discovery of novel non acidic mpges 1 inhibitors by virtual screening with a multistep protocol

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc Discovery of novel, non-acidic mPGES-1 inhibitors by virtual screening with a multistep protocol Stefan M Noha a, Katrin Fischer b, Andreas Koeberle b, Ulrike Garscha b, Oliver Werz b, Daniela Schuster a,⇑ a b Computer Aided Molecular Design (CAMD) Group, Institute of Pharmacy/Pharmaceutical Chemistry, University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University of Jena, Philosophenweg 14, D-07743 Jena, Germany a r t i c l e i n f o Article history: Received 18 February 2015 Revised 13 May 2015 Accepted 19 May 2015 Available online xxxx Keywords: Inflammation mPGES-1 Virtual screening 3D pharmacophore Kruskal–Wallis test a b s t r a c t Microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors are considered as potential therapeutic agents for the treatment of inflammatory pain and certain types of cancer So far, several series of acidic as well as non-acidic inhibitors of mPGES-1 have been discovered Acidic inhibitors, however, may have issues, such as loss of potency in human whole blood and in vivo, stressing the importance of the design and identification of novel, non-acidic chemical scaffolds of mPGES-1 inhibitors Using a multistep virtual screening protocol, the Vitas-M compound library ($1.3 million entries) was filtered and 16 predicted compounds were experimentally evaluated in a biological assay in vitro This approach yielded two molecules active in the low micromolar range (IC50 values: 4.5 and 3.8 lM, respectively) Ó 2015 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/) Introduction In the arachidonic acid cascade, the activity of the cytosolic phospholipase A2 is required for the release of arachidonic acid (AA), a critical precursor molecule for pro-inflammatory mediators Different enzymatic pathways convert AA into distinct eicosanoids These mediators regulate various physiological processes and also trigger multiple effects in various human diseases.1,2 Among these mediators, prostaglandin E2 (PGE2) is well recognized as critical bioactive molecule High PGE2 levels, typically occurring in inflammation, are relevant for swelling, fever, and inflammatory pain, and thus, pharmacological inhibition of PGE2 biosynthesis is considered a promising opportunity for the treatment of inflammatory pain, for example, in rheumatic diseases.3 Additionally, PGE2 synthesis is important in tumor growth and cancer progression.4–6 PGE2 is produced from the cyclooxygenase (COX)-derived prostaglandin H2 (PGH2) by PGE2 synthases (PGES) (EC 5.3.99.3).7 Among the three PGES isoenzymes, the microsomal PGES-1 (mPGES-1) displays a unique role because its expression is induced in the inflammatory response, similar to COX-2.8 By inhibiting mPGES-1 as the terminal synthase in PGE2 biosynthesis, mPGES-1 inhibitors are considered very promising regarding their side effect profile.9,10 The application of other anti-inflammatory agents, such as unspecific COX inhibitors, ⇑ Corresponding author Tel.: +43 (0)512 507 58253; fax: +43 (0)512 507 5269 E-mail address: Daniela.Schuster@uibk.ac.at (D Schuster) traditional nonsteroidal anti-inflammatory drugs (NSAIDs), or COX-2-selective inhibitors (coxibs), is associated with side effects concerning, among others, the renal function and effects on the gastrointestinal tract.11 In contrast, during prolonged inhibition of mPGES-1 in dogs, pronounced effects on the renal function were not observed.12 So far, there is no mPGES-1 inhibitor available for clinical use, although data from pre-clinical studies stressed the relevance of mPGES-1 inhibitors as potentially therapeutic agents Therefore, the development of mPGES-1 inhibitors is highly relevant.13 A series of mPGES-1 inhibitors is reported in the literature, of which several comprise an acidic functionality, such as an oxicam template,14 a sulfonamide group15 or a carboxylic acid moiety.16,17 Unfortunately, acidic molecules suppressing mPGES-1 activity may have inferior potency in human whole blood seemingly due to unspecific plasma protein binding.14,16 This suggests that the design and identification of novel, non-acidic chemical scaffolds is warranted As an overview, non-acidic chemical scaffolds of mPGES-1 inhibitors, which were reported so far, are shown in Figure 1.18–27 Previously, we reported the discovery of acidic mPGES-1 inhibitors using a pharmacophore-based virtual screening approach Using this screening protocol, acidic inhibitors from synthetic libraries were discovered The most potent inhibitors exhibited IC50 values in the sub-micromolar range.17 Additionally, mPGES-1 inhibitors with comparable potency from Lichen species were discovered using the previously reported pharmacophore model.28 http://dx.doi.org/10.1016/j.bmc.2015.05.045 0968-0896/Ó 2015 The Authors Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) Please cite this article in press as: Noha, S M.; et al Bioorg Med Chem (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.045 S M Noha et al / Bioorg Med Chem