Binding Mode Exploration of B1 Receptor Antagonists' by the Use of Molecular Dynamics and Docking Simulation-How Different Target Engagement Can Determine Different Biological Effects.

The kinin B1 receptor plays a critical role in the chronic phase of pain and inflammation. The development of B1 antagonists peaked in recent years but almost all promising molecules failed in clinical trials. Little is known about these molecules' mechanisms of action and additional information will be necessary to exploit the potential of the B1 receptor. With the aim of contributing to the available knowledge of the pharmacology of B1 receptors, we designed and characterized a novel class of allosteric non-peptidic inhibitors with peculiar binding characteristics. Here, we report the binding mode analysis and pharmacological characterization of a new allosteric B1 antagonist, DFL20656. We analyzed the binding of DFL20656 by single point mutagenesis and radioligand binding assays and we further characterized its pharmacology in terms of IC50, B1 receptor internalization and in vivo activity in comparison with different known B1 antagonists. We highlighted how different binding modes of DFL20656 and a Merck compound (compound 14) within the same molecular pocket can affect the biological and pharmacological properties of B1 inhibitors. DFL20656, by its peculiar binding mode, involving tight interactions with N114, efficiently induced B1 receptor internalization and evoked a long-lasting effect in an in vivo model of neuropathic pain. The pharmacological characterization of different B1 antagonists highlighted the effects of their binding modes on activity, receptor occupancy and internalization. Our results suggest that part of the failure of most B1 inhibitors could be ascribed to a lack of knowledge about target function and engagement.

Bifunctional ligands of the bradykinin B2 and B1 receptors: An exercise in peptide hormone plasticity.

Kinins are the small and fragile hydrophilic peptides related to bradykinin (BK) and derived from circulating kininogens via the action of kallikreins. Kinins bind to the preformed and widely distributed B2 receptor (B2R) and to the inducible B1 receptor (B1R). B2Rs and B1Rs are related G protein coupled receptors that possess natural agonist ligands of nanomolar affinity (BK and Lys BK for B2Rs, Lys-des-Arg9-BK for B1R). Decades of structure-activity exploration have resulted in the production of peptide analogs that are antagonists, one of which is clinically used (the B2R antagonist icatibant), and also non-peptide ligands for both receptor subtypes. The modification of kinin receptor ligands has made them resistant to extracellular or endosomal peptidases and/or produced bifunctional ligands, defined as agonist or antagonist peptide ligands conjugated with a chemical fluorophore (emitting in the whole spectrum, from the infrared to the ultraviolet), a drug-like moiety, an epitope, an isotope chelator/carrier, a cleavable sequence (thus forming a pro-drug) and even a fused protein. Dual molecular targets for specific modified peptides may be a source of side effects or of medically exploitable benefits. Biotechnological protein ligands for either receptor subtype have been produced: they are enhanced green fluorescent protein or the engineered peroxidase APEX2 fused to an agonist kinin sequence at their C-terminal terminus. Antibodies endowed with pharmacological actions (agonist, antagonist) at B2R have been reported, though not monoclonal antibodies. These findings define classes of alternative ligands of the kinin receptor of potential therapeutic and diagnostic value.

The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.

New insights into the stereochemical requirements of the bradykinin B1 receptor antagonists binding.

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 modeling using the chemokine CXC4 and bovine rhodopsin receptors as template. The model was further refined using molecular dynamics for 600ns 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.

Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.

Comparison of bioactive chemical space networks generated using substructure- and fingerprint-based measures of molecular similarity.

Chemical space networks (CSNs) have recently been introduced as a conceptual alternative to coordinate-based representations of chemical space. CSNs were initially designed as threshold networks using the Tanimoto coefficient as a continuous similarity measure. The analysis of CSNs generated from sets of bioactive compounds revealed that many statistical properties were strongly dependent on their edge density. While it was difficult to compare CSNs at pre-defined similarity threshold values, CSNs with constant edge density were directly comparable. In the current study, alternative CSN representations were constructed by applying the matched molecular pair (MMP) formalism as a substructure-based similarity criterion. For more than 150 compound activity classes, MMP-based CSNs (MMP-CSNs) were compared to corresponding threshold CSNs (THR-CSNs) at a constant edge density by applying different parameters from network science, measures of community structure distributions, and indicators of structure-activity relationship (SAR) information content. MMP-CSNs were found to be an attractive alternative to THR-CSNs, yielding low edge densities and well-resolved topologies. MMP-CSNs and corresponding THR-CSNs often had similar topology and closely corresponding community structures, although there was only limited overlap in similarity relationships. The homophily principle from network science was shown to affect MMP-CSNs and THR-CSNs in different ways, despite the presence of conserved topological features. Moreover, activity cliff distributions in alternative CSN designs markedly differed, which has important implications for SAR analysis.

