Design and synthesis of aryl sulfonamide-based nonsteroidal mineralocorticoid receptor antagonists

Hit-to-lead medicinal chemistry efforts are described starting from a screening hit 1, leading to a new class of aryl sulfonamide-based MR antagonist, exemplified by 17, that possesses favourable MR binding affinity, selectivity profile against closely related NHRs, physicochemical properties and metabolic stability.     

Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin

We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [相似文献   

Hydantoin based inhibitors of MMP13—Discovery of AZD6605

Piperidine ether and aryl piperazine hydantoins are reported as potent inhibitors of MMP13. A medicinal chemistry campaign focused on replacing the reverse hydroxamate zinc binding group associated with historical inhibitors with a hydantoin zinc binding group then optimising MMP13 potency, solubility and DMPK properties whilst maintaining good selectivity over MMP14. A number of high quality candidates were progressed and following rat and dog safety evaluation, AZD6605 (3m) was identified as a candidate drug.     

Apelin, the natural ligand of the orphan seven-transmembrane receptor APJ, inhibits human immunodeficiency virus type 1 entry

In addition to the CCR5 and CXCR4 chemokine receptors, a subset of primary human immunodeficiency virus type 1 (HIV-1) isolates can also use the seven-transmembrane-domain receptor APJ as a coreceptor. A previously identified ligand of APJ, apelin, specifically inhibited the entry of primary T-tropic and dualtropic HIV-1 isolates from different clades into cells expressing CD4 and APJ. Analysis of apelin analogues demonstrated that potent and specific antiviral activity was retained by a 13-residue, arginine-rich peptide. Antiviral potency was influenced by the integrity of methionine 75, which contributes to APJ-binding affinity, and by the retention of apelin residues 63 to 65. These studies demonstrate the ability of a small peptide ligand to block the function of APJ as an HIV-1 coreceptor, identify apelin sequences important for the inhibition, and provide new reagents for the investigation of the significance of APJ to HIV-1 infection and pathogenesis.     

Tricyclic imidazole antagonists of the Neuropeptide S Receptor

A new structural class of potent antagonists of the Neuropeptide S Receptor (NPSR) is reported. High-throughput screening identified a tricyclic imidazole antagonist of NPSR, and medicinal chemistry optimization of this structure was undertaken to improve potency against the receptor as well as CNS penetration. Detailed herein are synthetic and medicinal chemistry studies that led to the identification of antagonists 15 and NPSR-PI1, which demonstrate potent in vitro NPSR antagonism and central exposure in vivo.     

Ontogeny of apelin and its receptor in the rodent gastrointestinal tract

Apelin is the endogenous ligand for the APJ receptor and both apelin and APJ are expressed in the gastrointestinal (GI) tract. The aim of this study was to define ontogeny of apelin and APJ in the developing rodent GI tract by measuring expression levels and characterizing abundance and cellular localization at an embryonic stage (E18.5 or E21), two postnatal stages (P4, P16) and in the adult. Apelin and APJ mRNA levels were measured by real time RT-PCR, apelin and APJ-containing cells were identified by immunohistochemical (IHC) staining. Gastric, duodenal and colonic apelin and APJ mRNA levels were highest at birth and declined postnatally. In the postnatal rat stomach, few apelin peptide-containing cells were identified, the density of gastric apelin-containing cells increased progressively after weaning and into adulthood. A robust APJ immunostaining was observed postnatally in the epithelium, intestinal goblet cells and in smooth muscle cells. In the adult rat, APJ immunostaining in the surface epithelium and goblet cells decreased markedly. During the early postnatal period, in an apelin-deficient mouse, APJ expression and immunostaining in the gut were reduced suggesting that apelin regulates APJ. Together, our data support a role for the apelin–APJ system in the regulation of smooth muscle, epithelial and goblet cell function in the GI tract.     

Tumor Endothelial Cell-Specific Drug Delivery System Using Apelin-Conjugated Liposomes

Background

A drug delivery system specifically targeting endothelial cells (ECs) in tumors is required to prevent normal blood vessels from being damaged by angiogenesis inhibitors. The purpose of this study was to investigate whether apelin, a ligand for APJ expressed in ECs when angiogenesis is taking place, can be used for targeting drug delivery to ECs in tumors.

