Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Design and preparation of N-linked hydroxypyridine-based APJ agonists.

Agonism of the apelin receptor (APJ) has demonstrated beneficial effects in models of heart failure. We have previously disclosed compounds such as 4, which showed good APJ agonist activity but were metabolized to the mono-demethylated, non-interconverting atropisomer metabolites. Herein, we detail the design and optimization of a novel series of N-linked APJ agonists with good potency, metabolic stability, and rat pharmacokinetic profile, which are unable to undergo the same metabolic mono-demethylation cleavage.

Synthesis and characterization of an orally bioavailable small molecule agonist of the apelin receptor.

The apelin receptor (APJ) is a target for cardiovascular indications. Previously, we had identified a novel pyrazole-based agonist 1 ((S)-N-(1-(cyclobutylamino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamide hydrochloride) of this GPCR. Systematic modification of 1 was performed to produce compounds with improved potency and ADME properties. Orally bioavailable compound 47 with favorable agonist potency (Ca2+EC50 = 24 nM, cAMPi EC50 = 6.5 nM) and pharmacokinetic properties (clearance ∼20 mL/min/kg in rats) was identified. This compound has vastly reduced brain penetration and is devoid of significant off-target liability. In summary, a potent and selective APJ agonist suitable for in vivo studies of APJ in peripheral tissues after oral administration has been identified.

Apelin pathway in cardiovascular, kidney, and metabolic diseases: Therapeutic role of apelin analogs and apelin receptor agonists.

The apelin/apelin receptor (ApelinR) signal transduction pathway exerts essential biological roles, particularly in the cardiovascular system. Disturbances in the apelin/ApelinR axis are linked to vascular, heart, kidney, and metabolic disorders. Therefore, the apelinergic system has surfaced as a critical therapeutic strategy for cardiovascular diseases (including pulmonary arterial hypertension), kidney disease, insulin resistance, hyponatremia, preeclampsia, and erectile dysfunction. However, apelin peptides are susceptible to rapid degradation through endogenous peptidases, limiting their use as therapeutic tools and translational potential. These proteases include angiotensin converting enzyme 2, neutral endopeptidase, and kallikrein thereby linking the apelin pathway with other peptide systems. In this context, apelin analogs with enhanced proteolytic stability and synthetic ApelinR agonists emerged as promising pharmacological alternatives. In this review, we focus on discussing the putative roles of the apelin pathway in various physiological systems from function to dysfunction, and emphasizing the therapeutic potential of newly generated metabolically stable apelin analogs and non-peptide ApelinR agonists.

Identification of a Hydroxypyrimidinone Compound (21) as a Potent APJ Receptor Agonist for the Potential Treatment of Heart Failure.

This paper describes our continued efforts in the area of small-molecule apelin receptor agonists. Recently disclosed compound 2 showed an acceptable metabolic stability but demonstrated monodemethylation of the dimethoxyphenyl group to generate atropisomer metabolites in vitro. In this article, we extended the structure-activity relationship at the C2 position that led to the identification of potent pyrazole analogues with excellent metabolic stability. Due to the increased polarity at C2, the permeability for these compounds decreased. Further adjustment of the polarity by replacing the N1 2,6-dimethoxyphenyl group with a 2,6-diethylphenyl group and reoptimization for the potency of the C5 pyrroloamides resulted in potent compounds with improved permeability. Compound 21 displayed excellent pharmacokinetic profiles in rat, monkey, and dog models and robust pharmacodynamic efficacy in the rodent heart failure model. Compound 21 also showed an acceptable safety profile in preclinical toxicology studies and was selected as a backup development candidate for the program.

Identification of 6-hydroxy-5-phenyl sulfonylpyrimidin-4(1H)-one APJ receptor agonists.

Heart failure (HF) treatment remains a critical unmet medical need. Studies in normal healthy volunteers and HF patients have shown that [Pyr1]apelin-13, the endogenous ligand for the APJ receptor, improves cardiac function. However, the short half-life of [Pyr1]apelin-13 and the need for intravenous administration have limited the therapeutic potential for chronic use. We sought to identify potent, small-molecule APJ agonists with improved pharmaceutical properties to enable oral dosing in clinical studies. In this manuscript, we describe the identification of a series of pyrimidinone sulfones as a structurally differentiated series to the clinical lead (compound 1). Optimization of the sulfone series for potency, metabolic stability and oral bioavailability led to the identification of compound 22, which showed comparable APJ potency to [Pyr1]apelin-13 and exhibited an acceptable pharmacokinetic profile to advance to the acute hemodynamic rat model.

