thr47, F2 and F3 describe directionality of the hydrogenbridge formation. F1, F5, and F6 represent hydrophobic or aromatic features as derived from hotspot interactions for a dry probe as revealed by molecular interaction fieldanalysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
A. Voet et al. / Biochemical and Biophysical Research Communications 405 (2011) 527–532 529
PliI and PliG lysozyme inhibitors were similarly produced inE. coli BL21(DE3) using the pET expression system, and subse-quently isolated and purified by affinity chromatography asdescribed by Van Herreweghe et al. and Vanderkelen et al., respec-tively [11,12].
2.4. Determination of lysozyme inhibitory activity suppression
Suppression of lysozyme inhibitory activity was detected bymeasuring the recovery of lysozyme activity in the presence oflysozyme inhibitor, using a turbidity assay described by Callewaertet al. [26]. Thirty microliter inhibitor solution was preincubated for10 min with 30 ll of potential suppressor molecule (final concen-tration of 250 lg/ml). Subsequently, 210 ll of a substrate cell sus-pension, and finally 30 ll of lysozyme solution were added. For theHEWL and T. japonica lysozyme, lyophilized Micrococcus luteus(Sigma–Aldrich, Bornem, Belgium) in 10 mM potassium phosphate
buffer pH 7.0 (0.7 mg/ml) was used as a substrate, while salmonlysozyme activity was measured using cell wall material of Yersiniaenterocolitica ATCC9610, prepared by treatment of cells with chlo-roform-saturated 50 mM Tris pH 7.0 as described by Nakimbugweet al. [27]. The resulting OD600 decrease of this suspension, andcontrol suspensions (with and without inhibitor and potential sup-pressor molecule) was followed for 2 h using a Bioscreen C Micro-biology Reader (Labsystems Oy, Helsinki, Finland). Lysozyme andinhibitor concentrations were chosen such that lysozyme activityalone resulted in an OD600 decrease of 0.32 ± 0.04 over 2 h and that100% inhibition was achieved in the absence of suppressormolecule.
3. Results
Molecular modelling of the protein complex indicated thatthe majority of amino acids involved in the interaction of
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80 100 120Time (minutes)
OD
600
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80 100 120Time (minutes)
OD
600
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80 100 120Time (minutes)
OD
600
0.4
0.5
0.6
0.7
0.8
0.9
0 20 40 60 80 100 120Time (minutes)
OD
600
0.5
0.6
0.7
0.8
0.9
OD
600
0.5
0.6
0.7
0.8
0.9
OD
600
A
B
C
530 A. Voet et al. / Biochemical and Biophysical Research Communications 405 (2011) 527–532
S. typhimurium PliC and P. aeruginosa MliC as available from thetemplate crystal structure are conserved. The fact that the interac-tion interface is conserved justifies the homology model as a validstarting point for the structure based discovery of suppressors.
The PliC/MliC interactions with HEWL span a vast interface area(1170 Å2). For the drug design however, we focused on a pocketformed within the inhibitory protein such that the compoundscan inhibit the protein–protein interaction (PPI) without interfer-ing with HEWL catalytic activity. The key interaction in this pocketis formed by the HEWL Thr47 hydroxyl function with the con-served Tyr78 and Thr91 of the PliC inhibitory protein (see Fig. 2B).
A funnel based principle filtered the SPECS library of ±200.000compounds, resulting in 28 molecules with potential to interferewith the PliC–HEWL interaction. The first step was the pharmaco-phore filtering using two different pharmacophore queries. Thefirst query selected 1691 molecules, the second query 4123, result-ing in a combined 5367 different molecules. Subsequently, thesemolecules were docked using the GOLD algorithm with a maxi-mum of three different docking poses per compound, and thesedocked poses were subjected to the different pharmacophore que-ries, to select for compounds making the correcting interactions,resulting in 173 retained molecules. Out of these, 28 divergent bestscoring molecules were ordered and experimentally evaluated.
The in vitro suppressor activity of the HEWL–PliC interactionwas tested for the selected compounds. Four compounds (DDS-002, DDS-003, DDS-004 and DDS-027) partially restored HEWLactivity to a moderate to high degree (30%, 69%, 89%, and 91%,respectively) when added in a concentration of 250 lg/ml (seeFig. 3A). Dilution to lower concentrations (25 and 2.5 lg/ml) how-ever, abolished suppressor activity (data not shown). To verify thespecificity of this suppressor activity, the four active compoundswere subsequently tested against the SL–PliG and TjL–PliI complex(see Fig. 3B and C). Since no OD600 decrease could be observed inany of these tests, lysozyme activity remained inhibited, demon-strating that the compounds were not able to interfere with theSL–PliG and TjL–PliI interaction and are specifically targeting theHEWL–PliC interaction.