In vivo radioimaging of bradykinin receptor b1, a widely overexpressed molecule in human cancer.

The bradykinin receptor B1R is overexpressed in many human cancers where it might be used as a general target for cancer imaging. In this study, we evaluated the feasibility of using radiolabeled kallidin derivatives to visualize B1R expression in a preclinical model of B1R-positive tumors. Three synthetic derivatives were evaluated in vitro and in vivo for receptor binding and their ability to visualize tumors by PET. Enalaprilat and phosphoramidon were used to evaluate the impact of peptidases on tumor visualization. While we found that radiolabeled peptides based on the native kallidin sequence were ineffective at visualizing B1R-positive tumors, peptidase inhibition with phosphoramidon greatly enhanced B1R visualization in vivo. Two stabilized derivatives incorporating unnatural amino acids ((68)Ga-SH01078 and (68)Ga-P03034) maintained receptor-binding affinities that were effective, allowing excellent tumor visualization, minimal accumulation in normal tissues, and rapid renal clearance. Tumor uptake was blocked in the presence of excess competitor, confirming that the specificity of tumor accumulation was receptor mediated. Our results offer a preclinical proof of concept for noninvasive B1R detection by PET imaging as a general tool to visualize many human cancers.

Mechanisms involved in kinin-induced glioma cells proliferation: the role of ERK1/2 and PI3K/Akt pathways.

Gliomas are the most common malignant brain tumors in adults. Bradykinin (BK) displays an important role in cancer, although the exact role of kinin receptors in the glioma biology remains unclear. This study investigated the role of kinin B1 and B2 receptors (B1R and B2R) on cell proliferation in human glioblastoma cell lineages. The mRNA expression of B1R and B2R was verified by RT-qPCR, whereas the effects of kinin agonists (des-Arg(9)-BK and BK) were analyzed by cell counting, MTT assay and annexin-V/PI determination. The PI3K/Akt and ERK1/2 signaling activation was assessed by flow cytometry. Our results demonstrated that both human glioblastoma cell lines U-138MG and U-251MG express functional B1R and B2R. The proliferative effects induced by the incubation of des-Arg(9)-BK and BK are likely related to the activation of PI3K/Akt and ERK 1/2 pathways. Moreover, the pre-incubation of the selective PI3Kγ blocker AS252424 markedly prevented kinin-induced AKT phosphorylation. Noteworthy, the selective B1R and B2R antagonists SSR240612 and HOE-140 were able to induce cell death of either lineages, with mixed apoptosis/necrosis characteristics. Taken together, the present results show that activation of B1R and B2R might contribute to glioblastoma progression in vitro. Furthermore, PI3K/Akt and ERK 1/2 signaling may be a target for adjuvant treatment of glioblastoma with a possible impact on tumor proliferation.

Kinin B1 receptor homo-oligomerization is required for receptor trafficking to the cell surface.

The kinin B1 receptor (B1R) is a G protein-coupled receptor with pro-inflammatory activity that is latent in healthy tissues but induced by tissue insult. Here, we investigated if B1R homo-oligomerization is a possible mechanism regulating the presentation of this receptor at the level of maturation and trafficking to the cell surface. To this end, we used HEK293 cells stably expressing N-terminal FLAG and HA epitope-tagged wild-type human B1R and an N-terminal receptor fragment, B1stop135, which terminates at the C-terminal end of the third transmembrane domain and has previously been shown to oligomerize with B1R. Receptors were monitored by immunoblotting and immunoprecipitation, receptor function by agonist binding and agonist-promoted phosphoinositide hydrolysis, and receptor trafficking by confocal immunofluorescence microscopy. When expressed alone, B1R is core N-glycosylated and forms oligomers localized intracellularly and on the cell surface. B1stop135 also exists as core N-glycosylated oligomers but is localized exclusively intracellularly. When co-expressed, B1stop135 prevents specifically B1R homo-oligomerization by forming nonfunctional B1R-B1stop135 hetero-oligomers, retains B1R intracellularly at least in part in the endoplasmatic reticulum (ER), increases calnexin binding to the receptor, and increases receptor degradation. We conclude that B1R homo-oligomerization is necessary for B1R maturation and trafficking to the cell surface. Modulating this mechanism may be a novel therapeutic avenue in inflammatory disease.

