New insights into the stereochemical requirements of the bradykinin B1 receptor antagonist binding

Elsevier Editorial System(tm) for Journal of Molecular Graphics and Modelling Manuscript Draft Manuscript Number: JMGM-D-15-00517R2 Title: NEW INSIGHTS INTO THE STEREOCHEMICAL REQUIREMENTS OF THE BRADYKININ B1 RECEPTOR ANTAGONISTS BINDING Article Type: Full Length Article Keywords: Bradykinin B1 receptor; GPCR homology modeling; pharmacophore development; B1 antagonist Corresponding Author: Prof. Juan Jesus Perez, Corresponding Author's Institution: First Author: Juan Jesus Perez, Professor Order of Authors: Juan Jesus Perez, Professor; Cecylia Severin Lupala, Ph.D.; Patricia Gomez-Gutierrez, Ph.D. Abstract: Bradykinin (BK) is a nonapeptide involved in several pathophysiological conditions including among others, septic and haemorrhagic shock, anaphylaxis, arthritis, rhinitis, asthma, inflammatory bowel disease. Accordingly, BK antagonists have long been sought after for therapeutic intervention. Action of BK is mediated through two different G-protein coupled receptors known as B1 and B2. Although there are several B1 antagonists reported in literature, their pharmacological profile is not yet optimal so that new molecules need to be discovered. In the present work we have constructed an atomistic model of the B1 receptor and docked diverse available non-peptide antagonists in order to get a deeper insight into the structure-activity relationships involving binding to this receptor. The model was constructed by homology modelling using the chemokine CXC4 and bovine rhodopsin receptors as template. The model was further refined using molecular dynamics for 600 ns with the protein embedded in a POPC bilayer. From the refinement process we obtained an average structure that was used for docking studies using the Glide software. Antagonists selected for the docking studies include Compound 11, Compound 12, Chroman28, SSR240612, NPV-SAA164 and PS020990. The results of the docking study underline the role of specific receptor residues in ligand binding. The results of this study permitted to define a pharmacophore that describes the stereochemical requirements of antagonist binding, and can be used for the discovery of new compounds. Graphical Abstract Dear Prof.Madura, Thank you for your letter of last June 15th. Please find enclosed a revised version of the manuscript entitled: “NEW INSIGHTS INTO THE STEREOCHEMICAL REQUIREMENTS OF THE BRADYKININ B1 RECEPTOR ANTAGONISTS BINDING” for your consideration to be published in the Journal of Molecular Graphics and Computer Modeling. The manuscript deals with the development of a pharmacophore of the antagonist binding to the bradykinin B1 receptor. The model is based on the docking of five diverse antagonists to a model of the B1 receptor constructed by homology modeling and subsequently refined by molecular dynamics using the CXC4 chemokine receptor as template. The pharmacophore was subsequently used for virtual screening and a few of the compounds discovered are described. In this new revised version we have incorporated all the points raised by the referees and are discussed on a separate cover. I hope that the new revised version of the manuscript is now suitable for publication in JMGCM. Sincerely yours, Juan J. Perez Barcelona, June 17th 2016 *Cover Letter The present paper describes a pharmacophore for bradykinin B1 antagonism. The pharmacophore has been obtained by constructing the B1 receptor by homology modeling from the CXC4 receptor. The receptor was embedded in a lipid bilayer and refined by molecular dynamics. On the model constructed five antagonists were docked. Analysis of the superimposed ligands permitted to define a pharmacophore. Finally, as a proof of concept, the pharmacophoric hypothesis was sued as query for a in silico screening. We describe in the present manuscript some of the hits obtained. *Highlights (for review) Response to the reviewers: Reviewer #1: We have modified Figure 9 that erroneously included a dotted line indicating a non-existing interaction. According to the suggestion made by the reviewer, we have included the Ballesteros notation in the residue labelling in Figures 4-11. *Response to Reviewers 1 NEW INSIGHTS INTO THE STEREOCHEMICAL REQUIREMENTS OF THE BRADYKININ B1 RECEPTOR ANTAGONISTS BINDING Cecylia S. Lupala, Patricia Gomez-Gutierrez and Juan J. Perez Dept. of Chemical Engineering Universitat Politecnica de Catalunya ETSEIB. Av. Diagonal, 647; 08028 Barcelona, Spain *Revised Manuscript Click here to view linked References 2 Abstract Bradykinin (BK) is a nonapeptide involved in several pathophysiological conditions including among others, septic and haemorrhagic shock, anaphylaxis, arthritis, rhinitis, asthma, inflammatory bowel disease. Accordingly, BK antagonists have long been sought after for therapeutic intervention. Action of BK is mediated through two different G-protein coupled receptors known as B1 and B2. Although there are several B1 antagonists reported in literature, their pharmacological profile is not yet optimal so that new molecules need to be discovered. In the present work we have constructed an atomistic model of the B1 receptor and docked diverse available non-peptide antagonists in order to get a deeper insight into the structure-activity relationships involving binding to this receptor. The model was constructed by homology modelling using the chemokine CXC4 and bovine rhodopsin receptors as template. The model was further refined using molecular dynamics for 600 ns with the protein embedded in a POPC bilayer. From the refinement process we obtained an average structure that was used for docking studies using the Glide software. Antagonists selected for the docking studies include Compound 11, Compound 12, Chroman28, SSR240612, NPV-SAA164 and PS020990. The results of the docking study underline the role of specific receptor residues in ligand binding. The results of this study permitted to define a pharmacophore that describes the stereochemical requirements of antagonist binding, and can be used for the discovery of new compounds. 3 Introduction Bradykinin (BK) is a nonapeptide with sequence Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, involved in several pathophysiological conditions including among others, septic and hemorrhagic shock, anaphylaxis, arthritis, rhinitis, asthma, inflammatory bowel disease. Accordingly, BK antagonists have long been sought after for therapeutic intervention. Action of BK is mediated through two different G-protein coupled receptors known as B1 and B2. The former is up-regulated during inflammation episodes or tissue trauma whereas the latter, is constitutively expressed in a variety of cell types, mediating most physiological actions of bradykinin [1-3]. Most attempts in the past were devoted to develop antagonists of the B2 receptor [4], with a stride made after the market release of the peptide antagonist Icatibant [5,6]. More recently, the demonstrated implication of B1 in establishing and maintaining the signaling of chronic and inflammatory pain [3,7,8], as well as in hyperalgesia and leucocyte infiltration through the activation of a cytokine network, has increased the interest in finding small molecule B1 antagonists for the treatment of both pain and inflammation [9-13]. Despite of these efforts there is no drug currently in the market designed to antagonize the B1 bradykinin receptor. The goal of the present work is to carry out a structure-activity study of B1 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists described in the literature and understand how these features translate into ligand anchoring points and complementary regions of the receptor, through the analysis of the respective ligand-receptor complexes. For this purpose we selected a set of compounds from the literature covering the maximal structural diversity as shown in Figure 1. The six compounds selected for the present study include Merck compounds 11 (1) [14-16] and 12 (2) [17]; Novartis NPV-SAA164 (3) [18,19]; Amgen Chroman28 (4) [20,21]; Sanofi-Synthelabo SSR240612 (5) [22] and Pharmacopeia PS020990 (6) [23]. The selected compounds were docked onto a refined model of the BK B1 receptor constructed by homology modeling, following the procedure explained in the methods section, being the complexes further analyzed for their ligand-receptor interactions. The outcome of this study is summarized on a 3D pharmacophore that explains the observed structure-activity results and provides insights into the discovery of novel molecules with antagonistic profile at the B1 bradykinin receptor. Methodology Computational methods 4 A starting model of the human BK B1 receptor was constructed by homology modeling using the chemokine CXCR4 receptor as template (pdb entry code 3ODU) [24]. This template was selected among those GPCRs whose crystallographic structure is available based on its proximity to B1 receptor in the GPCRs phylogenetic tree. The sequences of the two receptors were