0 20 40 60 80 100 120Time (minutes)
0 20 40 60 80 100 120Time (minutes)
0.40.4
Fig. 3. Detection of suppressor activity of compounds DDS-002 (j), DDS-003 ( ),DDS-004 (s) and DDS-027 (�) on (A) HEWL–PliC, (B) salmon lysozyme–PliG and (C)Tapes japonica lysozyme–PliI interaction, measured as restored lysozyme activity.The OD600 decrease of the substrate suspension by lysozyme ( ), and the lack ofOD600 decrease of the substrate suspension in the presence of lysozyme andinhibitor ( ) functions as control curves.
4. Discussion
Protein–protein interactions are probably the most complexand diverse class of macromolecular interactions that are known.They are responsible for a multitude of biological effects and theirrole in a multitude of pathologies has become clear. There is anincreasing interest from the pharmaceutical industry in targetingprotein interfaces with small druglike molecules [28–30]. It is as-sumed that the number of pharmaceutically relevant PPIs exceedsthe number of classical drug targets exploited today.[31] AlthoughPPIs have long been considered as not drugable, many Small Mol-ecule Protein–Protein Interaction Inhibitors (SMPPIIs) have beenreported in recent years (for a recent review see Berg [31]).
In this context, c-type lysozyme is an interesting enzyme be-cause of its widespread occurrence in various human body fluids(and other animals) and its effectiveness as an antibacterial agent.
The interaction between the PliC/MliC inhibitor family and lyso-zyme is in part based on a lysozyme loop protruding in a pocket ofthe inhibitor proteins (Fig. 1B), potentially enabling high affinitysmall molecule binding to compete with the inhibitory bindingof the protein to lysozyme and thereby suppressing the lysozymeinhibitory activity of this protein. This explains the search for aSMPPII targeting the c-type lysozyme–PliC inhibitor interactionas a novel antibiotic agent.
Since the 3D structure of the S. typhimurium PliC in complexwith c-type lysozyme was unknown, our starting point was thestructure of the P. aeruginosa MliC HEWL complex [19]. Given that
the interacting amino acids of the MliC protein are conserved in thePliC sequence, we anticipated that the interaction of the PliC pro-tein would be similar and thus homology modelling of the complexwould yield a reliable model for structure-based drug design.
An analysis by fry pointed out that many of the SMPPIIs mimicone of the interface proteins of the complex [32]. This indicated thepossibility to exploit these properties in the design of pharmaco-phore queries. The above described hydroxyl function was there-fore implemented as an essential pharmacophore feature. Toobtain additional interactions, supplementing our pharmacophorequery, molecular interaction fields were calculated. This revealedhydrophobic hotspots in the binding cleft and the option for a car-bonyl function instead of a hydroxyl function. As such two separatequeries could be defined. Molecules which were selected usingthese pharmacophore queries could be docked into the samereceptor since rotational freedom of the hydroxyl functions wasincorporated during docking simulation.
Fig. 4. Biological validation identified four hit molecules, depicted in frame A as 2D structures. Frame B depicts the binding mode of DDS-002 as docked using the GOLDalgorithm into the PliC structure. This binding mode agrees with the interactions of pharmacophore query 1.
A. Voet et al. / Biochemical and Biophysical Research Communications 405 (2011) 527–532 531
However, in retrospect, the four molecules showing experimen-tally validated lysozyme inhibition suppressor activity (Fig. 4), allbear a hydroxyl function. This highlights the similarity of the pro-tein and the SMPPII. The hydroxyl function clearly mimics Thr47and thus follows the first pharmacophore query, rather than thesecond pharmacophore query which encodes for a carbonylfunction.
Besides sharing a hydroxyl function, the four active moleculesalso share a common atom connectivity around the hydroxyl func-tion. This is especially the case for DDS-002, DDS-003 and DDS-004, the three molecules -probably not coincidentally- showingthe highest HEWL suppressor activity (Fig. 3A).
Using molecular interaction field analysis, hydrophilic interac-tion hot spots could not be identified except for the hydrogenbridge donor/acceptor position on the hydroxyl functionality ofThr-47. This is inline with the hydrophobic character of protein–protein interaction interfaces and the hydrophobic character ofthe majority of the SMPPIIs [33] .
Since the identified suppressor molecules only showed clearactivity in a concentration of 250 lg/ml, the potency of these mol-ecules is still very low. This could be explained by the large differ-ence between the interface area of PliC in the protein–proteincomplex interface and that formed with the small molecules(±1170 and ±175 Å2, respectively). Furthermore, these suppressorswere discovered using a homology model. A more accurate exper-imentally determined 3D structure of the PliC – HEWL complexmight be necessary for an optimized pharmacophore query andoptimized molecular docking into the PliC cavity. In addition, thesemolecules have the potency for being optimized towards newderivatives with improved suppressor activity using classicalmedicinal chemical exploration of the molecules. An example ofsuch optimization could be a chemical modification which rigidi-fies the flexible core of the molecule.
