•Bradykinin-related peptides stimulate 2 GPCRs, the B1 and B2 receptors (B1R, B2R).•Peptide and non-peptide antagonists and fluorescent ligands are known for both.•The B2R, widely and constitutively ...expressed, is a validated target in HAE.•The B1R is mostly inducible under the effect of cytokines and immunopathology.•Signaling, adaptation and pathological roles of B1R and B2R are briefly reviewed.
Bradykinin-related peptides, the kinins, are blood-derived peptides that stimulate 2 G protein–coupled receptors, the B1 and B2 receptors (B1R, B2R). The pharmacologic and molecular identities of these 2 receptor subtypes will be succinctly reviewed herein, with emphasis on drug development, receptor expression, signaling, and adaptation to persistent stimulation. Peptide and non-peptide antagonists and fluorescent ligands have been produced for each receptor. The B2R is widely and constitutively expressed in mammalian tissues, whereas the B1R is mostly inducible under the effect of cytokines during infection and immunopathology. The B2R is temporarily desensitized by a cycle of phosphorylation/endocytosis followed by recycling, whereas the nonphosphorylable B1R is relatively resistant to desensitization and translocated to caveolae on activation. Both receptor subtypes, mainly coupled to protein G Gq, phospholipase C and calcium signaling, mediate the vascular aspects of inflammation (vasodilation, edema formation). On this basis, icatibant, a peptide antagonist of the B2R, is approved in the management of hereditary angioedema attacks. This disease is the therapeutic showcase of the kallikrein-kinin system, with an orally bioavailable B2R antagonist under development, as well as other agents that inhibit the kinin forming protease, plasma kallikrein. Other clinical applications are still elusive despite the maturity of the medicinal chemistry efforts applied to kinin receptors.
A major challenge to the treatment of advanced prostate cancer (PCa) is the development of resistance to androgen-deprivation therapy (ADT) and chemotherapy. It is imperative to discover effective ...therapies to overcome drug resistance and improve clinical outcomes. We have developed a novel class of silicon-containing compounds and evaluated the anticancer activities and mechanism of action using cellular and animal models of drug-resistant PCa. Five organosilicon compounds were evaluated for their anticancer activities in the NCI-60 panel and established drug-resistant PCa cell lines. GH1504 exhibited potent in vitro cytotoxicity in a broad spectrum of human cancer cells, including PCa cells refractory to ADT and chemotherapy. Molecular studies identified several potential targets of GH1504, most notably androgen receptor (AR), AR variant 7 (AR-v7) and survivin. Mechanistically, GH1504 may promote the protein turnover of AR, AR-v7 and survivin, thereby inducing apoptosis in ADT-resistant and chemoresistant PCa cells. Animal studies demonstrated that GH1504 effectively inhibited the in vivo growth of ADT-resistant CWR22Rv1 and chemoresistant C4-2B-TaxR xenografts in subcutaneous and intraosseous models. These preclinical results indicated that GH1504 is a promising lead that can be further developed as a novel therapy for drug-resistant PCa.
Very few antagonists have been identified for the human pregnane X receptor (PXR). These molecules may be of use for modulating the effects of therapeutic drugs, which are potent agonists for this ...receptor (e.g., some anticancer compounds and macrolide antibiotics), with subsequent effects on transcriptional regulation of xenobiotic metabolism and transporter genes. A recent novel pharmacophore for PXR antagonists was developed using three azoles and consisted of two hydrogen bond acceptor regions and two hydrophobic features. This pharmacophore also suggested an overall small binding site that was identified on the outer surface of the receptor at the AF-2 site and validated by docking studies. Using computational approaches to search libraries of known drugs or commercially available molecules is preferred over random screening. We have now described several new smaller antagonists of PXR discovered with the antagonist pharmacophore with in vitro activity in the low micromolar range S-p-tolyl 3',5-dimethyl-3,5'-biisoxazole-4'-carbothioate (SPB03255) (IC(50), 6.3 microM) and 4-(3-chlorophenyl)-5-(2,4-dichlorobenzylthio)-4H-1,2,4-triazol-3-ol (SPB00574) (IC(50), 24.8 microM). We have also used our computational pharmacophore and docking tools to suggest that most of the known PXR antagonists, such as coumestrol and sulforaphane, could also interact on the outer surface of PXR at the AF-2 domain. The involvement of this domain was also suggested by further site-directed mutagenesis work. We have additionally described an FDA approved prodrug, leflunomide (IC(50), 6.8 microM), that seems to be a PXR antagonist in vitro. These observations are important for predicting whether further molecules may interact with PXR as antagonists in vivo with potential therapeutic applications.