Methods and Results

Uptake of apelin via APJ stably expressed in NIH-3T3 cells was investigated using TAMRA (fluorescent probe)-conjugated apelin. Both long and short forms of apelin (apelin 36 and apelin 13) were taken up, the latter more effectively. To improve efficacy of apelin- liposome conjugates, we introduced cysteine, with its sulfhydryl group, to the C terminus of apelin 13, resulting in the generation of apelin 14. In turn, apelin 14 was conjugated to rhodamine-encapsulating liposomes and administered to tumor-bearing mice. In the tumor microenvironment, we confirmed that liposomes were incorporated into the cytoplasm of ECs. In contrast, apelin non-conjugated liposomes were rarely found in the cytoplasm of ECs. Moreover, non-specific uptake of apelin-conjugated liposomes was rarely detected in other normal organs.

Conclusions

ECs in normal organs express little APJ; however, upon hypoxic stimulation, such as in tumors, ECs start to express APJ. The present study suggests that apelin could represent a suitable tool to effectively deliver drugs specifically to ECs within tumors.     

Apelin protects myocardial injury induced by isoproterenol in rats

We aimed to explore the change in level of apelin and its receptor APJ during myocardial injury and the therapeutic effects of apelin in myocardial injury. Rat myocardial injury was induced by subcutaneous injection of a high dose of isoproterenol (ISO); apelin and APJ mRNA levels were determined by RT-PCR; APJ protein was determined by Western blot; EIA and RIA were used to measure the apelin content and receptor binding, respectively. Plasma lactate dehydrogenase (LDH) activity and myocardial and plasma malondialdehyde (MDA) contents were higher in ISO-treated hearts than that in controls. ISO-treated rats showed lower +/-LV dp/dt(max) values and higher LVEDP value (all P<0.01), which suggested severe heart failure. As well, the apelin content in plasma, atrial and ventricular myocardium was decreased by 27%, 30% and 25% (P<0.01), respectively. The mRNA levels of apelin and APJ in myocardia were also markedly reduced; but the APJ protein level in myocardia was increased. However, administration of apelin significantly ameliorated myocardial injury and ISO-induced heart failure. Compared with the ISO-alone group, the group given low-dosage apelin (5 nmol/kg/day) had 39% and 66% higher +LV dp/dt(max) and -LV dp/dt(max) values, and 40.7% lower LVEDP value (P<0.01), and the leakage of myocardial LDH and increased MDA content were attenuated (all P<0.01). Interestingly, bolus injections of apelin (10 nmol/kg/day) resulted in potent inotropic effects in ISO-treated rats. ISO-induced myocardial injury resulted in hypoexpression of apelin and its receptor APJ, and the administration of exogenous apelin ameliorated heart failure and myocardial injury. Apelin could have a cardioprotective effect, and the apelin-APJ system may be a new therapeutic target in myocardial injury and heart failure.     

Apelin suppresses apoptosis of human osteoblasts

Objectives: Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. Osteoblast can express apelin and APJ. The aim of this study was to investigate the action of apelin on apoptosis of human osteoblasts. Results: Apelin inhibited human osteoblasts apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Our study also showed an increased Bcl-2 protein expression and decreased Bax protein expression under the treatment of apelin. Apelin decreased cytochrome c release and caspase-3 activation in human osteoblasts. Apelin activated phosphatidylinositol-3 kinase (PI-3 kinase) and Akt. The apelin-induced activation of Akt was blocked by suppression of APJ with siRNA. LY294002 (a PI-3 kinase inhibitor) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO; an Akt inhibitor) abolished apelin induced activation of Akt, and, LY294002 or HIMO abolished the anti-apoptotic activity of apelin. Furthermore, apelin protects against apoptosis induced by the glucocorticoid dexamethasone. Conclusions: Apelin suppresses serum deprivation-induced apoptosis of human osteoblasts and the anti-apoptotic action is mediated via the APJ/PI-3 kinase/Akt signaling pathway. Hui Xie and Ling-Qing Yuan both authors contributed equally to this work.     

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ～330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure–activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.     