xxx (2015) xxx–xxx Figure Chemical series of non-acidic mPGES-1 inhibitors are depicted with 2D structures Comparably, other groups have reported virtual screening approaches to find novel mPGES-1 inhibitors For instance, Rörsch et al applied a multistep ligand-based virtual screening protocol to discover novel and non-acidic mPGES-1 inhibitors.29 In addition, several active compounds were discovered by applying docking-based screening strategies, of which some even elicited high potency.30–33 Docking-based virtual screening campaigns towards mPGES-1 have been facilitated as a 3D electron crystallography structure was reported in 2008.34 In 2013, a high-resolution X-ray crystal structure of mPGES-1 has been resolved.35 Very recently an X-ray crystal structure of mPGES-1 with a co-crystallized ligand has been reported.36 In this study, a novel concept for the validation of the 3D pharmacophore model was applied using the Kruskal–Wallis test.37 This test was suggested as a robust investigation of the discriminatory power of distinct virtual screening methods, and was previously used for the comparative assessment of docking and scoring functions.38,39 The analysis with the Kruskal–Wallis test is characterized as less artifact-prone and also enables a post hoc test, rendering this analysis an attractive method in the validation also for pharmacophore-based virtual screening.38,39 Figure Overview of the virtual screening protocol 2.2 Software specifications The computational studies were performed on a workstation running Microsoft Windows 7, which was employed for the work with the molecular modeling package Discovery Studio version 3.540 and PipelinePilot 8.0.1.41 In parallel, the calculations for the work with Maestro suite 9.2.11242 were performed on a workstation running OpenSuse 12.1 The statistical evaluation was performed within Microsoft Excel 2010 and its add-in Analyse-it Method Evaluation version 2.26.43 Materials and methods 2.3 Validation 2.1 Study design In brief, we consecutively performed forward filtering, using 2D similarity screening, and pharmacophore-based virtual screening The most interesting molecules which were retained thereof, accounting in addition pharmacophore fit evaluation and diversity clustering, were submitted to molecular docking Finally, this protocol was applied to prospective virtual screening of the Vitas-M library (http://www.vitasmlab.com/) The hit-list was visually inspected to select compounds for a biological evaluation to discover novel and non-acidic mPGES-1 inhibitors (Fig 2) 2.3.1 Concept We assessed the discriminatory power of the 3D pharmacophore model by following the workflow reported by Seifert et al.38,39 In this work, the discriminatory power of docking and scoring functions was assessed by ANOVA (analysis of variance) or a nonparametric version of it, that is, the Kruskal–Wallis test.37 Because this concept can also be useful for the development of 3D pharmacophore models, this analysis was included in the model validation and conducted as an extension to the validation with benchmarking experiments So a validation set, set_1, was assembled and used for screening experiments with the hypotheses The Please cite this article in press as: Noha, S M.; et al Bioorg Med Chem (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.045 S M Noha et al / Bioorg Med Chem xxx (2015) xxx–xxx statistical evaluation of the results was accomplished with the Kruskal–Wallis test and a post hoc test Furthermore, benchmarking experiments were conducted by screening a second validation set, set_2, and calculating well-established performance metrics 2.3.2 Validation sets and calculations Set_1 comprised highly active (IC50 60.5 lM), medium active (IC50: 0.5–5 lM), and confirmed inactive molecules (IC50 >5 lM) from several congeneric series of non-acidic mPGES-1 inhibitors, with 14 molecules in each group It consisted, in total, of 42 molecules For more details on set_1, see Supporting information In the validation, we screened set_1, followed by the statistical evaluation of the results obtained thereof with the Kruskal–Wallis test Furthermore, we included in this analysis Bonferroni’s post hoc test, employing the confirmed inactive molecules in the post hoc test as control group, and accounting the results of this evaluation significant with p 10 lM, Fig S3) However, effectively inhibited 5-lipoxygenase in a cell-free assay with an IC50 value of about lM, whereas was less active (IC50 >10 lM, Fig S4) Note that dual inhibition of mPGES-1 and 5-lipoxygenase is common to many structural classes of inhibitors of natural synthetic origin.62 Together, we conclude that and rather specifically interact with mPGES-1 and inhibit its activity To obtain a more profound insight on potential binding modes, the virtual screening hits were submitted to molecular docking Accounting the docking poses of compounds and (Fig 6A), these novel bioactive molecules were predicted to be involved in hydrogen-bonding to Ser127, which is assumed to serve as a key residue in the catalytic activity.35 Furthermore, a hydrogen-bond was predicted