Characterization of a novel and selective CB1 antagonist as a radioligand for receptor occupancy studies

Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease. CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affinity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radioligand was used to conduct ex vivo receptor occupancy studies.     

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 EC₅₀ of 10.3 nM in a rabbit biochemical challenge model. The practical syntheses of chiral arylsulfonylated oxopiperazine acetic acids are also described.     

Calreticulin enhances B2 bradykinin receptor maturation and heterodimerization

In different native tissues and cells the receptor for the vasodepressor bradykinin, B₂, forms dimers with the receptor for the vasopressor angiotensin II, AT₁. Because AT₁/B₂ heterodimers may contribute to enhanced angiotensin II-stimulated signaling under pathophysiological conditions, we analyzed mechanisms of AT₁/B₂ heterodimerization. We found that efficient B₂ receptor maturation was a prerequisite for heterodimerization because only the fully mature B₂ receptor was capable to interact with AT₁. To identify chaperones involved in B₂ receptor maturation and heterodimerization we performed microarray gene expression profiling of human embryonic kidney (HEK293) cells. The expression of the chaperone calreticulin was up-regulated in cells with efficient B₂ receptor maturation. Vice versa, upon down regulation of calreticulin expression by RNA interference, B₂ receptor maturation and AT₁/B₂ receptor heterodimerization were significantly impaired. Concomitantly, the B₂ receptor-mediated enhancement of AT₁-stimulated signaling was reduced. Thus, calreticulin enhances B₂ receptor maturation and heterodimerization with AT₁.     

The neuropeptide bradykinin stimulates phosphoinositide turnover in HSDM1C1 cells: B2-antagonist-sensitive responses and receptor binding studies

Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC₅₀) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg⁹-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg⁰-HYP³-Thienyl^5,8-D-Phe⁷]-BK (HYP³-antagonist), [D-Arg⁰-HYP³-Thienyl,^5,8-D-Phe⁷]-BK (Thienyl antagonist) and Des-Arg⁹-Leu⁸-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B₂-type of BK receptors. This was confirmed further by the fact that both the B₂-selective Hyp³- and Thienyl-antagonists inhibited BK-induced PI turnover with K_BS of 369±51 nM and 368±118 nM respectively while the B₁-selective antagonist, Des-Arg⁹-Leu⁸-BK, was inactive at 1 M. [³H]BK receptor binding studies revealed two binding sites, one with high affinity (K_d=0.24±0.06 nM; B_max=1.4±0.4 pmol/g tissue) and the other with low affinity (K_d=18.5±0.95 nM; B_max=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [³H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B₂-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [³H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A₂.     

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

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 bradykinin B1 receptor antagonist R-954 inhibits Ehrlich tumor growth in rodents

The present study investigated the effects of a new bradykinin B₁ receptor antagonist, R-954, on the development of Ehrlich ascitic tumor (EAT) induced by the intraperitoneal inoculation of EAT cells in mice and the formation of a solid tumor by the subcutaneous injection of the cells in rat paw. The development of the tumor was associated with an increase in mouse total cell counts in bone marrow (10.8-fold), ascitic fluid (14.6-fold), and blood (12.6-fold). R-954 (2 mg/kg, s.c.) significantly reduced the ascitic fluid volume (63.7%) and the mouse weight gain (30.5%) after 10 consecutive days of treatment. The B₁ antagonist as well as the anti-neoplasic drug vincristine also significantly inhibited the increase in total cell count in bone marrow, ascitic fluid, and blood. R-954 reduced significantly the total protein extravasation (57.3%), the production of nitric oxide (56%), PGE₂ production (82%), and TNFα release (85.7%) in mice peritoneal cavity whereas vincristine reduced the release of these inflammatory mediators by 84-94%. The increase in paw edema after intraplantar injection of EAT cells was reduced by approximately 52% by either R-954 or vincristine treatment. In conclusion, this study presents for the first time the antitumoral activity of a new bradykinin B₁ receptor antagonist on ascitic and solid tumors induced by Ehrlich cell inoculation in mice and rats.     

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus

A transgenic mouse model, deficient in kinin B₁ receptor (B₁^−/−) was used to evaluate the role of B₂ receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B₁^−/− fundus. However, ACE activity detected by the convertion of Ang I to Ang II was inhibited by captopril in both types of gastric tissues. Taking into account the hypothesis that captopril and ACE bind to the B₂ receptor, we suggest that this complex was not formed in the stomach deficient in B₁ receptor. Therefore, our finding strongly support the hypothesis that in smooth muscles that constitutively express the kinin B₁ and B₂ receptors, an interaction between captopril and ACE, B₁ and B₂ receptors should occur forming a complex protein interaction for the potentiating effect of ACE on kinin receptors.     