aligned, taking into account the conserved motifs found in all GPCRs, as well as the location of the disulfide bridges. These motifs, together with salt bridges are important factors in constraining the conformation of the extracellular and transmembrane domains of the receptor. Sequences were found to share about 25% sequence identity. Additionally, since the chemokine CXCR4 lacks the expected helical TM8, residues of the B1 receptor known to be in the TM8, starting from the highly conserved NPXXY motif [25] were modeled using the corresponding segment of the crystal structure of bovine rhodopsin (pdb entry code 1GZM) [26]. From the aligned sequences and the template backbone, a starting model of the receptor was constructed using the Modeller 9 version 8 (9v8) software [27]. Model was validated using the Molecular Operating Environment (MOE) program [28] after checking diverse steric parameters such as side chain rotamers, inter-residue contacts or backbone conformations. In a subsequent step, compound 11 (1 in Figure 1) was docked into the starting model for receptor refinement using the GLIDE software [29]. The choice of this ligand was due to the abundant information available on receptor residues involved in its binding from site directed mutagenesis experiments [14-15]. Finally, the ligand-receptor complex was refined using molecular dynamics of the ligand-receptor complex embedded in a lipid bilayer. Specifically, the protein was embedded in a box consisting in 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) lipids and water molecules generated and equilibrated according to the procedure described previously [30]. The box had an initial size of 10.3 x 8.0 x 10.2 nm3 (XYZ), organized in such a way that the bilayer plane was oriented on the XY plane. Before protein insertion, the box contained 256 lipids (corresponding to an area per lipid of 0.64 nm2) and circa 17,000 water molecules. The protein was placed in the center of the box, and the overlapping molecules were removed. Specifically, all water molecules with oxygen atoms closer than 0.40 nm to a non-hydrogen atom of the protein and similarly, all lipid molecules with at least one atom closer than 0.25 nm to a non-hydrogen atom of the protein were removed. This resulted in a final system containing 197 lipids and circa 16,000 water molecules. Removal of these atoms introduced small voids between the protein and water or lipid molecules that disappeared during the first part of the MD simulation, in 11 Chroman 28 Chroman-28 developed by Amgen, is a potent and selective antagonist of human, non-human primate, rat, and rabbit bradykinin B1 receptors with anti-inflammatory activity and Ki between 0.7 nM at human B1 receptors [20]. Chroman 28 was developed from extensive medicinal chemistry studies devoted to optimize the spatial placement of a lipophilic sulfonamide and a basic amine moiety, previously noted to be important for high binding affinity to the B1 receptor [40]. Figure 7 shows the proposed bound conformation of Chroman 28 to the human B1 receptor. Analysis of the ligand-receptor interactions starting from the piperidine ring reveals that this moiety sits in a negatively charged region at the mouth of the receptor displaying a hydrogen bond interaction with the sidechain of Asp297.32. SAR studies reveal that the piperidine ring can be replaced by diverse amines, being the pyrrolidine or butylamine the most active analogs. In contrast, replacement by a hydroxyl group leads to a complete loss of the affinity [41]. These results support the positioning of the piperidine ring described in the present model, since an amine can interact adequately with the diverse acid residues of the region including Asp-2917.32, Glu-2736.58 or Glu-2877.28. Replacement of an amine by a hydroxyl group generates a phenol derivative that at neutral pH will be in its basic form. This results agree well with the proposed pharmacophoric requirement for B1 antagonism mentioned above [40].Further down the molecule, the chroman group is located close to the side chain of His1995.35 interacting through a hydrogen bond between the oxygen of the chroman moiety and one of the nitrogens of the imidazole group and, also through a quadrupole-quadrupole interaction between the two rings. The model clearly justifies the differential affinity observed between the R and S enantiomers in SAR studies [41]. Further down the molecule, the amide carbonyl group exhibits a hydrogen bond with the side chain of Arg-2025.38 and one