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Background and purpose Adenosine is a local mediator that regulates physiological and pathological processes via activation of four GPCRs (A1 , A2A , A2B , and A3 ). We have investigated the effect of two A1 -receptor-selective agonists and the novel A1 -receptor bitopic ligand VCP746 on the rat cardiovascular system. Experimental approach The regional haemodynamic responses of these agonist was investigated in conscious rats. Male Sprague-Dawley rats (350-450 g) were chronically implanted with pulsed Doppler flow probes on the renal, mesenteric arteries and the descending abdominal aorta and the jugular vein and caudal artery catheterized. Cardiovascular responses were measured following intravenous infusion (3 min each dose) of CCPA (120, 400, and 1,200 ng·kg-1 ·min-1 ), capadenoson or adenosine (30, 100, and 300 μg·kg-1 ·min-1 ), or VCP746 (6, 20, and 60 μg·kg-1 ·min-1 ) following pre-dosing with DPCPX (0.1 mg·kg-1 , i.v.) or vehicle. Key results CCPA produced a significant A1 -receptor-mediated decrease in heart rate that was accompanied by vasoconstrictions in the renal and mesenteric vascular beds but an increase in hindquarters vascular conductance. The partial agonist capadenoson also produced an A1 -receptor-mediated bradycardia. In contrast, VCP746 produced increases in heart rate and renal and mesenteric vascular conductance that were not mediated by A1 -receptors. In vitro studies confirmed that VCP746 had potent agonist activity at both A2A - and A2B -receptors. Conclusions and implications These results suggest VCP746 mediates its cardiovascular effects via activation of A2 rather than A1 adenosine receptors. This has implications for the design of future bitopic ligands that incorporate A1 allosteric ligand moieties.

Animal models are essential for assessing cardiovascular responses to novel therapeutics. Cardiovascular safety liabilities represent a leading cause of drug attrition and better preclinical measurements are essential to predict drug-related toxicities. Presently, radiotelemetric approaches recording blood pressure are routinely used in preclinical

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the virus responsible for the COVID-19 pandemic. Patients may present as asymptomatic or demonstrate mild to severe and life-threatening symptoms. Although COVID-19 has a respiratory focus, there are major cardiovascular complications (CVCs) associated with infection. The reported CVCs include myocarditis, heart failure, arrhythmias, thromboembolism and blood pressure abnormalities. These occur, in part, because of dysregulation of the Renin–Angiotensin–Aldosterone System (RAAS) and Kinin–Kallikrein System (KKS). A major route by which SARS-CoV-2 gains cellular entry is via the docking of the viral spike (S) protein to the membrane-bound angiotensin converting enzyme 2 (ACE2). The roles of ACE2 within the cardiovascular and immune systems are vital to ensure homeostasis. The key routes for the development of CVCs and the recently described long COVID have been hypothesised as the direct consequences of the viral S protein/ACE2 axis, downregulation of ACE2 and the resulting damage inflicted by the immune response. Here, we review the impact of COVID-19 on the cardiovascular system, the mechanisms by which dysregulation of the RAAS and KKS can occur following virus infection and the future implications for pharmacological therapies.

Receptor internalization in response to prolonged agonist treatment is an important regulator of G protein-coupled receptor (GPCR) function. The adenosine A1 receptor (A1AR) is one of the adenosine receptor family of GPCRs, and evidence for its agonist-induced internalization is equivocal. The recently developed NanoBiT technology uses split NanoLuc Luciferase to monitor changes in protein interactions. We have modified the human A1AR on the N-terminus with the small high-affinity HiBiT tag. In the presence of the large NanoLuc subunit (LgBiT), complementation occurs, reconstituting a full-length functional NanoLuc Luciferase. Here, we have used complemented luminescence to monitor the internalization of the A1AR in living HEK293 cells. Agonist treatment resulted in a robust decrease in cell-surface luminescence, indicating an increase in A1AR internalization. These responses were inhibited by the A1AR-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), with an antagonist affinity that closely matched that measured using ligand binding with a fluorescent A1 receptor antagonist (CA200645). The agonist potencies for inducing A1AR internalization were very similar to the affinities previously determined by ligand binding, suggesting little or no amplification of the internalization response. By complementing the HiBiT tag to exogenous purified LgBiT, it was also possible to perform NanoBRET ligand-binding experiments using HiBiT-A1AR. This study demonstrates the use of NanoBiT technology to monitor internalization of the A1AR and offers the potential to combine these experiments with NanoBRET ligand-binding assays.

Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β2 -adrenoceptor (β2 AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β2 AR (pKD >7.0) with good selectivity over the β1 AR (pKD

To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs., Stoddart et al. propose rational design of a covalent and selective ligand probe to fluorescently label GPCR in transiently transfected and endogenous systems. Using the adenosine A2A receptor as a model system, they show fluorescent labelling in an endogenous system, without impeding access to the orthosteric binding site. This study is useful to selectively and non-invasively study localisation and functions of GPCRs.

Summary Camelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15-kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognizes the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C terminus to the 11-amino-acid HiBiT tag (VUN400-HiBiT) which complements LgBiT protein, forming a full-length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect allosteric interactions with CXCR4. These data demonstrate that the high specificity offered by extracellular targeted nanobodies can be utilized to probe receptor pharmacology., Graphical Abstract, Highlights • An extracellular targeted nanobody against CXCR4 was modified with the HiBiT tag • Complemented luminescence was used to monitor nanobody-receptor interactions • Nanobody-mediated complemented luminescence detected endogenously expressed CXCR4 • CXCL12 and AMD3100 inhibition of nanobody binding was consistent with allosterism, Soave et al. have used a single-domain HiBiT-tagged antibody fragment to monitor the pharmacology of CXCR4 and demonstrate that this can be used to monitor receptor allosterism. They also demonstrate its ability to study endogenous CXCR4s. Taken together, this is a powerful approach to study GPCRs in living cells.

Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.

Background: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis, primarily signalling via VEGF Receptor 2 (VEGFR2). Endothelial cells also express the co-receptor Neuropilin-1 (NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real-time ligand binding kinetics when monitored using Bioluminescence Resonance Energy Transfer (BRET). We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explore differences between VEGF-A isoforms in living cells that co-expressed both receptors. Experimental Approach: Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using a membrane-impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerization is required for luminescence emissions. Binding affinities and kinetics of VEGFR2-selective VEGF165b-TMR and non-selective VEGF165a-TMR were monitored using BRET from this defined complex. Key Results: Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 minutes. VEGF165a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 had no impact on the binding kinetics or affinity of VEGF165a-TMR. Conclusions and Implications: This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor tyrosine kinase and its co-receptor for the first time.

Here, we describe rational design of a compound that covalently and selectively labels a G protein-coupled receptor (GPCR) in living cells with a fluorescent moiety. Using wild-type adenosine A2A receptor as a model system, we show fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

SummaryCamelid single-domain antibody fragments (nanobodies) offer the specificity of an antibody in a single 15kDa immunoglobulin domain. Their small size allows for easy genetic manipulation of the nanobody sequence to incorporate protein tags, facilitating their use as biochemical probes. The nanobody VUN400, which recognises the second extracellular loop of the human CXCR4 chemokine receptor, was used as a probe to monitor specific CXCR4 conformations. VUN400 was fused via its C-terminus to the 11-amino acid HiBiT tag (VUN400-HiBiT) which complements to LgBiT protein, forming a full length functional NanoLuc luciferase. Here, complemented luminescence was used to detect VUN400-HiBiT binding to CXCR4 receptors expressed in living HEK293 cells. VUN400-HiBiT binding to CXCR4 could be prevented by orthosteric and allosteric ligands, allowing VUN400-HiBiT to be used as a probe to detect specific conformations of CXCR4. These data demonstrate that the high specificity offered by extracellular-targeted nanobodies can be utilised to probe receptor pharmacology.

Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β1-adrenoceptor (β1AR-m23 StaR), on β1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ1-AR, but not its human homologue. Specific binding of mAb3 to tβ1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β1-AR conformational states and oligomeric complexes.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF165a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF165a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF165a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane.

Background and Purpose: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis. A striking feature of the binding of a fluorescent analogue of VEGF165a to NanoLuciferase-tagged VEGF Receptor 2 (VEGFR2) in living cells is that the bioluminescence resonance energy transfer (BRET) signal is not sustained and declines over time. This may be secondary to receptor internalisation. Here we have compared the binding of three fluorescent VEGF-A isoforms to VEGFR2 in cells and isolated membrane preparations. Experimental Approach: Ligand binding kinetics were monitored in both intact HEK293T cells and membranes (expressing NanoLuciferase tagged VEGFR2) using BRET between the tagged receptor and fluorescent analogues of VEGF165a, VEGF165b and VEGF121a. VEGFR2 endocytosis in intact cells expressing VEGFR2 was monitored by following the appearance of fluorescent ligand-associated receptors in intracellular endosomes using automated quantitative imaging. Key Results: Quantitiative analysis of the effect of fluorescent VEGF-A isoforms on VEGFR2 endocytosis in cells demonstrated that they produced a rapid and potent translocation of ligand-bound VEGFR2 into intracellular endosomes. NanoBRET can be used to monitor the kinetics of the binding of fluorescent VEGF-A isoforms to VEGFR2. In isolated membrane preparations, ligand binding association curves were maintained for the duration of the 90 minute experiment. Measurement of koff at pH 6.0 in membrane preparations indicated shorter ligand residence times than those obtained at pH 7.4. Conclusions and Implications: These studies suggest that rapid VEGF-A isoform-induced receptor endocytosis shortens agonist residence times on the receptor (1/koff) as VEGFR2 moves from the plasma membrane to intracellular endosomes.