Discovery of novel diarylketoxime derivatives as selective and orally active melanin-concentrating hormone 1 receptor antagonists

Optimization of the lead 2a led to the identification of a novel diarylketoxime class of melanin-concentrating hormone 1 receptor (MCH-1R) antagonists. Our focus was directed toward improvement of hERG activity and metabolic stability. The representative derivative 4b showed potent and dose-dependent body weight reduction in diet-induced obese (DIO) C57BL/6J mice after oral administration. The synthesis and structure–activity relationships of the novel diarylketoxime MCH-1R antagonists are described.     

Aryl sulfones as novel bradykinin B1 receptor antagonists for treatment of chronic pain

We report the development of aryl sulfones as Bradykinin B1 receptor antagonists. Variation of the linker region identified diol 23 as a potent B1 antagonist, while modifications of the aryl moiety led to compound 26, both of which were efficacious in rabbit biochemical challenge and pain models.     

Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening

G protein-coupled receptors (GPCRs) are involved in a large variety of physiological disorders, and are thus important pharmaceutical drug targets. Here, we describe the development and characterization of a beta-lactamase reporter gene assay as a functional readout for the ligand-induced activation of the human bradykinin B1 receptor, expressed recombinantly in CHO cells. The beta-lactamase reporter gene assay provides high sensitivity due to the absence of endogenous beta-lactamase activity in mammalian cells. The cell-permeable fluorogenic substrate allows single-cell cloning of cells expressing functional BK1 receptors. Pharmacological characterization reveals comparable sensitivity and potency of known BK1 receptor agonists and antagonists between the beta-lactamase assay, competition-binding assay, and other direct measurements of second messengers. The beta-lactamase assay has been optimized for cell density, time of agonist stimulation, and DMSO sensitivity. This CHO-hBK1-beta-lactamase assay is well suited to automation and miniaturization required for high-throughput screening.     

Structure-activity relationships of trout bradykinin ([Arg(0),Trp(5),Leu(8)]-bradykinin)

Trout bradykinin ([Arg⁰,Trp⁵,Leu⁸]-BK) produces sustained and concentration–dependent contractions of isolated longitudinal smooth muscle from trout stomach, although mammalian BK is without effect. Circular dichroism studies have demonstrated that trout BK, unlike mammalian BK, does not adopt a stable β-turn conformation, even in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol. The myotropic actions of a series of analogs in which each amino acid in trout BK was replaced by either alanine or the corresponding D-isomer were investigated. The peptides with Ala⁴, D-Pro³, D-Trp⁵, D-Ser⁶, and D-Pro⁷ substitutions were inactive and did not act as antagonists of trout BK. The analog with [Ala⁵] was a weak partial agonist. The substitution (Arg⁰ → Ala) led to >50-fold decrease in potency but, in contrast to the importance of Phe⁸ in both BK and desArg⁹-BK in activating the mammalian B₂ and B₁ receptors respectively, substitutions at Leu⁸ in trout BK had only a minor effect on potency. Antagonists to the mammalian B₂ receptor generally contain a D-aromatic amino acid at position 7 of BK but the analog [Arg⁰,Trp⁵,D-Phe⁷,Leu⁸]-BK was a weak agonist at the trout receptor. Similarly, the potent nonpeptide mammalian B₂ receptor antagonist FR173657 was without effect on the action of trout BK. These data suggest the hypothesis that the receptor binding conformation of trout BK is defined by the central region (residues 3–7) of the peptide but is adopted only upon interaction with the receptor. The bioactive conformation is probably stabilized by an ionic interaction between Arg⁰ in the peptide and an acidic residue in the receptor.     

Direct protein-protein interaction between PLCgamma1 and the bradykinin B2 receptor--importance of growth conditions

Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)gamma1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCgamma1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCgamma1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCgamma1. Finally we also identified bradykinin-induced PLCgamma1 recruitment and activation in primary culture renal mesangial cells.     

Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay

The folate receptor (FR) is a valuable therapeutic target that is highly expressed on a variety of cancers. The current development of folate-targeted cancer therapies has created the need for quantitating functional FRs in clinical specimens. In this article, we report on the creation of a highly sensitive radioactive binding method for quantitatively measuring FR expression in frozen tissue homogenates. Expression was positive in approximately 89% of human ovarian carcinomas but was negligible in both mucinous ovarian carcinomas and normal ovary. Expression was also significant in carcinomas of the kidney, endometrium, lung, breast, bladder, and pancreas. Normal tissues from humans and six different laboratory species were also analyzed; surprisingly, some interspecies variability in FR expression (especially in kidney, spleen, and lung tissue) was found. Interestingly, normal human lung tissue displayed high expression levels, whereas expression in normal lung of the other species was negligible. However, considering that folate-drug conjugates fail to accumulate in the lungs of patients, the consequence of this finding was not considered to be of clinical concern. Overall, this new methodology is reliable for determining functional FR expression levels in tissues, and it could possibly be a useful clinical test to determine patient candidacy for FR-targeted therapeutics.     

