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7. Thimerosal. Al-Bakri and Waleed A.8.10. positive and neg- ative ionizable groups and aromatic planes. University of brominated furanones were reported to have anti-QS 0960-894X/$ . This set of analogues represents a highly active anti- Keywords: HipHop-Refine. 1). The three compounds represent a new class of QS inhibitors.12. The fact that this compound contained tetravalent lead (Pb) prompted us to evaluate the activities of phenyl mercuric nitrate and thimerosal. All rights reserved. judiciously prepared to emphasize rep- facilitate the establishment of infection. both fit the binding pharmacophore. doi:10.19–23 query to screen the NCI database for possible QS inhibitors.08.1–9. resentative coverage over a specified energy range. Quorum sensing (QS) is a cell-density-dependent inter. aeru.see front matter  2006 Elsevier Ltd. aliphatic and aromatic hydro- o on  2006 Elsevier Ltd.2 The Las mon to the most potent training molecules. Tel.40 CATALYST models ligand–receptor interaction using information derived only from the ligand structure. However. The al model was employed as 3D search query to screen the National Cancer Institute (NCI) database. Phenyl mercuric nitrate. probably via similar mechanism. pyoverdin and pyocynin. QS family that inhibit QS in several bacterial strains.40 Despite that many 5339649.24–30. that is.9 There are two QS systems HipHop-Refine identifies a set of chemical features com- in P.13 phobes.1–6 combined with the lack of crystallographic represent the steric constraints of the proposed recep- structure for LasR. HipHop-Refine utilizes the conformational space of itors as potential agents for reducing bacterial viru. e-mail: mutasem@ju. fax: +00962 6 QS transcription factors. The chemical features by LasR to become active transcription factor. Tetravalent lead. One of the hits illustrated nano- molar QS inhibitory activity.: +00962 6 5355000x2505.1016/j. LasI is essential for the for each input molecule consistent with their binding to production of AI signal molecule. generated and used as queries for database The model was subsequently utilized as 3D search searching. We employed the HipHop-Refine module of CATA- rs cellular signaling mechanism that enables bacteria to LYST software to build a plausible binding hypothesis. flexibility of training ligands is modeled by creating mul- ginosa regulates a group of virulence factors that tiple conformers. The two mercurials illustrated nanomolar to low micromolar IC50 inhibitory values against pseudomonal QS. prompted us to develop a phar. AHL) tionalities arranged in 3D space. accepted 10 August 2006 Available online 30 August 2006 Abstract—HipHop-Refine was employed to derive a binding hypothesis for pseudomonal quorum sensing (QS) antagonists.17. th The great recent interest in developing new QS inhib. revised 10 August 2006. tor.bmcl. The conformational for their cell signaling.11 considered can be hydrogen bond donors and acceptors The Rhl system seems to be regulated by the Las (HBDs and HBAs). Amman.2006. Jordan Received 14 May 2006. Pseudomonal quorum sensing antagonists.069 . for example. Bioorganic & Medicinal Chemistry Letters 16 (2006) 5902–5906 Discovery of potent inhibitors of pseudomonal quorum sensing via py pharmacophore modeling and in silico screening Mutasem O. we employed four brominated furanones as training set for pharmacophore modeling (Fig.7–9 The QS signaling of P.g. which is required a proposed common receptor site.* Amal G. positive and negative charges. In this project.. Taha. aeruginosa: the Las and Rhl systems. inhibition of * Corresponding author. This 3D system consists of LasR transcriptional regulator array of chemical features provides a relative alignment r's and LasI synthase protein.18 Many CATALYST-derived models have been Au macophoric model for LasR-based QS inhibitors. inactive molecules to construct excluded volumes that lence. Zalloum co Faculty of Pharmacy.14–16 pe Gram negative bacteria rely on small auto-inducer Molecules are described as collection of chemical func- (AI) molecules (e. coordinate the expression of certain genes. All rights reserved. acyl homoserine lactones.