Apelin/APJ system: A novel therapeutic target for locomotor system diseases.

Apelin is an endogenous ligand of G protein-coupled receptor APJ. Apelin/APJ system is widely expressed in abundant tissues, especially bone, joint and muscle tissue. This review focus on the effects of apelin/APJ system on locomotor system. An increasing number of evidence suggests that apelin/APJ system plays a crucial role in many physiological and pathological processes of locomotor system. Physiologically, apelin/APJ system promotes bone formation, muscle metabolism and skeletal muscle production. Pathologically, apelin/APJ system exacerbates osteoarthritis pathogenesis, whereas it alleviates osteoporosis. Besides, the level of apelin expression is regulated by different training modes, including continuous aerobic exercise, high-intensity interval training and resistance exercises. More importantly, exercise-induced apelin may be a potent pharmacological agent for the treatment of diseases and the regulation of physiological processes. Considering the pleiotropic effects of apelin on locomotor system, apelin/APJ system may be an important therapeutic target for locomotor system diseases.

Molecular mechanism of apelin-13 regulation of colonic motility in rats.

Apelin is a novel neuropeptide identified as the endogenous ligand for the apelin receptor. Apelin and its receptor are widely distributed in the gastrointestinal tract. Studies have reported that apelin-13 is involved in modulating gastrointestinal motility; however, the evidence is insufficient and the relevant mechanism is still not fully clear. Consequently, our study designed to explore the effect induced by exogenous apelin-13, to analyze the mechanism of action in isolated rat colons and colonic smooth muscle cells. The spontaneous contractions of colonic smooth muscle strips from rat were measured in an organ bath system. L-type calcium currents and large conductance Ca2+-activated K+ (BKCa) currents in rat colonic smooth muscle cells were investigated using the electrophysiological patch-clamp technique. Apelin-13 decreased the spontaneous contractile activity of colonic smooth muscle strips in a dose-dependent manner, and the inhibitory effect was not abolished by tetrodotoxin. The electrophysiological recordings revealed that apelin-13 reduced the crest currents of L-type calcium in a concentration-dependent manner in colonic smooth muscle cells at the test potential of 0 mV. Moreover, apelin-13 moved the current-voltage (I-V) curves of L-type calcium channels upward, but did not change their contour. Furthermore, the characteristics of L-type calcium channels with steady-state activation and steady-state inactivation were not significantly changed. Similarly, application of apelin-13 also significantly decreased BKCa currents in a concentration-dependent manner. In conclusion, apelin-13 inhibited the spontaneous contractile activity of isolated rat colons via the suppression of L-type calcium channels and BKCa channels in colonic smooth muscle cells.

Discovery of a Hydroxypyridinone APJ Receptor Agonist as a Clinical Candidate.

Apelin-13 is an endogenous peptidic agonist of the apelin receptor (APJ) receptor with the potential for improving cardiac function in heart failure patients. However, the low plasma stability of apelin-13 necessitates continuous intravenous infusion for therapeutic use. There are several approaches to increase the stability of apelin-13 including attachment of pharmacokinetic enhancing groups, stabilized peptides, and Fc-fusion approaches. We sought a small-molecule APJ receptor agonist approach to target a compound with a pharmacokinetic profile amenable for chronic oral administration. This manuscript describes sequential optimization of the pyrimidinone series, leading to pyridinone 14, with in vitro potency equivalent to the endogenous ligand apelin-13 and with an excellent oral bioavailability and PK profile in multiple preclinical species. Compound 14 exhibited robust pharmacodynamic effects similar to apelin-13 in an acute rat pressure-volume loop model and was advanced as a clinical candidate.

Pyrazole Agonist of the Apelin Receptor Improves Symptoms of Metabolic Syndrome in Mice.

Apelin receptor agonism improves symptoms of metabolic syndrome. However, endogenous apelin peptides have short half-lives, making their utility as potential drugs limited. Previously, we had identified a novel pyrazole-based agonist scaffold. Systematic modification of this scaffold was performed to produce compounds with improved ADME properties. Compound 13 with favorable agonist potency (cAMPi EC50 = 162 nM), human liver microsome stability (T1/2 = 62 min), and pharmacokinetic profile in rodents was identified. The compound was tested in a mouse model of diet-induced obesity (DIO) and metabolic syndrome for efficacy. Treatment with 13 led to significant weight loss, hypophagia, improved glucose utilization, reduced liver steatosis, and improvement of disease-associated biomarkers. In conclusion, a small-molecule agonist of the apelin receptor has been identified that is suitable for in vivo investigation of the apelinergic system in DIO and perhaps other diseases where this receptor has been implicated to play a role.