of the sulfonyl oxygens with the side chain of residue Gln-2957.36. Interestingly, the other sulfonyl oxygen interacts with Arg-1764.64 located at the C-terminus of TM4 via a hydrogen bond. Moreover, the phenyl group of the aryl sulfonamide core interacts with Tyr-2666.51, albeit at a bit longer distance. Improvement of this interaction led to the discovery of a sulfonamide containing a 2-oxopiperazine [42]. Finally, the naphthalene group sits in the aromatic pocket where quadruple-quadruple interactions with Trp-932.60, as well as Phe-101ECL1 are formed. SAR studies show that this moiety can be replaced by a trifluorobenzene without affinity loss, supporting the environment where the naphtyl group is located in the present model [42]. 12 SSR240612 SSR240612 is a high affinity antagonist of Lys-des-Arg9 in HEK cells expressing human B1 receptor and MRC5 human lung fibroblast B1 expressing cells. Developed by Sanofi, SSR240612 was the first B1 receptor antagonist with proven efficacy and oral bioavailability in models of pain and inflammation [22, 43]. The proposed bound conformation of SSR240612 to the BK B1 receptor is shown in Figure 8. Similarly as found in Chroman 28, the nitrogen of the piperidine moiety exhibits a hydrogen bond with Asp-2917.32. Further down the molecule the isopropylamide group sits in a region with several hydrophobic residues including Leu191ECL2 and Leu-193ECL2. In the center of the molecule, the amide group exhibits a hydrogen bond with Gln2957.36. Close to it the sulfonamide group exhibits a hydrogen bond between one of the thionyl groups and the side chain of residues Arg-1764.64, whereas the other exhibits a hydrogen bond with the amide hydrogen of Leu191ECL2 backbone. Attached to the sulfonamide there is a benzodioxole moiety that sits in a region with several aromatic residues including Trp-932.60 and Phe-101ECL1. On the other side of the molecule, the oxygen of the diaxole moiety interacts with Asn-96ECL1 via hydrogen bond. Finally, the methoxynaphthyl group interacts with residue Tyr-2666.51and Asn-2987.39. PS020990 Discovered by Pharmacopeia from a throughput screening program using encoded combinatorial libraries, PS020990 exhibits a potent antagonist profile on human BK B1, inhibiting 3H-Des-Arg10-Kalidin at IMR90 cells with a K1 of 1nM [23]. PS020990 is a high selective BK B1 antagonist with a >1000-fold selectivity reported over B2R [23]. The proposed bound conformation of PS020990 is shown in Figure 9. Present docking studies reveal the imidazole ring sitting at the polar top end of the binding pocket interacting with the His-1995.35 and exhibits a hydrogen bond with Glu-2736.58. Next to the imidazole group there is a pyridine ring that acts as spacer. It has attached a 3-chlorobenzylamine moiety that exhibits two hydrogen bond interactions, including the amine nitrogen with Arg-2025.38 and the chlorine of the 3-chlorobenzyl moiety with the sidechain of Asn-1143.27. Further down the molecule, the carbonyl oxygen of the central amide exhibits a hydrogen bond with Gln-2957.36, while the propylcyclohexane moiety sits close to Ile-190ECL2. At the terminal end of the molecule the 4-chlorobenzyl group rests at the center of the aromatic site, coordinating the π-π interactions with Trp-932.60 that also involves Phe- 13 2997.40. Definition of a pharmacophore for recognition to the bradykinin B1 receptor Comparison of the ligand-receptor complexes of the diverse antagonists used for the present study allows the definition of a pharmacophore that explains the observed structure-activity relationship observed. To define a pharmacophore of the ligand-receptor interaction, we superimposed the proposed bound structures as found in the ligand-receptor complex resulted from molecular docking studies. Analysis of the superimposed structures permitted the identification of common chemical features in similar regions of the space namely, the pharmacophoric points. For each of these pharmacophoric points an average geometric center was computed using the atomic centers of the participating atoms among the diverse antagonists. No exclusion volumes were incorporated in the pharmacophore, although the virtual screening procedure included a docking process after searching hits for pharmacophore fulfilment in order to eliminate the molecules that crashed with the receptor. The proposed pharmacophore is shown in Figure 10. It consists of five pharmacophoric points that all the ligands studied in the present study fulfil. The first point, at