Apela Regulates Fluid Homeostasis by Binding to the APJ Receptor to Activate Gi Signaling

Apela (APJ early endogenous ligand, also known as elabela or toddler) is a recently discovered peptide hormone. Based on genetic studies in zebrafish, apela was found to be important for endoderm differentiation and heart development during embryogenesis. Although common phenotypes of apela and APJ-null zebrafish during embryonic development suggested that apela interacts with the APJ receptor, kinetics of apela binding to APJ and intracellular signaling pathways for apela remain unknown. The role of apela in adults is also uncertain. Using a chimeric apela ligand, we showed direct binding of apela to APJ with high affinity (K_d = 0.51 nm) and the ability of apelin, the known peptide ligand for APJ, to compete for apela binding. Apela, similar to apelin, acts through the inhibitory G protein pathway by inhibiting forskolin-stimulated cAMP production and by inducing ERK1/2 phosphorylation. In adult rats, apela is expressed exclusively in the kidney, unlike the wide tissue distribution of apelin. In vivo studies demonstrated the ability of apela to regulate fluid homeostasis by increasing diuresis and water intake. Dose-response studies further indicated that apela induces 2- and 5-fold higher maximal responses than apelin in ERK1/2 phosphorylation and diuresis/water intake, respectively. After designing an apela antagonist, we further demonstrated the role of endogenous ligand(s) in regulating APJ-mediated fluid homeostasis. Our results identified apela as a potent peptide hormone capable of regulating fluid homeostasis in adult kidney through coupling to the APJ-mediated G_i signaling pathway.     

Synthesis and structure–activity relationship of dihydrobenzofuran derivatives as novel human GPR119 agonists

Through appropriate medicinal chemistry design tactics and computer-assisted conformational modeling, the initial lead A was evolved into a series of dihydrobenzofuran derivatives 3 as potent GPR119 agonists. This Letter describes the optimization of general structure 3, including the substituent(s) on dihydrobenzofuran, the R¹ attachment on right-hand piperidine nitrogen, and the left-hand piperidine/piperazine and its attachment R². The efforts led to the identification of compounds 13c and 24 as potent human GPR119 modulators with favorable metabolic stability, ion channel activity, and PXR profiles.     

Identifying structural determinants of potency for analogs of apelin-13: Integration of C-terminal truncation with structure–activity

Apelin peptides function as endogenous ligands of the APJ receptor and have been implicated in a number of important biological processes. While several apelinergic peptides have been reported, apelin-13 (Glu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe) remains the most commonly studied and reported ligand of APJ. This study examines the effect of C-terminal peptide truncations and comprehensive structure–activity relationship (SAR) for a series of analogs based on apelin-13 in an attempt to develop more potent and stable analogs. C-terminal truncation studies identified apelin-13 (N-acetyl 2–11) amide (9) as a potent agonist (EC₅₀ = 4.4 nM). Comprehensive SAR studies also determined that Arg-2, Leu-5, Lys-8, Met-11, were key positions for determining agonist potency, whereas the hydrophobic volume of Lys-8 was a specific determinate of activity. Plasma stability studies on the truncated 10-mer peptide 28 (EC₅₀ = 33 nM) indicated the primary sites of cleavage occurred between Nle-3 and Leu-4 and also between Ala-5 and Ala-6. These new ligands represent the shortest known apelin peptides with good functional potency.     

Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo

APJ is a G-protein-coupled receptor with seven transmembrane domains, and its endogenous ligand, apelin, was identified recently. They are highly expressed in the cardiovascular system, suggesting that APJ is important in the regulation of blood pressure. To investigate the physiological functions of APJ, we have generated mice lacking the gene encoding APJ. The base-line blood pressure of APJ-deficient mice is equivalent to that of wild-type mice in the steady state. The administration of apelin transiently decreased the blood pressure of wild-type mice and a hypertensive model animal, a spontaneously hypertensive rat. On the other hand, this hypotensive response to apelin was abolished in APJ-deficient mice. This apelin-induced response was inhibited by pretreatment with a nitric-oxide synthase inhibitor, and apelin-induced phosphorylation of endothelial nitric-oxide synthase in lung endothelial cells from APJ-deficient mice disappeared. In addition, APJ-deficient mice showed an increased vasopressor response to the most potent vasoconstrictor angiotensin II, and the base-line blood pressure of double mutant mice homozygous for both APJ and angiotensin-type 1a receptor was significantly elevated compared with that of angiotensin-type 1a receptor-deficient mice. These results demonstrate that APJ exerts the hypotensive effect in vivo and plays a counterregulatory role against the pressor action of angiotensin II.     