Vandetanib and pazopanib are clinically available, multi‐targeted inhibitors of vascular endothelial growth factor (VEGF) and platelet‐derived growth factor (PDGF) receptor tyrosine kinases. Short‐term VEGF receptor inhibition is associated with hypertension in 15%‐60% of patients, which may limit the use of these anticancer therapies over the longer term. To evaluate the longer‐term cardiovascular implications of treatment, we investigated the “on”‐treatment (21 days) and “off”‐treatment (10 days) effects following daily administration of vandetanib, pazopanib, or vehicle, in conscious rats. Cardiovascular variables were monitored in unrestrained Sprague‐Dawley rats instrumented with radiotelemetric devices. In Study 1, rats were randomly assigned to receive either daily intraperitoneal injections of vehicle (volume 0.5 mL; n = 5) or vandetanib 25 mg/kg/day (volume 0.5 mL; n = 6). In Study 2, rats received either vehicle (volume 0.5 mL; n = 4) or pazopanib 30 mg/kg/day (volume 0.5 mL; n = 7), dosed once every 24 hours for 21 days. All solutions were in 2% Tween, 5% propylene glycol in 0.9% saline solution. Vandetanib caused sustained increases in mean arterial pressure (MAP), systolic blood pressure (SBP), and diastolic blood pressure (DBP) compared to baseline and vehicle. Vandetanib also significantly altered the circadian cycling of MAP, SBP, and DBP. Elevations in SBP were detectable 162 hours after the last dose of vandetanib. Pazopanib also caused increases in MAP, SBP, and DBP. However, compared to vandetanib, these increases were of slower onset and a smaller magnitude. These data suggest that the cardiovascular consequences of vandetanib and pazopanib treatment are sustained, even after prolonged cessation of drug treatment.

Item does not contain fulltext The last frontier for a complete understanding of G-protein-coupled receptor (GPCR) biology is to be able to assess GPCR activity, interactions, and signaling in vivo, in real time within biologically intact systems. This includes the ability to detect GPCR activity, trafficking, dimerization, protein-protein interactions, second messenger production, and downstream signaling events with high spatial resolution and fast kinetic readouts. Resonance energy transfer (RET)-based biosensors allow for all of these possibilities in vitro and in cell-based assays, but moving RET into intact animals has proven difficult. Here, we provide perspectives on the optimization of biosensor design, of signal detection in living organisms, and the multidisciplinary development of in vitro and cell-based assays that more appropriately reflect the physiologic situation. In short, further development of RET-based probes, optical microscopy techniques, and mouse genome editing hold great potential over the next decade to bring real-time in vivo GPCR imaging to the forefront of pharmacology.

BACKGROUND AND PURPOSE: Receptor tyrosine kinase inhibitors (RTKIs) targeted at VEGF receptor 2 (VEGFR2) have proved to be attractive approaches to cancer therapy based on their ability to reduce angiogenesis. Here we have undertaken a quantitative analysis of the interaction of RTKIs and two VEGF splice variants, VEGF165a and VEGF165b, with VEGFR2 by studying nuclear factor of activated T-cells (NFAT) reporter gene activity in live HEK-293 cells. EXPERIMENTAL APPROACH: HEK-293 cells expressing the human VEGFR2 and a firefly luciferase reporter gene regulated by an NFAT response element were used for quantitative analysis of the effect of RTKIs on VEGF165a- and VEGF165b-stimulated luciferase gene expression. KEY RESULTS: VEGF165a produced a concentration-dependent activation of the NFAT-luciferase reporter gene in living cells that was inhibited in a non-competitive fashion by four different RTKIs (cediranib, pazopanib, sorafenib and vandetanib). The potency obtained for each RTKI from this analysis was similar to those obtained in binding studies using purified VEGFR2 kinase domains. VEGF165b was a lower-efficacy agonist of the NFAT-luciferase response when compared with VEGF165a. Analysis of the concentration–response data using the operational model of agonism indicated that both VEGF165 isoforms had similar affinity for VEGFR2. CONCLUSIONS AND IMPLICATIONS: Quantitative pharmacological analysis of the interaction of VEGF165 isoforms and RTKIs with VEGFR2 in intact living cells has provided important insights into the relative affinity and efficacy of VEGF165a and VEGF165b for activation of the calcineurinNFAT signalling pathway by this tyrosine kinase receptor.