The fact that these molecules are selectively active against theinteraction of HEWL with the PliC/MliC type inhibitors but notagainst the i-type, nor g-type lysozyme–inhibitor interactions sup-ports the hypothesis that they act by binding to PliC/MliC.
While the inhibitors are only weakly active, they do suppresslysozyme inhibitor activity, demonstrating the feasibility for thisnovel antibacterial approach.
More potent suppressor molecules may eventually find a widerange of potential applications, since lysozymes are broadly dis-tributed in both humans and animals.
These molecules, together with our recently reported SMPPIIswith antiretroviral activity, discovered using a similar approach,represent another case showing the possibility to rationally dis-cover SMPPIIs using pharmacophores based on the protein inter-faces [34].
Acknowledgments
A.V. wishes to thank the scientific research council of the uni-versity for the postdoctoral funding. L.V. acknowledges the FlemishInstitute for the Promotion of Scientific Technological Research(IWT) for the doctoral fellowship. L.C. is supported by a postdoc-toral and J.M.V.H. by a doctoral fellowship of the Research Founda-tion-Flanders (FWO-Vlaanderen). The work was further supportedby research grants from FWO-Vlaanderen (G.0363.08) and fromKULeuven (IOF-HB/08/005 and METH/07/03). Inge W. Nilsen (NOF-IMA, Tromsø, Norway) is acknowledged for donating the strainBL21 GOLD (DE3) PQM64.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bbrc.2011.01.053.
References
[1] L. Callewaert, C.W. Michiels, Lysozymes in the animal kingdom, J. Biosci. 35(2010) 127–160.
[2] P. Markart, T.R. Korfhagen, T.E. Weaver, H.T. Akinbi, Mouse lysozyme M isimportant in pulmonary host defense against Klebsiella pneumoniae infection,Am. J. Respir. Crit. Care Med. 169 (2004) 454–458.
[3] H.T. Akinbi, R. Epaud, H. Bhatt, T.E. Weaver, Bacterial killing is enhanced byexpression of lysozyme in the lungs of transgenic mice, J. Immunol. 165 (2000)5760–5766.
[4] A.M. Cole, D.R. Thapa, V. Gabayan, et al., Decreased clearance of Pseudomonasaeruginosa from airways of mice deficient in lysozyme M, J. Leukoc. Biol. 78(2005) 1081–1085.
[5] D.C. Phillips, Crystallographic studies of enzymic properties of lysozyme, ActaCrystallogr. 21 (1966) A163.
[6] B.A. Malcolm, S. Rosenberg, M.J. Corey, et al., Site-directed mutagenesis of thecatalytic residues Asp-52 and Glu-35 of chicken egg-white lysozyme, Proc.Natl. Acad. Sci. USA 86 (1989) 133–137.
[7] I.G. Boneca, O. Dussurget, D. Cabanes, et al., A critical role for peptidoglycan N-deacetylation in listeria evasion from the host innate immune system, Proc.Natl. Acad. Sci. USA 104 (2007) 997–1002.
[8] A. Bera, R. Biswas, S. Herbert, F. Gotz, The presence of peptidoglycan O-acetyltransferase in various Staphylococcal species correlates with lysozymeresistance and pathogenicity, Infect. Immun. 74 (2006) 4598–4604.
532 A. Voet et al. / Biochemical and Biophysical Research Communications 405 (2011) 527–532
[9] V. Monchois, C. Abergel, J. Sturgis, et al., Claverie, Escherichia coli ykfE ORFangene encodes a potent inhibitor of C-type lysozyme, J. Biol. Chem. 276 (2001)18437–18441.
[10] L. Callewaert, A. Aertsen, D. Deckers, et al., A new family of lysozyme inhibitorscontributing to lysozyme tolerance in gram-negative bacteria, PLoS Pathog. 4(2008) e1000019.
[11] J.M. Van Herreweghe, L. Vanderkelen, L. Callewaert, et al., Lysozyme inhibitorconferring bacterial tolerance to invertebrate type lysozyme, Cell Mol. Life Sci.67 (2010) 1177–1188.
[12] L. Vanderkelen, J.M. Van Herreweghe, K.G. Vanoirbeek, et al. Identification of abacterial inhibitor against g-type lysozyme, Cell Mol. Life Sci. (2010) doi10.1007/s00018-010-0507-3.