3-Oxo-2-piperazinyl acetamides as potent bradykinin B1 receptor antagonists for the treatment of pain and inflammation.

The discovery of novel and highly potent oxopiperazine based B1 receptor antagonists is described. Compared to the previously described arylsulfonylated (R)-3-amino-3-phenylpropionic acid series, the current compounds showed improved in vitro potency and metabolic stability. Compound 17, 2-((2R)-1-((4-methylphenyl)sulfonyl)-3-oxo-2-piperazinyl)-N-((1R)-6-(1-piperidinylmethyl)-1,2,3,4-tetrahydro-1-naphthalenyl)acetamide, showed EC(50) of 10.3 nM in a rabbit biochemical challenge model. The practical syntheses of chiral arylsulfonylated oxopiperazine acetic acids are also described.

Discovery of dihydroquinoxalinone acetamides containing bicyclic amines as potent Bradykinin B1 receptor antagonists.

Replacement of the core beta-amino acid in our previously reported piperidine acetic acid and beta-phenylalanine-based Bradykinin B1 antagonists by dihydroquinoxalinone acetic acid increases the in vitro potency and metabolic stability. The most potent compounds from this series have IC(50)s<0.2 nM in a human B1 receptor functional assay. A molecular modeling study of the binding modes of key compounds, based on a B1 homology model, explains the structure-activity relationship (SAR) for these analogs.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins.

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B1 and B2 (kinin receptors) and AT1 and AT2 (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B1 agonists Des-Arg9-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg9-BK or Des-Arg10-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT1 agonist (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT1 receptor. To investigate this hypothesis, we replaced Arg212 for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B1 receptor by the B2 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg212 in the TM V and a region of TM VI of rat B1 receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII.

Identification of the critical residues of bradykinin receptor B1 for interaction with the kinins guided by site-directed mutagenesis and molecular modeling.

We report the critical residues for the interaction of the kinins with human bradykinin receptor 1 (B1) using site-directed mutagenesis in conjunction with molecular modeling of the binding modes of the kinins in the homology model of the B1 receptor. Mutation of Lys118 in transmembrane (TM) helix 3, Ala270 in TM6, and Leu294 in TM7 causes a significant decrease in the affinity for the peptide agonists des-Arg10kallidin (KD) and des-Arg9BK but not the peptide antagonist des-Arg10Leu9KD. In contrast, mutations in TM2, TM3, TM6, and TM7 cause a significant decrease in the affinity for both the peptide agonists and the antagonist. These data indicate that the B1 bradykinin binding pocket for agonists and antagonists is similar, but the manners in which they interact with the receptor do not completely overlap. Therefore, there is a potential to influence the receptor's ligand selectivity.

Uneven evolutionary rates of bradykinin B1 and B2 receptors in vertebrate lineages.

Bradykinin acts through two receptor subtypes in mammals and generates a variety of responses including pain, inflammation and hypotension. The evolutionary history of the bradykinin system has been unclear due to shortage of information outside mammals. We describe here two receptor subtypes and the bradykinin precursor in three species of bony fish (the zebrafish Danio rerio, the Japanese pufferfish Takifugu rubripes, and the green spotted pufferfish Tetraodon nigroviridis) and chicken and analyze the relationships to mammals by a combination of phylogeny, conserved synteny and exon-intron organization. All of these species have two receptor genes located close to each other in a tandem formation, with the B2 gene 5' to the B1 gene, in chromosomal regions displaying conserved synteny between the species (albeit conservation of synteny in zebrafish is still unclear due to poor genome assembly). The evolutionary rate differs between the two genes as well as between lineages leading to differing pharmacological properties for both B1 and B2 across vertebrate classes. Also the bradykinin precursor gene was identified in all of these species in a chromosome region with conserved synteny. The tissue distribution of mRNA in T. rubripes is similar for B1 and B2, suggesting more similar regulation for the two genes than in mammals. In conclusion, the receptor tandem duplication predates the divergence of ray-finned fish and tetrapods and no additional duplicates of the receptors or bradykinin seem to have survived the ray-finned fish tetraploidization.