The bradykinin B(2) receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 ...(d-Arg-Hyp(3),Igl(5),D-Igl(7),Oic(8)-bradykinin) is a second generation peptide antagonist found to be competitive at the human B(2) receptor and insurmountable at the rabbit B(2) receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (D-Arg-Hyp(3),Igl(5),Oic(7),D-Igl(8)-bradykinin; inverted sequence Oic(7), D-Igl(8)) and B-9972 (D-Arg-Hyp(3),Igl(5),Oic(7),Igl(8)-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B(2) receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B(2) receptors: conjugate of the B(2) receptor with green fluorescent protein (B(2)R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B(2) receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B(2) receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B(2)R-GFP desensitization ((3)Hbradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B(2) receptors by the agonists. The results illustrate the agonist-antagonist transition in B(2) receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.
Survivin overexpression has been associated with an unfavorable outcome in human PCa; however, its role in metastasis remains elusive. We aim to (a) evaluate the clinical implications of survivin ...expression in PCa bone metastasis; (b) determine in vivo efficacy of BKM1740, a small-molecule compound, against PCa skeletal growth and survival; and (c) investigate molecular mechanism by which BKM1740 augments apoptosis in bone metastatic PCa cells.
Survivin expression was analyzed in PCa specimens and experimental models. Bone metastatic C4-2 and ARCaP(M) cell lines were used to evaluate the in vitro effects of BKM1740 and molecular mechanism for the induction of apoptosis. C4-2 cells were grown intratibially in athymic nude mice to evaluate the in vivo efficacy of BKM1740. Tumor growth in mouse bone was assessed by serum prostate-specific antigen and radiography and confirmed by immunohistochemical analyses.
Survivin expression is positively associated with clinical PCa bone metastasis. BKM1740 induced apoptosis in PCa cells by repressing survivin. Mice with established C4-2 tumors in tibia showed a marked decrease in serum prostate-specific antigen and much improved bone architecture radiographically after treatment with BKM1740. Immunohistochemical assays of mouse tumor samples confirmed that the in vivo effects were mediated by inhibition of survivin and induction of apoptosis.
Survivin expression is associated with PCa bone metastasis. BKM1740 treatment specifically inhibited survivin and induced apoptosis in vitro and was efficacious in retarding PCa skeletal growth in a mouse model. BKM1740 is a promising small-molecule compound that could be used to treat PCa bone metastasis.
We have designed de novo and synthesized ten 26-residue D-conformation amphipathic α-helical cationic antimicrobial peptides (AMPs), seven with “specificity determinants”, which provide specificity ...for prokaryotic cells over eukaryotic cells. The ten AMPs contain five or six positively charged residues (d-Arg, d-Lys, d-Orn, l-Dab, or l-Dap) on the polar face to understand their role in hemolytic activity against human red blood cells and antimicrobial activity against seven Acinetobacter baumannii strains, resistant to polymyxin B and colistin, and 20 A. baumannii worldwide isolates from 2016 and 2017 with antibiotic resistance to 18 different antibiotics. AMPs with specificity determinants and with l-Dab and l-Dap residues on the polar face have essentially no hemolytic activity at 1000 μg/mL (380 μM), showing for the first time the importance of these unusual amino acid residues in solving long-standing hemolysis issues of AMPs. Specificity determinants maintained excellent antimicrobial activity in the presence of human sera.
The inducible kinin B1 receptor is emerging as an attractive therapeutic target for the treatment of pain and inflammation. Although many studies described its regulation at the transcriptional ...level, little is known about the maturation of the B1 receptor. Using two human embryonic kidney (HEK) 293 cell lines stably expressing rabbit B1 receptors tagged with the yellow fluorescent protein at the C terminus (B1R-YFP) or the N-terminal myc epitope (myc-B1R), we showed that receptors are mainly retained in a perinuclear compartment and detectable as low-glycosylated species under control conditions. Interference with the ubiquitin-proteasome pathway function (proteasome inhibitors, coexpression with dominant-negative ubiquitin) blocked B1 receptor degradation and amplified its intracellular accumulation. A potent nonpeptide antagonist specifically increased the abundance of highly glycosylated B1R-YFP forms at the cell surface (accessible to chymotrypsin digestion in intact cells); this compound augmented low-glycosylated receptors in brefeldin A-treated cells, supporting the hypothesis that it reaches a newly synthesized receptor in the endoplasmic reticulum. Cell-impermeant peptide or low-affinity nonpeptide B1 receptor antagonists failed to influence the level of highly glycosylated receptors. Chemical chaperones stabilized all B1R-YFP species and up-regulated endogenous B1 receptors expressed at the surface of rabbit smooth muscle cells. Although myc-B1Rs behaved similarly to B1R-YFP in most aspects, antibody-based detection assays failed to reveal highly glycosylated species of this construct. Taken together, these results show that B1 receptors overexpressed in HEK 293 cells are degraded by the proteasome. Furthermore, a pharmacological chaperone highlights the existence of a highly N-glycosylated form of the rabbit kinin B1 receptor at the cell surface.