40 On the other We employed this model as 3D-search query against the hand.35 Their anti-QS activities were assessed through their effects Obviously.e. only 19 hits were actually available and an energy range of 20 kcal/mol). SAR studies comprised of two HBAs. before conducting the 3D search we fore. the software was configured to allow a minimal inter-feature distance of 10 pm to generate hypothesis co that reflected the small size of the inhibitors.40. activities. O. The structures of the training compounds. based on Br Br the assumption that 1 and 2 are inactive due to their bu- Br O O tyl tails. 4 was allowed to miss one fea. features within the pharmacophoric model.25.819 compounds) using the on side chains are active pseudomonal QS inhibitors.001 hits.24. 16 (2006) 5902–5906 5903 Br Br them Principal values of zero. which was th reported to exert potent inhibitory actions against LasR-based pseudomonal QS (IC50 = 2. sulfur containing AHL analogues generated pharmacophore model.34. However.18 However. 3 and 4). Furanone 3.17.28 5 6 7 was assigned a Principal value of 1 to ensure that its fea- tures will be considered when generating hypotheses and O that it will be mapped at least once by each generated HO hypothesis. Med. ‘fast flexible search’ approach implemented within CAT- gesting they bind to a tighter binding pocket.24.18 However.0 lM. prompting us to limit our These settings yielded one pharmacophore hypothesis training set to 1–4. they were assigned O O MaxOmitFeat values of zero. a conformational database was generated using (see the fit equation in the footnote of Table 1) and the ‘best’ option and default CATALYST conformation the highest ranking 40 compounds were requested from generation parameters (a maximum of 250 conformers the NCI. The hits es similar conformers.. hits. Figure 4 shows that brominated furanones function via distinct binding overlay alignment of the training compounds onto the site(s). However. which was O O O O reported to be 17 times less active than 3 (in vivo data). were subsequently fitted against the pharmacophore pounds. ALYST. / Bioorg.40.24–30.17. The structures of some evaluated in silico hits.28 was assigned a Principal value of 2 to en- sure that all of the chemical features in the compound Cl N S O Au HN O O O P will be considered in building the pharmacophore P N P S O space. o After several preliminary trials. . Finally. two hydrophobic features.17. compounds of molecular weights >500D and <100D to conform to the molecular size of active brominated fura- rs The conformational space of each inhibitor was sampled nones.41 There. which seem to clash with certain steric bound- aries within the binding site. only few were found to be active specifically against pseudomonal QS. O 10 ities against pseudomonal LasR (despite their potent ac. 8 9 (Thiomersal) (Phenyl mercuric nitrate) tions against QS of other bacterial strains).31–33 from the NCI and evaluated as QS inhibitors. al with long hydrophobic tails (C10 and C12) were found to have potent anti-LasR-based QS. fit der them more flexible (their numbers of conformers are values. in vitro data). sug.25. Hits utilizing the poling algorithm of CATALYST. we excluded other classes of inhibitors (other than reduced the database to 219.37 Table 1 shows the NCI codes. The pharmacophore captured 86.40. it was decided to config- ure HipHop-Refine as follows. pyoverdin. 3).17. Hg ture in any generated model by assigning it a MaxOmit. while Figure 2 shows their corresponding chemical structures. while the butyl tails of 1 and 2 ren.18 On the other hand.24.240 structures by removing halogenated furanones) from modeling. O S O S O Hg O O Feat parameter of one.41 and therefore they were considered inactive by assigning Figure 2. Lett. Figure 1. Chem. which are defined as those compounds that possess chemical promotes conformational variation via employing functionalities that spatially overlap with corresponding pe molecular mechanical force field algorithm that penaliz. This value instructs Hip- 1 2 Hop-Refine to force the two inactive compounds to Br Br Br map all the pharmacophoric features of the binding model in order to identify spaces occupied by the two O O inactive compounds and free from the active inhibitors py O O (i. and biological activities of some of the tested 15 and 18.31–33 For each of the training com. Taha et al.40 For example. M.18 Compounds 1 and 2 were Pb N qualitatively reported to be of minimal inhibitory activ. inhibitors 3 and 4 are rigid structures (one on pseudomonal production of pyocyanin and r's conformer for each). respectively). of other classes of pseudomonal QS inhibitors suggest and seven excluded volumes (Fig.41 Furthermore. These regions are subsequently filled with 3 (C 30) 4 (C56) excluded volumes. only furanones with short (or no) hydrophobic NCI structure database (238. compound 4.36.

(D) 8. ous inhibition theory was further weakened by the . it can act as phore. However. that is. Most of the tested hits were inactive. Two possible mechanisms were considered. 16 (2006) 5902–5906 A B py co C D al on rs pe E F o r's th Au Figure 3. / Bioorg. (B) with added excluded volumes (gray spheres). one of namely. Taha et al.5904 M. Lett. (i) nonspecific (promiscuous) inhibition38 and the hits illustrated impressive QS inhibition. Med. Chem. Table 1 and Figs. (IC50 values < 800 nM. This conduct prompted us to suspect that the a non-specific bacterial toxicant against variety of tar- inhibitory action of 8 is related to the lead atom and gets. The promiscu- of LasR. 8 (ii) active-site directed irreversible inhibition. This contradiction is quite intriguing. O. since the tested Tetravalent lead is known to form stable complexes hits illustrated similar fit values against the pharmaco. the fact that 8 was devoid of any anti- mediated by an additional mechanism that complements bacterial actions within its anti-QS concentration pharmacophore recognition within the binding pocket range casts doubt on this mechanism. (E) 9 and (F) 10. (A) Pharmacophore features. with thiol (R–SH) groups. However. Mapping against (C) 3. 2 and 3D).39 and thus.