[13] C. Abergel, V. Monchois, D. Byrne, et al., Structure and evolution of the Ivyprotein family, unexpected lysozyme inhibitors in gram-negative bacteria,Proc. Natl. Acad. Sci. USA 104 (2007) 6394–6399.
[14] D. Deckers, D. Vanlint, L. Callewaert, et al., Role of the lysozyme inhibitor Ivy ingrowth or survival of Escherichia coli and Pseudomonas aeruginosa bacteria inhen egg white and in human saliva and breast milk, Appl. Environ. Microbiol.74 (2008) 4434–4439.
[15] F. Daigle, J.E. Graham, R. Curtiss 3rd, Identification of Salmonella typhi genesexpressed within macrophages by selective capture of transcribed sequences,SCOTS, Mol. Microbiol. 41 (2001) 1211–1222.
[16] J. Humann, L.L. Lenz, Bacterial peptidoglycan-degrading enzymes and theirimpact on host muropeptide detection, J. Innate Immun. 1 (2009) 88–97.
[17] J.W. Park, C.H. Kim, J.H. Kim, et al., Clustering of peptidoglycan recognitionprotein-SA is required for sensing lysine-type peptidoglycan in insects, Proc.Natl. Acad. Sci. USA 104 (2007) 6602–6607.
[18] K.A. Cloud-Hansen, S.B. Peterson, E.V. Stabb, et al., Breaching the great wall:peptidoglycan and microbial interactions, Nat. Rev. Microbiol. 4 (2006) 710–716.
[19] S. Yum, M.J. Kim, Y. Xu, et al., Structural basis for the recognition of lysozymeby MliC, a periplasmic lysozyme inhibitor in gram-negative bacteria, Biochem.Biophys. Res. Commun. 378 (2009) 244–248.
[20] D. Deckers, B. Masschalck, A. Aertsen, et al., Periplasmic lysozyme inhibitorcontributes to lysozyme resistance in Escherichia coli, Cell Mol. Life Sci. 61(2004) 1229–1237.
[21] R.A. Laskowski, M.W. Macarthur, D.S. Moss, J.M. Thornton, Procheck – aprogram to check the stereochemical quality of protein structures, J. Appl.Crystallogr. 26 (1993) 283–291.
[22] J. Bostrom, J.R. Greenwood, J. Gottfries, Assessing the performance of OMEGAwith respect to retrieving bioactive conformations, J. Mol. Graph. Model. 21(2003) 449–462.
[23] P.J. Goodford, A computational procedure for determining energeticallyfavorable binding sites on biologically important macromolecules, J. Med.Chem. 28 (1985) 849–857.
[24] G. Jones, P. Willett, R.C. Glen, et al., Development and validation of a geneticalgorithm for flexible docking, J. Mol. Biol. 267 (1997) 727–748.
[25] M.D. Eldridge, C.W. Murray, T.R. Auton, et al., Empirical scoring functions: I.The development of a fast empirical scoring function to estimate the bindingaffinity of ligands in receptor complexes, J. Comput. Aided Mol. Des. 11 (1997)425–445.
[26] L. Callewaert, B. Masschalck, D. Deckers, et al., Purification of Ivy, a lysozymeinhibitor from Escherichia coli, and characterisation of its specificity for variouslysozymes, Enzyme Microb. Technol. (2005) 205–211.
[27] D. Nakimbugwe, B. Masschalck, D. Deckers, et al., Cell wall substrate specificityof six different lysozymes and lysozyme inhibitory activity of bacterialextracts, FEMS Microbiol. Lett. 259 (2006) 41–46.
[28] T. Berg, Modulation of protein–protein interactions with small organicmolecules, Angew. Chem., Int. Ed. Engl. 42 (2003) 2462–2481.
[29] L. Pagliaro, J. Felding, K. Audouze, et al., Emerging classes of protein–proteininteraction inhibitors and new tools for their development, Curr. Opin. Chem.Biol. 8 (2004) 442–449.
[30] H. Yin, A.D. Hamilton, Strategies for targeting protein–protein interactionswith synthetic agents, Angew. Chem., Int. Ed. Engl. 44 (2005) 4130–4163.
[31] T. Berg, Small-molecule inhibitors of protein–protein interactions, Curr. Opin.Drug Discov. Devel. 11 (2008) 666–674.
[32] D.C. Fry, Drug-like inhibitors of protein–protein interactions: a structuralexamination of effective protein mimicry, Curr. Protein Pept. Sci. 9 (2008)240–247.
[33] R. Bourgeas, M.J. Basse, X. Morelli, P. Roche, Atomic analysis of protein–proteininterfaces with known inhibitors: the 2P2I database, PLoS One 5 (2010) e9598.
[34] F. Christ, A. Voet, A. Marchand, et al., Nat. Chem. Biol. 6 (2010) 442–448.