Graphical abstract, Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β1-adrenoceptor (β1AR-m23 StaR), on β1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ1-AR, but not its human homologue. Specific binding of mAb3 to tβ1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β1-AR conformational states and oligomeric complexes.

Keywords:adenosine;allosterism;receptor;GPCR;dimerization;biased signalling The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs-protein-coupled adenosine receptors (A2A and A2B), or inhibit AC activity, in the case of Gi/o-coupled adenosine receptors (A1 and A3). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1- and A3-receptor allosteric modulators on in vivo pharmacology.

Graphical abstract, Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF165a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF165a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF165a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane.

VEGF inhibitors, including receptor tyrosine kinase inhibitors, are used as adjunct therapies in a number of cancer treatments. An emerging issue with these drugs is that most cause hypertension. To gain insight into the physiological mechanisms involved, we evaluated their regional hemodynamic effects in conscious rats. Male Sprague Dawley rats (350–450 g) were chronically implanted with pulsed Doppler flow probes (renal and mesenteric arteries, and the descending abdominal aorta) and catheters (jugular vein, peritoneal cavity, and distal abdominal aorta). Regional hemodynamics were measured over 4 d, before and after daily administration of cediranib (3 and 6 mg/kg, 3 and 6 mg/kg/h for 1 h, i.v.), sorafenib (10 and 20 mg/kg, 10 and 20 mg kg/h for 1 h, i.v.), pazopanib (30 and100 mg/kg, i.p.), or vandetanib (12.5 and 25 mg/kg, i.p.). All drugs evoked significant increases (P < 0.05; n = 7–8) in mean arterial pressure, which were generally accompanied by significant mesenteric and hindquarters, but not renal, vasoconstrictions. The hypertensive effects of cediranib were unaffected by losartan (10 mg/kg/h), bosentan (20 mg/kg/h), or a combination of phentolamine and propranolol (each 1 mg/kg/h), suggesting a need for new strategies to overcome them.—Carter, J. J., Fretwell, L. V., Woolard, J. Effects of 4 multitargeted receptor tyrosine kinase inhibitors on regional hemodynamics in conscious, freely moving rats.

β-Blockers reduce mortality and improve symptoms in people with heart disease; however, current clinically available β-blockers have poor selectivity for the cardiac β1-adrenoceptor (AR) over the lung β2-AR. Unwanted β2-blockade risks causing life-threatening bronchospasm and reduced efficacy of β2-agonist emergency rescue therapy. Thus, current life-prolonging β-blockers are contraindicated in patients with both heart disease and asthma. Here, we describe NDD-713 and -825, novel highly β1-selective neutral antagonists with good pharmaceutical properties that can potentially overcome this limitation. Radioligand binding studies and functional assays that use human receptors expressed in Chinese hamster ovary cells demonstrate that NDD-713 and -825 have nanomolar β1-AR affinity >500-fold β1-AR vs. β2-AR selectivity and no agonism. Studies in conscious rats demonstrate that these antagonists are orally bioavailable and cause pronounced β1-mediated reduction of heart rate while showing no effect on β2-mediated hindquarters vasodilatation. These compounds also have good disposition properties and show no adverse toxicologic effects. They potentially offer a truly cardioselective β-blocker therapy for the large number of patients with heart and respiratory or peripheral vascular comorbidities.—Baker, J. G., Gardiner, S. M., Woolard, J., Fromont, C., Jadhav, G. P., Mistry, S. N., Thompson, K. S. J., Kellam, B., Hill, S. J., Fischer, P. M. Novel selective β1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease.

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β1-adrenergic (β1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β1AR. Immunization with the β1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β1AR to which the mAbs bind. The anti-β1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β1AR agonism.