Unsurprisingly. f Mapping against the pharmacophore misses one feature.0 in HipHop-generated models.27 N/Ac N/A 6 132963 3.30 The fact that 8–10 combine the ability to fit onto the which seem to expel and replace reversible binders with- r's binding pharmacophore and to form stable covalent in the binding pocket. / Bioorg. Overlay alignment of the training compounds: 1 (yellow). that is.70 0.50 3.g. both compounds fitted omonal QS via an analogous mechanism. good leaving groups.355d (0.794e) 9 Thimerosal 3. initial selective and reversible binding within the binding pocket (i. the two mercurials exhibited by their Michael acceptor functionalities and bromo significant QS inhibitory actions (Table 1). in the color intensity of pyocynin and pyoverdin in the presence of 8–10 is most probably due to the inhibition The fact that mercury and lead have similar chemical of LasR and not simple chemical degradation or reactivities and biological profiles39 prompted us to eval. . Lett. On the other hand. 3 (green) and 4 (blue) onto the generated pharmacophore response to the added auto-inducers. b Fit values against the pharmacophore. site-directed irreversible inhibition. 16 (2006) 5902–5906 5905 complexes with thiol groups strongly supports the active site-directed irreversible inhibition proposition. e IC50 against a lab strain (ATCC: 9027). Taha et al.18 c Not active. probably mediated pe 3E and 3F).21 N/A N/A Au 8 203113 3. other screening hits and lac- with their anti-QS activities (Table 1). tone analogues24) is related to the nanomolar affinities of the natural QS auto-inducers (AHL) to LasR. in the production of pyoverdin and pyocyanin in 2 (red). M. Med. Fig. uate the anti-QS actions of two known mercurial bio- rs cides: thimerosal and phenyl mercuric nitrate (9 and Our results suggest that furanones 3 and 4 inhibit pseud- 10. The presence of co 8 inhibited the production of pyocyanin and pyoverdin regardless of the level of the added auto-inducers. Interestingly. calculated as in the following: Fit = Rmapped hypothesis features · W [1 R (disp/tol)2]. Figs.. disp is the distance between the center of a particular pharmacophoric sphere and the center of the corresponding superimposed chemical moiety of the fitted compound.019d (3. R(disp/tol)2 is the summation of (disp/tol)2 values for all pharmacophoric features that successfully superimpose corresponding chemical functionalities in the fitted compound.74 N/A N/A 7 133009 3. exhibited trivial anti-QS Therefore.708d (3. pharmacophore recognition) followed by covalent bonding that connects certain nucleophilic center within the binding pocket (proba- bly a thiol group) with the metallic core of the inhibitors.282d (0. Chem. interaction. respectively. This value is fixed to 1. which does or pyocyanin upon mixing and incubation overnight. active- tightly onto the pharmacophoric model (Table 1. Table 1. Finally.603d (3. the fit values We believe the reason for the poor QS inhibitory actions of 8–10 against the pharmacophoric model correlate well of reversible binders (e. W is the weight of the corresponding hypothesis feature spheres. O. The results of some evaluated in silico hits o Hitsa NCI code or chemical name Best Fit valuesb IC50 Assay (lM) Pyocyanin Pyoverdin th 5 43465 3. tol is the radius of the pharmacophoric feature sphere (known as Tolerance. they were devoid of any antibacterial activities within their QS inhibitory ranges. each value is the average of three trials.e. d IC50 against a pseudomonal clinical isolate from Jordan University Hospital.158e) 0. model.6 Å by default). 2)..166e) 10 Phenyl mercuric nitratef 2. that is. not fit the pharmacophore. where Rmapped hypothesis features is the number of pharmacophore features that successfully superimpose corresponding chemical moieties within the fitted compound.162d (3.236e) 3. it remains to be mentioned that none of the al compounds 8–10 altered the color intensity of pyoverdin fact that tetraethyl-lead (Pb(CH2CH3)4).981e) a The compounds and their numbers are as in Figure 2. each value is the average of three trials. absence of 8 allowed dramatic increase Figure 4. py Further evidence on the validity of this mechanism comes from investigating the effects of variable concentrations of QS auto-inducers (N-acyl homoserine lactones) on the production of pyocyanin and pyover- din in the presence and absence of 8. Luckily. one can assume that the dramatic reduction on actions.162e) 0. Furthermore.90 0. equals 1.

1712.. Foglino.. 92. 8. for the evaluation of pyocyanin production. Chem. Curr. 25. J.. 149.00. 29.A. The 9th European CATALYST User Group Meeting. Sci. 127. A. W.. Micro- 3. Van de Waterbeemd. Comput. Lett. 1997. 1995. Smellie. Pat. Expert Opin. U. o 19. 2005. 1996. py E.: San Diego. 2006. Taha et al. J. 1977.. J.. K. 41. Annu. 5.. Güner. The cultures were then diluted to OD600 = 0. 701. Iglewski. P. 16. 15. J. Cockayne. 2. 2003. 325. 1995. Chem. J. Microbiol. Sci. Sprague. K. J. H. J. withdrawn from each culture to count the viable cells 13. Iglewski. Bacteriol. 2005. H. S. K. I. G. D. centrifuged for 30 min at 5000g to harvest bacterial cells. S. Welch. L-Y. T. J. Qandil. M. 13.. H. Development. Langer. 14. 4235. Smellie. Suga. Z. fresh sterile nutrient broth (ca. 18. Bacteriol. Eberl. J. 1721. Clin. M. 2001. Givskov. A 1. 3127. M.. M. N.. B. Mol. Stintzi..05 with on P. Hansen. 40. L.. for kindly providing the tested hits. 10. J. Wood. 1.. Chuaqui. 1054. Inf. 24.. 2005. Passador. 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