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ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched., This research was funded in whole, or in part by: Wellcome Trust (WT107715/Z/15/Z, A.P.D., and J.J.M.); Wellcome Trust Programme in Metabolic and Cardiovascular Disease (203814/Z/16/A, T.L.W., D.N.), Wellcome Trust Major Award (208363/Z/17/Z) for Imaging Core (G.S.); British Heart Foundation (FS/17/61/33473 A.P.D., R.G.C.M; TG/18/4/33770, A.P.D., J.J.M.; FS/18/46/33663, S.S.). Cambridge Biomedical Research Centre Biomedical Resources Grant (University of Cambridge, Cardiovascular Theme, RG64226). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated., Williams et al. confirm that human embryonic stem cell-derived cardiomyocytes express the protein machinery critical for SARS-CoV-2 infection and are susceptible to SARS-CoV-2 spike protein pseudotyped virus infection. They further use this platform as a screen to identify inhibitors of SARS-CoV-2 infection, reporting benztropine (targeting B0AT1/ACE2 complex) and DX600 (targeting ACE2) as potential inhibitors.

Introduction Apelin receptor activation promotes beneficial vasodilation and positive inotropy1 and is critical for early cardiac development.2 In cardiovascular disease states, including heart failure, expression of apelin receptor endogenous peptide ligands, apelin and Elabela, are downregulated.1 We aim to use human embryonic stem cell (hESC)-derived cardiomyocytes (hESC-CMs) as a model to interrogate the role of the apelin receptor in the cardiovascular system and the potential of targeting the receptor therapeutically. Here, we have generated a system to inducibly knockdown the apelin receptor in hESC-CMs to examine the effects on differentiation of hESC to cardiomyocyte, and the effects on function of the resulting hESC-CMs. Methods A short hairpin RNA based apelin receptor (shAPLNR) inducible knockdown system was generated in pluripotent hESCs, utilising the sOPTiKD system as described previously,3 before differentiating to hESC-CMs. Inclusion of tetracycline in the culture medium was used to induce knockdown, with knockdown efficiency determined by qRT-PCR and radioligand binding. RNA-sequencing analysis and a panel of phenotypic assays were performed to examine effects of apelin receptor knockdown on cardiomyocyte differentiation and function. Results We have previously demonstrated that hESC-derived cardiomyocytes express the apelinergic system at a similar level to adult cardiomyocytes (Bmax hESC-CMs 21 fmol/mg versus adult 14 fmol/mg). Apelin receptor expression was significantly reduced in hESCs and hESC-CMs by tetracycline inclusion at both RNA and protein level (84.2% ± 4.3 and 82.8% ± 3.9 compared to control in hESCs and hESC-CMs, respectively). Interestingly, efficiency of differentiation to cardiomyocyte was reduced compared to control cells (77.8% ± 5.5 cardiomyocyte control vs 22.2% ± 7.4 cardiomyocyte knockdown). RNA-sequencing analysis revealed 272 differentially expressed genes involved in pathways related to electrophysiological signalling, adhesion and the cytoskeleton. Discussion We have successfully shown knockdown of apelin receptor expression in hESC-CMs. To our knowledge, this is the first use of this system to knockdown expression of a GPCR. This system allows detailed characterisation of apelin receptor activation in cardiovascular development and disease pathogenesis, providing an innovative approach to develop strategies to target the apelin receptor therapeutically in cardiovascular disease, where novel treatments are urgently needed. Conflict of Interest None References Yang et al. Circulation 2017;135:1160–1173. Chng et al. Developmental Cell 2013;27(6):672–680. Bertero et al. Development 2016;143:4405–4418.

The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan receptor but was subsequently paired with a novel peptide ligand, apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the apelin receptor, including apelin-17, apelin-13, and [Pyr1]apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the apelin receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the apelin receptor and are blocked by apelin receptor antagonists. This review summarizes the pharmacology of these ligands and the apelin receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the apelin receptor protein.

Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection.

Introduction The apelin receptor is expressed throughout the cardiovascular system, including in cardiomyocytes. Receptor activation by either of its endogenous peptide ligands, apelin or Elabela, has a positive inotropic effect and promotes vasodilatation. Human embryonic stem cell (hESC)-derived cells have the potential for use in translational research to investigate cellular signalling, disease pathogenesis and potential novel treatments. Our aim was to determine if hESC-derived cardiomyocytes express apelin receptor protein and to quantify receptor density to ascertain if this cell type can be used as a phenotypic model for human diseases associated with apelin receptor mutations. Methods H9 hESCs were cultured to induce differentiation to beating cardiomyocytes. Saturation radioligand binding experiments were performed using [Glp65,Nle75,Tyr77][125I]apelin-13 and [Pyr1]apelin-13 to define non-specific binding. Bound radioactivity was counted and data analysed using iterative curve fitting programs to obtain values of receptor affinity (KD) and density (BMAX). Immunocytochemistry was carried out using anti-apelin receptor or anti-cardiac cell marker antibodies. Results Previous work demonstrated expression of the apelin receptor in hESC-derived cardiomyocytes at the gene level by qRT-PCR at similar levels to adult cells (figure 1A). Here, radioligand binding studies have confirmed receptor protein expression in beating hESC-derived cardiomyocytes. Binding was saturable and [125I]apelin-13 bound with sub-nanomolar affinity (0.12 nM) and receptor density found to be 21 fmol/mg (figure 1B), comparable to that found in human adult heart. Hill slope was close to 1 consistent with a single binding site for apelin in these cells. Furthermore, beating hESC-derived cardiomyocytes stained positive for apelin receptor, in addition to the standard cardiac markers including cardiac troponin T (figure 2). Conclusion These data importantly confirm that hESC-derived cardiomyocytes express apelin receptor protein at similar levels to adult human heart. Apelin receptor mutations have been identified in the 100,000 Genomes Bridge Project that are associated with rare diseases, including pulmonary arterial hypertension. Our ongoing experiments aim to pharmacologically characterise the apelinergic signalling pathway in the beating hESC-derived cardiomyocytes and we propose to generate hESC-derived phenotypic models by introducing selected apelin receptor mutations via CRISPR/Cas-9 gene editing. Conflict of interest None