the polar mouth of the binding pocket, is represented by a proton donor center that complements an interaction with Asp-2917.32 and/or Glu-2736.58 and His-1995.35; point 2 is either a proton donor/proton acceptor center that will interact with either Gln-2957.36; point 3 is a hydrophobic ring that will interact with Trp-932.60 and/or Phe-2997.40; point 4 is a proton accepting center that will interact with Asn1143.27 and/or Arg-1764.64; point 5 is a proton donor and /or proton acceptor site that will interact with Arg-2025.38, Asn-2987.39 and the side chain of Tyr-2666.51. All the compounds used for pharmacophore development (Figure 1) fulfil all five receptor points. It what follows we specify how specific moieties of the diverse compounds fulfil the pharmacohpric hypothesis. Thus, Compound 11 fulfils point 1 by means of the 2-phenyldihydroimidazole; point 2 by the sulfonamide oxygen; point 3 by means of the chlorophenyl moiety; point 4 by the oxygen of the dihydroquinoxalinone group and point 5 by means of by the oxygen of the amide linker group. For Compound 12, point 1 is fulfilled by the 4pyridinepiperazine moiety; point 2 is fulfilled by means of the amide linker moiety; point 4 by both the cyclohexyl group and the benzyl moiety of the benzodiazepine and point 4 and 5 are fulfilled by the carbonyl group of the diazepine moiety. In the case of the NPV-SAA164 point 1 is fulfilled by the methyl-piperazine group nitrogen; 14 point 2 by means of the carbonyl group linking to the morpholine group that fulfills point 4; point 3 by both the phenyl group providing maximum coverage of the pocket side, point 5 by means of the sulfonyl oxygens of the sulfonamide. Chroman-28 covers the pharmacophore point 1 by using the chroman-7-methylpiperidine, point 2 and point 4 by means of the oxygens of the sulfonamide group; point 3 is fulfilled by the naphthalene rings and point 5 by the linker amide carbonyl group. In SSR240612 point 1 is fulfilled by the dimethyl-piperidine group; point 2 is fulfilled by the carbonyl group of the amide linker; point 3 is fulfilled by the benzodioxole moiety; while the sulphonyl oxygen fulfils point 4 and point 5 is fulfilled by the methyl-propanamide group. Finally, in the case of compound PS020990 point 1 is fulfilled by the imidazole group; point 2 by the carbonyl group of the cyclohexyl propanamide moiety; point 3 by means of the chlorophenyl group adjacent to the cyclohexyl propanamide moiety and point 5 by means of the nitrogen of the pyrimidine ring. Proof of concept The pharmacophore described above was later used in the discovery of novel structures with antagonistic activity for BK B1 by virtual screening. For this purpose a search was made in few databases of 3D structures of compounds including the Available Chemical Directory (ACD), the Derwent World Drug Index, the National Cancer Institute (NCI) and Maybridge for approximately 500,000 compounds. A separated searches for compounds fulfilling five, four and three pharmacophoric points yielding a large set of compounds was carried out. A selected group of them were purchased and tested for their BK B1 antagonistic activity. Biological assays permitted to identify new highly diverse hits with structures that do not resemble those used for pharmacophore development. The success rate achieved to identify new hits with an antagonistic activity by virtual screening was approximately one third of the selected molecules, as previously found by other authors [44]. Table 1 shows the structures as well as the antagonistic activity to the human bradykinin BK B1 of a selected group of hits that are disclosed to give support to the pharmacophoric hypothesis developed in this work. These molecules were docked onto the receptor model and inspected for fulfilment of the pharmacophore. Information regarding the number of pharmacophoric points fulfilled by each of the hits is also included in Table 1. Interestingly, the antagonistic activity observed experimentally correlates well with the number of pharmacophoric points fulfilled by these molecules. As an example, Figure 11 shows compound #10 bound to the BK B1 showing the fulfilment of the 15 pharmacophoric points. The ligand is shown to fulfil 3 pharmacophoric points 1, 3 and 5. The charged amine moiety interacts with the Asp-2917.32, and thus fulfilling point 1, the indole group nest deep in the hydrophobic pocket and fulfils point 3 and finally the nitrile group fulfils point 5 by interacting via hydrogen bond with both the sidechain of