Pulmonary hypertension (PH) is a serious condition that affects mainly young and middle-aged women, and its etiology is poorly understood. A prominent pathological feature of PH is accumulation of ...macrophages near the arterioles of the lung. In both clinical tissue and the SU5416 (SU)/athymic rat model of severe PH, we found that the accumulated macrophages expressed high levels of leukotriene A4 hydrolase (LTA4H), the biosynthetic enzyme for leukotriene B4 (LTB4). Moreover, macrophage-derived LTB4 directly induced apoptosis in pulmonary artery endothelial cells (PAECs). Further, LTB4 induced proliferation and hypertrophy of human pulmonary artery smooth muscle cells. We found that LTB4 acted through its receptor, BLT1, to induce PAEC apoptosis by inhibiting the protective endothelial sphingosine kinase 1 (Sphk1)-endothelial nitric oxide synthase (eNOS) pathway. Blocking LTA4H decreased in vivo LTB4 levels, prevented PAEC apoptosis, restored Sphk1-eNOS signaling, and reversed fulminant PH in the SU/athymic rat model of PH. Antagonizing BLT1 similarly reversed established PH. Inhibition of LTB4 biosynthesis or signal transduction in SU-treated athymic rats with established disease also improved cardiac function and reopened obstructed arterioles; this approach was also effective in the monocrotaline model of severe PH. Human plexiform lesions, one hallmark of PH, showed increased numbers of macrophages, which expressed LTA4H, and patients with connective tissue disease-associated pulmonary arterial hypertension exhibited significantly higher LTB4 concentrations in the systemic circulation than did healthy subjects. These results uncover a possible role for macrophage-derived LTB4 in PH pathogenesis and identify a pathway that may be amenable to therapeutic targeting.
The B(1) receptor for kinins is selectively stimulated by bradykinin-related fragments lacking the C-terminal arginine, des-arginine(9)-bradykinin (des-Arg(9)-BK), and Lys-des-Arg(9)-BK. The latter ...peptide is the optimal agonist at the human and rabbit receptor. The B(1) receptor is inducible as a function of inflammatory conditions in the vasculature. We studied the effect of endogenously expressed peptidases on the potency of ligands of this receptor in an established bioassay, the rabbit aorta contractility. The potency measured for agonists (EC(50)) or antagonists (pA(2) scale) in this assay was compared with the affinity of each agent determined using (3)HLys-des-Arg(9)-BK binding competition in cultured aortic smooth muscle cells and with the competition K(i) for the hydrolysis of the aminopeptidase chromogenic substrate L-Ala-p-nitroanilide by smooth muscle cell membranes. The contractile potency of the agonist Lys-des-Arg(9)-BK is decreased by in situ metabolism, and aminopeptidase N mediates most of the distortion (inhibited by amastatin but not efficiently by puromycin). At the other end of the spectrum, the fully protected agonist Sar-D-Phe(8)des-Arg(9)-BK is not significantly potentiated by peptidase inhibitors. A similar distortion of apparent potency was observed for some peptide antagonists used in the contractility assay, B-10350 (Lys-Lys-Hyp(3), Igl(5), d-Tic(7), CpG(8)des-Arg(9)-BK) and Lys-Leu(8)des-Arg(9)-BK being intensely potentiated by amastatin treatment and effective L-Ala-p-nitroanilide competitors. N-Protected peptide antagonists or a nonpeptide antagonist of the B(1) receptor were not potentiated by amastatin. The coexpression of aminopeptidase N and the kinin B(1) receptor in rabbit arterial tissue is of interest for the inactivation of the high-affinity agonist Lys-des-Arg(9)-BK and for the design of hydrosoluble antagonist drugs.
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