Introduction Fluorescent ligands are a promising tool in the study of GPCR pharmacology as they are safe and more cost effective than conventional probes such as radioligands. In this study, we investigated the use of a novel fluorescent ligand, apelin-488, to successfully show qualitative and quantitative apelin receptor internalisation, which may help further our understanding of apelin receptor pharmacology. The apelin receptor, a class A GPCR that unusually binds two endogenous peptide ligands, apelin and elabela/toddler, is expressed and functional in the cardiovascular system. Mutations in the human apelin receptor have been identified in rare diseases and our aim is to identify tool compounds to explore the potential functional consequence of these mutations. Methods CHO-K1 cells stably expressing the wild-type apelin receptor were treated with apelin-488, fixed, and stained with Hoechst 33342 and a wheat germ agglutinin-AlexaFluor-594 conjugate as nuclear and membrane markers respectively. Triple fluorescence confocal images were generated using Opera PhenixTM High Content Screening System and data were quantified using Harmony High Content Imaging and Analysis Software. Fluorescent ligand intensity at the membrane or in the cytoplasm was determined, providing cellular localisation for the apelin receptor following ligand binding. Activity of the fluorescent ligand at the apelin receptor in comparison to the endogenous ligand, [Pyr1]apelin-13 or CMF-019, a G protein biased small molecule apelin receptor ligand, was determined using a dynamic mass redistribution assay and radioligand binding assays. Results The fluorescent ligand, apelin-488 binds specifically to the apelin receptor in stably transfected CHO-K1 cells. High content screening revealed that in these cells, the apelin receptor is internalised over time and was still present at the cell membrane at an early time point, 30 secs (fig 1A). However, by 60 mins, apelin-488 membrane localisation was reduced while internal localisation had increased, suggesting substantial apelin receptor internalisation had occurred. (fig 1B). Untransfected CHO-K1 cells showed no apelin-488 signal, supporting specific binding of the fluorescent ligand to the apelin receptor. A dynamic mass redistribution assay demonstrated that apelin-488 is functional at the apelin receptor, at a similar potency as the endogenous ligand [Pyr1]apelin-13 and the G protein biased small molecule apelin receptor ligand CMF-019 (Fig 2). Conclusion Apelin-488 shows specific binding to the apelin receptor while retaining activity in a dynamic mass redistribution and binding assays. This work provides evidence for apelin-488 as a novel fluorescent tool for assessing internalisation of the apelin receptor following receptor binding. A new method for studying apelin receptor internalisation is particularly interesting due to the implication of apelin receptor desensitisation in pulmonary arterial hypertension, mediated by arrestin and internalisation pathways. Additionally, apelin-488 may provide insight into binding and receptor trafficking in mutated apelin receptors previously identified in patients with rare cardiovascular diseases. As such, apelin-488 and other fluorescent ligands may help streamline development of drugs that could be beneficial in clinical settings. Conflict of interest none

Introduction The apelin receptor is a class A GPCR that binds two endogenous peptide ligands, apelin and elabela/toddler, to regulate the cardiovascular system. Apelin peptide levels are downregulated in pulmonary arterial hypertension (PAH) but the receptor remains present and clinically targetable. The NIHR BioResource BRIDGE study, a prospective component of the Genomics England 100,000 Genomes Project, has performed case-control genomic analysis of ∼7423 patients with rare cardiovascular diseases including PAH, and has identified a number of mutations in the apelin receptor in this cohort. Of these, 11 were selected for further assessment using high content imaging, following previous saturation binding studies that showed mutational effects on receptor expression and ligand binding. Methods The Opera PhenixTM High Content Screening System was used to generate high-throughput triple fluorescence confocal images in fixed CHO-K1 cells transiently transfected with eGFP-tagged wild-type or mutant apelin receptor. Cells were stained with wheat germ agglutinin-AlexaFluor-594 and Hoechst 33342 as membrane and nuclear markers respectively. Harmony High Content Imaging and Analysis Software was used to empirically quantify fluorescence intensities of the eGFP-tagged apelin receptor in the cytoplasm and membrane of transfected cells. Results Images showed high expression of wild-type receptor with some degree of co-localisation with the membrane stain (fig 1A; fig 2). Mutation 2 showed visibly lower total protein and reduced membrane expression (fig 1B; fig 2). However, mutations 6 and 9 (Fig 1C and 1E respectively) showed similar expression and no significant difference in membrane co-localisation compared to wild-type (fig 2). Mutation 8 showed a trend for reduced membrane co-localisation (fig 1D) compared to wild-type. Note that membrane co-localisation differences closely matched differences in overall eGFP signal. Future work will aim to use machine learning to accurately isolate cell populations that show definite apelin receptor-eGFP signal at the membrane before re-running analysis. Conclusion We report the successful use of high content imaging to quantify differences in expression and membrane co-localisation of the apelin receptor when naturally occurring mutations identified in the NIHR BioResource BRIDGE study are introduced into the protein. When considered with receptor affinities and densities in previous saturation binding data, this work suggests that certain mutations may exhibit an overall impact on receptor expression (i.e. mutation 2), whilst others may directly alter ligand binding without significantly influencing protein levels (i.e. mutant 8). This work may provide great insight on the effects of real human apelin receptor mutations that may be contributing to disease phenotypes. Conflict of interest none