Tyr-2666.51 and Arg-2025.38. While the molecule have other polar groups such as carbonyl that could interact with Gln-2957.36 and Asn-1143.27, the size and conformation of these moieties and the compound in general, are not adequate enough to orient in a suitable manner for proper interactions. This emphasizes the reason why most of the BK B1 antagonists published are big as they need to span out to all pharmacophoric points to suitably interact. Conclusions Models of the bound conformation of diverse non-peptide human BK B1 antagonists were constructed and the stereochemical features of the complexes were analyzed with the aim to find common trends. To accomplish this, an atomistic model of the receptor was constructed by homology modeling, using the human CXC4 chemokine and bovine rhodopsin receptor as template (chimeric template). Antagonists selected for the present study included Compound 11, Compound 12, NPV-SAA164, Chroman-28, SSR240612 and PS020990, covering the maximum possible diversity. Complexes with the bound conformation of each of the antagonists were constructed by docking the molecules into the receptor. Due to the flexibility of the ligands and the size of the orthosteric site of the receptor, several docking attempts were carried out for each of the molecules. The final conformation was selected by the scoring function and the results of both SAR and site directed mutagenesis studies available in the published literature. Our results suggest that there are certain anchoring points that are found in more than one compound permitting the definition of a common pharmacophore. This consist of five points that defined on the features of the ligand include proton donor (point 1); a proton acceptor/proton donor center (point 2); a hydrophobic ring (point 3); proton acceptor/proton donor center (point 4) and proton donor/proton acceptor. The pharmacophore was used in a subsequent study to guide a virtual screening process. The results permitted to identify a set of compounds some of which were purchased and in vitro tested for their capability to antagonize the BK B1. 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Pietrovski E F, Otuki M F, Regoli D, Bader M, Pesquero J B, Cabrini D A, Zampronio A R (2009) The non-peptide kinin receptor antagonists FR 173657 and SSR 240612: preclinical evidence for the treatment of skin inflammation Regul Peptides 152: 67-72. 44. Krishna S, Singh D K, Meena S, Datta D, Siddiqi M I, Banerjee D (2014) Pharmacophore-Based Screening and Identification of Novel Human Ligase I Inhibitors with Potential Anticancer Activity J Chem Inf Model 54: 781−792. 20 Captions to the Figures Figure 1: Chemical structures of the B1 antagonists studied in the present work. Compound 11 (1), Compound 12 (2), NPV-SAA164 (3), Chroman28 (4), SSR240612 (5) and PS020990 (6). Figure 2: Sequence alignment of human B1 receptor (bottom) with human CXC4 chemokine receptor and bovine receptors (for part in TM7 and TM8). Transmembrane segments are inserted in boxes colored in green. Figure 3: (a) Lateral view of the orthosteric binding pocket of the human B1 bradykinin receptor modeled in complex with compound 11, ( b) same as (a) viewed from the extracellular side (aerial). Figure 4. Pictorial view of the proposed binding mode of Compound 11 bound to the BK B1 receptor. Figure 5: Pictorial view of the proposed binding mode of Compound 12 bound to the BK B1 receptor. Figure 6: Pictorial view of the proposed binding mode of NPV-SAA164 bound to the BK B1 receptor. Figure 7: Pictorial view of the proposed binding mode of Chroman 28 bound to the BK B1 receptor. Figure 8: Pictorial view of the proposed binding mode of SSR240612 bound to the BK B1 receptor. Figure 9: Pictorial view of the proposed binding mode of PS020990 bound to the BK B1 receptor. Figure 10: Proposed pharmacophore for the BK B1 antagonism. Distance between pharmacophoric points are: d(1,2)= 9.2 A; d(1,3)=13.1; d(1,4)= 1.5; d(1,5)=9.5; d(2,3)=5.1; d(2,4)=7.6.; d(2,5)=9.3; d(3,4)=7.8; d(3,5)=9.5; d(4,5)=5.7 Figure 11: Pictorial view of compound #9 of Table 4.2 bound to the BK B1 receptor with the pharmacophore points represented as spheres of different colors. Figure 6 Click here to download high resolution image Figure 7 Click here to download high resolution image Figure 8 Click here to download high resolution image Figure 9 Click here to download high resolution image Figure 10 Click here to download high resolution image Figure 11 Click here to download high resolution image