In Vitro and In Vivo Evaluation of a Small-Molecule APJ (Apelin Receptor) Agonist, BMS-986224, as a Potential Treatment for Heart Failure.

BACKGROUND: New heart failure therapies that safely augment cardiac contractility and output are needed. Previous apelin peptide studies have highlighted the potential for APJ (apelin receptor) agonism to enhance cardiac function in heart failure. However, apelin's short half-life limits its therapeutic utility. Here, we describe the preclinical characterization of a novel, orally bioavailable APJ agonist, BMS-986224. METHODS: BMS-986224 pharmacology was compared with (Pyr1) apelin-13 using radio ligand binding and signaling pathway assays downstream of APJ (cAMP, phosphorylated ERK [extracellular signal-regulated kinase], bioluminescence resonance energy transfer-based G-protein assays, ß-arrestin recruitment, and receptor internalization). Acute effects on cardiac function were studied in anesthetized instrumented rats. Chronic effects of BMS-986224 were assessed echocardiographically in the RHR (renal hypertensive rat) model of cardiac hypertrophy and decreased cardiac output. RESULTS: BMS-986224 was a potent (Kd=0.3 nmol/L) and selective APJ agonist, exhibiting similar receptor binding and signaling profile to (Pyr1) apelin-13. G-protein signaling assays in human embryonic kidney 293 cells and human cardiomyocytes confirmed this and demonstrated a lack of signaling bias relative to (Pyr1) apelin-13. In anesthetized instrumented rats, short-term BMS-986224 infusion increased cardiac output (10%-15%) without affecting heart rate, which was similar to (Pyr1) apelin-13 but differentiated from dobutamine. Subcutaneous and oral BMS-986224 administration in the RHR model increased stroke volume and cardiac output to levels seen in healthy animals but without preventing cardiac hypertrophy and fibrosis, effects differentiated from enalapril. CONCLUSIONS: We identify a novel, potent, and orally bioavailable nonpeptidic APJ agonist that closely recapitulates the signaling properties of (Pyr1) apelin-13. We show that oral APJ agonist administration induces a sustained increase in cardiac output in the cardiac disease setting and exhibits a differentiated profile from the renin-angiotensin system inhibitor enalapril, supporting further clinical evaluation of BMS-986224 in heart failure.

Gαi-biased apelin analog protects against isoproterenol-induced myocardial dysfunction in rats.

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and ß-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the ß-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels.NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.

Structure-Activity Relationship and Bioactivity of Short Analogues of ELABELA as Agonists of the Apelin Receptor.

ELABELA (ELA) is the second endogenous ligand of the apelin receptor (APJ). Although apelin-13 and ELA both target APJ, there is limited information on structure-activity relationship (SAR) of ELA. In the present work, we identified the shortest bioactive C-terminal fragment ELA23-32, which possesses high affinity for APJ (Ki 4.6 nM) and produces cardiorenal effects in vivo similar to those of ELA. SAR studies on conserved residues (Leu25, His26, Val29, Pro30, Phe31, Pro32) show that ELA and apelin-13 may interact differently with APJ. His26 and Val29 emerge as important for ELA binding. Docking and binding experiments suggest that Phe31 of ELA may bind to a tight groove distinct from that of Phe13 of Ape13, while the Phe13 pocket may be occupied by Pro32 of ELA. Further characterization of signaling profiles on the Gαi1, Gα12, and ß-arrestin2 pathways reveals the importance of aromatic residue at the Phe31 or Pro32 position for receptor activation.

Half-life extension of peptidic APJ agonists by N-terminal lipid conjugation.

Agonism of the endothelial receptor APJ (putative receptor protein related to AT1; AT1: angiotensin II receptor type 1) has the potential to ameliorate congestive heart failure by increasing cardiac output without inducing hypertrophy. Although the endogenous agonist, pyr-apelin-13 (1), has shown beneficial APJ-mediated inotropic effects in rats and humans, such effects are short-lived given its extremely short half-life. Here, we report the conjugation of 1 to a fatty acid, providing a lipidated peptide (2) with increased stability that retains inotropic activity in an anesthetized rat myocardial infarction (MI) model. We also report the preparation of a library of 15-mer APJ agonist peptide-lipid conjugates, including adipoyl-γGlu-OEG-OEG-hArg-r-Q-hArg-P-r-NMeLeuSHK-G-Oic-pIPhe-P-DBip-OH (17), a potent APJ agonist with high plasma protein binding and a half-life suitable for once-daily subcutaneous dosing in rats. A correlation between subcutaneous absorption rate and lipid length/type of these conjugates is also reported.