Blockade of bradykinin B2 receptor more effectively reduces postischemic blood-brain barrier disruption and cytokines release than B1 receptor inhibition

Blood-brain barrier disruption and brain edema are detrimental in ischemic stroke. The kallikrein-kinin system appears to play an important role in the regulation of vascular permeability and is invoked in edema formation. The effects of kinins are mediated by bradykinin receptors B1R and B2R. However, little is known about the exact roles of bradykinin receptors in the early stage of cerebral ischemia. In this study, we demonstrated that ischemia upregulated the level of B1R and B2R at 24 h after reperfusion by immunofluorescence assays, mainly expressed in astrocytes and neurons, respectively, in the ischemic penumbra. Moreover, B2R inhibition more effectively reduced neurological severity scores, blood-brain barrier permeability and cytokines release than B1R inhibition did. Additionally, B2R inhibition also significantly suppressed B1R protein level. Therefore, blockade of B2R may be a more effective strategy for the treatment of ischemic brain injury than B1R inhibition within 24 h after reperfusion.     

Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury

Phospholipase A₂ is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A₂ (cPLA₂)-related inflammatory responses after TBI. We found that cPLA₂ and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA₂ expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA₂ inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA₂ in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA₂-related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA₂ activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA₂ activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA₂-related inflammatory response from the PKC pathway.     

N-methylthioacetylation of RYYRIK-NH2 with enhanced specific binding affinity and high antagonist activity for nociceptin ORL1 receptor

Antagonists of the neuropeptide nociceptin are expected to be potential analgesic and antineuropathic drugs acting on ORL1 GPCR receptors. The peptide library-based antagonist Ac-RYYRIK-NH₂ inhibits the nociceptin activity mediated through ORL1, but preserves a considerably high level of agonist activity. We previously reported that the N-terminal acyl group is important for interaction with specific receptors, and developed isovarelyl-RYYRIK-NH₂, which exhibits strong antagonist activity with negligible agonist activity. In the present study, in order to obtain a more potent antagonist, we further modified the isovarelyl group by replacing its Cβ atom with an oxygen, nitrogen, or sulfur atom to give the methyl group improved interaction ability. The methyl group bound to such heteroatoms was expected to enhance the hydrophobic interaction between the peptide and the ORL1 receptor. The RYYRIK-NH₂ peptide with a methylthioacetyl group, CH₃SCH₂CO, revealed a higher receptor-binding affinity with strong antagonist activity, and the results suggested the presence of a receptor aromatic group as a complementary residue of this CH₃S group.     

Discovery of potent transient receptor potential vanilloid 1 antagonists: Design and synthesis of phenoxyacetamide derivatives

We aimed to discover a novel type of transient receptor potential vanilloid 1 (TRPV1) antagonist because such antagonists are possible drug candidates for treating various disorders. We modified the structure of hit compound 7 (human TRPV1 IC₅₀ = 411 nM) and converted its pyrrolidino group to a (hydroxyethyl)methylamino group, which substantially improved inhibitory activity (15d; human TRPV1 IC₅₀ = 33 nM). In addition, 15d ameliorated bladder overactivity in rats in vivo.     

Discovery of a series of acrylic acids and their derivatives as chemical leads for selective EP3 receptor antagonists

A series of acrylic acids and their structurally related compounds were evaluated for their binding affinity to four EP receptor subtypes (EP1-4). Starting from the initial hit 3, which was discovered in our in-house library, compounds 4 and 5 were identified as new chemical leads as candidates for further optimization towards a selective EP3 receptor antagonist. The identification process of these compounds and their pharmacokinetic profiles are presented.     

5-Hydroxy-2-(2-phenylethyl)chromone (5-HPEC): A novel non-nitrogenous ligand for 5-HT2B receptor

Chromones are a class of natural products found in almost every known terrestrial plant with over 4000 naturally occurring derivatives having been isolated and structurally elucidated. Recently, 5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC), isolated from Imperata cylindrical, showed neuroprotective activity against glutamate induced excitotoxicity in primary cultures of rat cortical cells. In comparison to other naturally occurring neuroprotective chromones, 5-HPEC contains fewer hydroxyl groups. Here we report our most recent characterization on this interesting natural product against a number of CNS receptors for the purpose to identify the potential molecular targets that may be related to its biological activity. Based on our studies, including radiobinding assays, calcium flux functional assays and molecular modeling studies, 5-HPEC may represent a type of novel nonnitrogenous ligands to the 5-HT2B receptor.