Dual antagonists of the bradykinin B1 and B2 receptors based on a postulated common pharmacophore from existing non-peptide antagonists.

We have recently drawn attention to the fact that most non-peptide antagonists of the kinin B1 receptor reported so far are structurally related, possessing the core motif phenyl-SO2-NR-(spacer(2-4))-CO-NRR. This is found in compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2- yl)phenyl]ethyl] - 2- [(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide), a very potent and selective B1 receptor antagonist. A subset of specific bradykinin B2 receptor antagonists (LF16-0687, bradyzide and derivatives) possesses a similar 'scaffold' (phenyl-SO2-NR-CRR-CO-NRR). We investigated whether simple molecules mimicking the postulated pharmacophores could be identified in two public chemical databases. Receptor binding to B1 and B2 receptors expressed by rabbit cultured smooth-muscle cells was confirmed for some of these newly identified agents, with a loss of receptor subtype selectivity. For instance, compound 3[2-(3-oxo-1-(toluene-4-sulfonyl)-1,2,3,4-4H-quinoxalin-2-yl)-N-phenyl-acetamide] exhibits IC50 values of 2.13 and 126 microM in the radioligand competition assays for B1 and B2 receptors, respectively, and a pA2 of 6.27 at the rabbit B1 receptor in a functional test (Lys-des-Arg9-bradykinin-induced contractility of the isolated aorta). Compound 5 (a close analog of compound 3) is a more balanced dual antagonist of low potency (IC50 values of 30 and 117 microM, respectively). As predicted, compounds modeled on a postulated pharmacophore common to some non-peptide B1 or B2 receptor antagonists exhibit measurable binding with decreased receptor subtype selectivity. Dual B1/B(2) receptor antagonists are of possible therapeutic interest and should be developed.

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

The role of helix 8 and of the cytosolic C-termini in the internalization and signal transduction of B(1) and B(2) bradykinin receptors.

Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B(2) bradykinin receptor (B(2)wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B(1) bradykinin receptor (B(1)wt) containing these B(2)wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B(1)wt C-terminus with corresponding B(2)wt regions either at C330(7.71) following putative helix 8 (B(1)CB(2)) or at the preceding Y312(7.53) in the NPXXY sequence (B(1)YB(2)) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B(1)KB(2)). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B(1)wt to serine (as in B(2)wt) as being responsible for this effect. The slowly internalizing chimera as well as a B(1)wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B(1)wt or the other chimeras. The slow internalization of B(1)KB(2) was also accompanied by a lack of agonist-induced phosphorylation, that in contrast was observed for B(1)YB(2) and B(1)CB(2), suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.

B1 bradykinin receptor homo-oligomers in receptor cell surface expression and signaling: effects of receptor fragments.

The human B(1) bradykinin receptor is an inducible and constitutively active G protein-coupled receptor that is involved in the inflammatory and pain responses to injury. Here, we investigated the role of B(1) receptor homo-oligomerization in cell surface receptor expression. B(1) receptors tagged with either the FLAG or hemagglutinin epitope were monitored immunologically and by radio-ligand binding, biotinylation, and phosphoinositide hydrolysis in human embryonic kidney 293 cells. Selective immunoprecipitation, immunoblotting, and immunoelectron microscopy with epitope-specific antibodies together provided evidence for constitutively formed cell surface receptor homo-oligomers. Truncation of the receptor from the N- and C-terminal ends indicated that the epitope for oligomerization seems to be located between Leu(26) on top of transmembrane helix 1 and Val(71) at the bottom of helix 2. A receptor construct terminating at Asp(134) at the bottom of helix 3, B1stop135, was expressed in the cell. It is interesting that this construct behaved as a dominant-negative mutant by competitively preventing formation of intact B(1) receptor homo-oligomers, and redistributing B(1) receptors from the cell surface to a common intracellular compartment. In contrast, expression of a construct containing the residues downstream of Asp(134), B1del(2-134), was inactive in this regard. Together, these results are consistent with a mechanism where constitutive B(1) receptor homooligomerization is required for expression of receptors on the cell surface and subsequent constitutive receptor signaling. This may be a novel mechanism by which the cell regulates the presentation of this constitutively highly active receptor at various stages of injury.