Identification of potent pyrazole based APELIN receptor (APJ) agonists

The apelinergic system comprises the apelin receptor and its cognate apelin and elabela peptide ligands of various lengths. This system has become an increasingly attractive target for pulmonary and cardiometabolic diseases. Small molecule regulators of this receptor with good drug-like properties are needed. Recently, we discovered a novel pyrazole based small molecule agonist 8 of the apelin receptor (EC₅₀ = 21.5 µM, K_i = 5.2 µM) through focused screening which was further optimized to initial lead 9 (EC₅₀ = 0.800 µM, K_i = 1.3 µM). In our efforts to synthesize more potent agonists and to explore the structural features important for apelin receptor agonism, we carried out structural modifications at N1 of the pyrazole core as well as the amino acid side-chain of 9. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC₅₀ values of <100 nM. Recruitment of β-arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β-arrestin recruitment. These compounds may be suitable as tools for in vivo studies of apelin receptor function.     

Exercise training promotes expression of apelin and APJ of cardiovascular tissues in spontaneously hypertensive rats

Because apelin may play an important regulatory role in human cardiac dysfunction, we investigated alterations in cardiovascular content of apelin and its receptor, APJ, during hypertension and the effect of exercise training on the cardiovascular apelin/APJ system in hypertensive animals. Spontaneously hypertensive rats (SHRs) underwent swimming training consisting of 54 swimming sessions of 60 min each (6 days/week for 9 weeks). Systolic blood pressure (SBP) was verified weekly by tail-cuff plethysmography. Apelin levels in plasma and cardiovascular tissues were determined by radioimmunoassay. The level of apelin/APJ mRNA was determined by RT-PCR. SHRs showed severe hypertension and pathological cardiomegaly. The level of apelin immunoreactivity (apelin-ir) in plasma and ventricular and aortic tissues was lower, by 40%, 40% and 42% (all P<0.01), respectively, in SHRs than in control Wistar-Kyoto rats, and the mRNA level of apelin and APJ in myocardium and aorta was markedly decreased. Compared with sedentary SHRs, swimming-trained SHRs showed decreased SBP and elevated mRNA expression of apelin and APJ in cardiovascular tissues and elevated apelin-ir level in plasma, myocardium and aorta (all P<0.01). SBP and level of apelin-ir in plasma and cardiovascular tissues were negatively correlated. Long-term swimming training relieved the pathogenesis of hypertension and reversed the downregulation of the cardiovascular apelin/APJ system induced by hypertension, which suggests that the improving effect of exercise training on hypertension could be mediated by upregulating the cardiovascular apelin/APJ system.     

Regulation of apelin mRNA expression by insulin and glucocorticoids in mouse 3T3-L1 adipocytes

The novel 36-amino acid peptide, apelin, is the endogenous ligand for the orphan receptor APJ. Apelin may play important roles in the regulation of the cardiovascular system and the hypothalamic-pituitary axis. It is a potent hypotensive agent and one of the most potent stimulators of cardiac contractility. In this study, we investigated the roles of apelin derived from adipocytes in the regulation of cardiovascular homeostasis. We found that both apelin and APJ mRNAs were expressed in isolated mouse adipocytes and that apelin mRNA levels increased during the differentiation of 3T3-L1 cells to adipocytes. We also found that the administration of insulin (1 nM-100 nM) increased, while that of dexamethasone (0.1 nM-100 nM) decreased the apelin mRNA levels in 3T3-L1 adipocytes in a dose-dependent manner, suggesting that insulin and glucocorticoids regulate apelin gene expression in adipocytes. We speculate that high glucocorticoid levels suppress apelin production and stimulate angiotensin II production in adipocyte, decreasing the counter-regulatory activity of apelin against the pressor action of angiotensin II, which might partly be involved in the mechanism underlying the development of obesity-related hypertension.     

Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation

In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells.     

Biology of the apelin-APJ axis in vascular formation

Apelin is a bioactive peptide with diverse physiological actions on many tissues mediated by its interaction with its specific receptor APJ. Since the identification of apelin and APJ in 1998, pleiotropic roles of the apelin/APJ system have been elucidated in different tissues and organs, including modulation of the cardiovascular system, fluid homeostasis, metabolic pathway and vascular formation. In blood vessels, apelin and APJ expression are spatiotemporally regulated in endothelial cells (ECs) during angiogenesis. In vitro analysis revealed that the apelin/APJ system regulates angiogenesis by the induction of proliferation, migration and cord formation of cultured ECs. Moreover, apelin seems to stabilize cell-cell junctions of ECs. In addition, genetically engineered mouse models suggest that apelin/APJ regulates vascular stabilization and maturation in physiological and pathological angiogenesis. In this review, we summarize the current understanding of the apelin/APJ system for vascular formation and maturation.