to be formed between and Gln36 Interestingly, both inhibitors were predicted to adopt a conformation, where these ligands complement the surface of the binding site adjacent to glutathione In case of 6, a substituted benzene ring was predicted to be orientated close to Phe44 and Leu39, which formed a hydrophobic contact to this ligand moiety In case of 7, the benzene ring, which was predicted to bind next to Phe44, has a chlorine substituent attached, which could be involved in a hydrophobic interaction with Leu39 The substituted benzene moieties of and 7, which were oriented towards the opposite site of the mPGES-1 binding site, were predicted to be embedded in a hydrophobic site formed by mainly aromatic or hydrophobic amino acids (e.g., Tyr28, Tyr130, Thr131, Ala31, and Ile32) In comparison, LVJ (Fig 6B), which has inhibitory potency on mPGES-1 activity in the low nanomolar range, adopts a position and orientation in the binding pocket which is slightly shifted towards mainly hydrophobic amino acids (e.g., Ala123, Val128, and Leu132), while several hydrogen-bonds are formed to the key residue Ser127 and other residues (e.g., Gln36 and His53) Discussion We herein report the discovery of four novel molecules suppressing mPGES-1 activity, of which the two most active ones showed the desired activity in the low micromolar range When only regarding the most active compounds, a hit rate of 12.5% (two virtual screening hits out of 16 tested molecules) was achieved Interestingly, this is comparable to other studies, in which prospective virtual library screening for the discovery of novel and non-acidic mPGES-1 inhibitors was conducted For instance, He et al attained good results by employing a molecular dynamics simulation to obtain an altered conformation of the 3Dstructure of mPGES-1, which was modified towards an active state conformation and utilized in a docking-based screening strategy Following the in silico approach, 21 molecules of 142 tested molecules showed the desired activity in the experimental evaluation (hit rate: 14.8%).32 Furthermore, Rörsch et al applied basically ligand-based methods in the search for mPGES-1 inhibitors Following the experimental evaluation of 17 molecules, three novel bioactive molecules were discovered and for one of those an IC50 value was determined, showing that this compound exerts potency in the sub-micromolar range.29 Fortunately, very recently the 3D-structure of mPGES-1 with a co-crystallized inhibitor became available.36 We therefore utilized this 3D-structure in a docking study of the two most active molecules, yielded in this study, compounds and 7, in order to surmise binding modes of these novel mPGES-1 inhibitors We thereby predicted that these molecules are accommodated nicely in the site adjacent to the cofactor glutathione, and could exhibit molecular interactions to the key residue Ser127 Together, the compounds and showed inhibitory potency on mPGES-1 activity in the low micromolar range, making them interesting starting points for optimization efforts Basically, virtual screening techniques are usually validated by screening (a) validation set(s) in benchmarking experiments In cases like this study, where very similar models perform with quite comparable results, an additional validation with the Kruskal– Wallis test can be helpful in the selection of the screening model, Please cite this article in press as: Noha, S M.; et al Bioorg Med Chem (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.045 S M Noha et al / Bioorg Med Chem xxx (2015) xxx–xxx especially as this test is considered to serve as robust investigation of the model quality.38,39 Conclusion In summary, a multistep virtual screening protocol is presented, which included a novel concept in the validation of the 3D pharmacophore Following a virtual screening campaign the results of the experimental evaluation confirmed the protocol quality, while the two most active molecules which inhibited mPGES-1 in a cell-free mPGES-1 activity assay, compounds and 7, may serve as promising starting points for further optimization The results may be considered as a case study; however, the modified concept applied in the pharmacophore model validation may be useful for further studies on other targets Acknowledgments We are grateful for the financial support by the Tyrolean Science Foundation (TWF), granted in 2011 (ID 134311) and the Austrian Science Fund (FWF, 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Hypo09 Hypo10 %Y GH EF1% EF0.5% Kruskal–Wallis’ statistic p 4.45 1. 54 1. 55 0.99 0.99 2.36 2.58 1. 37 1. 58 1. 61 0 .15 0 .12 0 .12 0 .10 0 .10 0 .12 0 .15 0 .16 0 .15 0 .15 32 .14 17 .86 17 .86 14 .29 14 .29 25.00... approaches to find novel mPGES- 1 inhibitors For instance, Rörsch et al applied a multistep ligand-based virtual screening protocol to discover novel and non- acidic mPGES- 1 inhibitors. 29 In addition,... functions was assessed by ANOVA (analysis of variance) or a nonparametric version of it, that is, the Kruskal–Wallis test.37 Because this concept can also be useful for the development of 3D pharmacophore