Apelin-potential therapy for COVID-19?

We believe that, in parallel to the attempts for direct blockade of the SARS-CoV-2 penetration into host cell and repurposing drugs, finding new therapeutic strategies for patients with lung injury or cardiovascular complications/coagulopathies associated with COVID-19 should be paid particular attention. Apelin or its receptor agonists are of great potential treatment for COVID-19 through suppressing angiotensin-converting enzyme (ACE) and angiotensin II (Ang-II) production, as well as, down-regulating angiotensin receptor 1 (AT1R) and ACE2 up-regulation. These drugs have potential to improve acute lung injury and cardiovascular/coagulopathy complications in COVID-19 which are associated with elevated Ang-II/Ang(1-7) ratio.

Apelin/APJ system: an emerging therapeutic target for respiratory diseases.

Apelin is an endogenous ligand of G protein-coupled receptor APJ. It is extensively expressed in many tissues such as heart, liver, and kidney, especially in lung tissue. A growing body of evidence suggests that apelin/APJ system is closely related to the development of respiratory diseases. Therefore, in this review, we focus on the role of apelin/APJ system in respiratory diseases, including pulmonary arterial hypertension (PAH), pulmonary embolism (PE), acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), obstructive sleep apnoea syndrome (OSAS), non-small cell lung cancer (NSCLC), pulmonary edema, asthma, and chronic obstructive pulmonary diseases. In detail, apelin/APJ system attenuates PAH by activating AMPK-KLF2-eNOS-NO signaling and miR424/503-FGF axis. Also, apelin protects against ALI/ARDS by reducing mitochondrial ROS-triggered oxidative damage, mitochondria apoptosis, and inflammatory responses induced by the activation of NF-κB and NLRP3 inflammasome. Apelin/APJ system also prevents the occurrence of pulmonary edema via activating AKT-NOS3-NO pathway. Moreover, apelin/APJ system accelerates NSCLC cells' proliferation and migration via triggering ERK1/2-cyclin D1 and PAK1-cofilin signaling, respectively. Additionally, apelin/APJ system may act as a predictor in the development of OSAS and PE. Considering the pleiotropic actions of apelin/APJ system, targeting apelin/APJ system may be a potent therapeutic avenue for respiratory diseases.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

Function-based high-throughput screening for antibody antagonists and agonists against G protein-coupled receptors.

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding. However, for complex membrane proteins, such as G protein-coupled receptors (GPCRs), binding-based screening rarely results in functional antibodies. Here we describe a function-based high-throughput screening method for quickly identifying antibody antagonists and agonists against GPCRs by combining glycosylphosphatidylinositol-anchored antibody cell display with ß-arrestin recruitment-based cell sorting and screening. This method links antibody genotype with phenotype and is applicable to all GPCR targets. We validated this method by identifying a panel of antibody antagonists and an antibody agonist to the human apelin receptor from an immune antibody repertoire. In contrast, we obtained only neutral binders and antibody antagonists from the same repertoire by phage display, suggesting that the new approach described here is more efficient than traditional methods in isolating functional antibodies. This new method may create a new paradigm in antibody drug discovery.

Discovery and SAR of aryl hydroxy pyrimidinones as potent small molecule agonists of the GPCR APJ.

This article describes the discovery of aryl hydroxy pyrimidinones and the medicinal chemistry efforts to optimize this chemotype for potent APJ agonism. APJ is a G-protein coupled receptor whose natural agonist peptide, apelin, displays hemodynamic improvement in the cardiac function of heart failure patients. A high throughput screen was undertaken to identify small molecule hits that could be optimized to mimic the apelin in vitro response. A potent and low molecular weight aryl hydroxy pyrimidinone analog 30 was identified through optimization of an HTS hit and medicinal chemistry efforts to improve its properties.

Structure-guided discovery of a single-domain antibody agonist against human apelin receptor.

Developing antibody agonists targeting the human apelin receptor (APJ) is a promising therapeutic approach for the treatment of chronic heart failure. Here, we report the structure-guided discovery of a single-domain antibody (sdAb) agonist JN241-9, based on the cocrystal structure of APJ with an sdAb antagonist JN241, the first cocrystal structure of a class A G protein-coupled receptor (GPCR) with a functional antibody. As revealed by the structure, JN241 binds to the extracellular side of APJ, makes critical contacts with the second extracellular loop, and inserts the CDR3 into the ligand-binding pocket. We converted JN241 into a full agonist JN241-9 by inserting a tyrosine into the CDR3. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for finding agonistic antibodies against class A GPCRs.