Background and Purpose Retigabine is a recently approved antiepileptic agent which activates Kv7.2–7.5 potassium channels. It is emerging that these channels have an important role in vascular regulation, but the vascular effects of retigabine in the conscious state are unknown. Hence, in the present study we assessed the regional haemodynamic responses to retigabine in conscious rats. Experimental Approach Male Sprague Dawley rats were chronically instrumented with pulsed Doppler flow probes to measure regional haemodynamic responses to retigabine under control conditions and during acute hypertension induced by infusion of angiotensin II and arginine vasopressin. Further experiments were performed, using the β-adrenoceptor antagonists CGP 20712A, ICI 118551 and propranolol, to elucidate the roles of β-adrenoceptors in the responses to retigabine in vivo and in vitro. Key Results Under normotensive conditions, retigabine induced dose-dependent hypotension and hindquarters vasodilatation, with small, transient renal and mesenteric vasodilatations. In the acutely hypertensive state, the renal and mesenteric, but not hindquarters, vasodilatations were enhanced. The response of the hindquarters vascular bed to retigabine was mediated, in part, by β2-adrenoceptors. However, in vitro experiments confirmed that retigabine did not act as a β-adrenoceptor agonist. Conclusions and Implications We demonstrated that retigabine causes regionally specific vasodilatations, which are different under normotensive and hypertensive conditions, and are, in part, mediated by β2-adrenoceptors in some vascular beds but not in others. These results broadly support previous findings and further indicate that Kv7 channels are a potential therapeutic target for the treatment of vascular diseases associated with inappropriate vasoconstriction.

Previous research has indicated that allosteric interactions across the dimer interface of β 1‐adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pKi values at the human β 1‐adenoceptor, which may result from such allosterism interactions. Three fluorescent β 1‐adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor‐bound fluorescent ligand and the N‐terminal NanoLuc tag of a human β 1‐adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high‐affinity‐specific binding to the NanoLuc‐ β 1‐adrenoceptor with each of the three fluorescent ligands yielding K D values of 87.1 ± 10 nmol/L (n = 8), 38.1 ± 12 nmol/L (n = 7), 13.4 ± 2 nmol/L (n = 14) for propranolol‐Peg8‐BY630, propranolol‐ β(Ala‐Ala)‐BY630 and CGP‐12177‐TMR, respectively. Parallel radioligand‐binding studies with 3H‐CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc‐ β 1‐adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein−1). Following a 1 h incubation with fluorescent ligands and β 1‐adrenoceptor competing antagonists, there were significant differences (P

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF(165), and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF(165)b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF(165) and less VEGF(165)b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.

The vascular endothelial growth factor (VEGF) family of proteins regulates blood flow, growth, and function in both normal physiology and disease processes. VEGF-A is alternatively spliced to form multiple isoforms, in two subfamilies, that have specific, novel functions. Alternative splicing of exons 5-7 of the VEGF gene generates forms with differing bioavailability and activities, whereas alternative splice-site selection in exon 8 generates proangiogenic, termed VEGF(xxx), or antiangiogenic proteins, termed VEGF(xxx)b. Despite its name, emerging roles for VEGF isoforms on cell types other than endothelium have now been identified. Although VEGF-A has conventionally been considered to be a family of proangiogenic, propermeability vasodilators, the identification of effects on nonendothelial cells, and the discovery of the antiangiogenic subfamily of splice isoforms, has added further complexity to their regulation of microvascular function. The distally spliced antiangiogenic isoforms are expressed in normal human tissue, but downregulated in angiogenic diseases, such as cancer and proliferative retinopathy, and in developmental pathologies, such as Denys Drash syndrome and preeclampsia. Here, we examine the molecular diversity of VEGF-A as a regulator of its biological activity and compare the role of the pro- and antiangiogenic VEGF-A splice isoforms in both normal and pathophysiological processes.

Item does not contain fulltext Vascular endothelial growth factor-A is widely regarded as the principal stimulator of angiogenesis required for tumour growth. VEGF is generated as multiple isoforms of two families, the pro-angiogenic family generated by proximal splice site selection in the terminal exon, termed VEGFxxx, and the anti-angiogenic family formed by distal splice site selection in the terminal exon, termed VEGFxxxb, where xxx is the amino acid number. The most studied isoforms, VEGF165 and VEGF165b have been shown to be present in tumour and normal tissues respectively. VEGF165b has been shown to inhibit VEGF- and hypoxia-induced angiogenesis, and VEGF-induced cell migration and proliferation in vitro. Here we show that overexpression of VEGF165b by tumour cells inhibits the growth of prostate carcinoma, Ewing's sarcoma and renal cell carcinoma in xenografted mouse tumour models. Moreover, VEGF165b overexpression inhibited tumour cell-mediated migration and proliferation of endothelial cells. These data show that overexpression of VEGF165b can inhibit growth of multiple tumour types in vivo indicating that VEGF165b has potential as an anti-angiogenic, anti-tumour strategy in a number of different tumour types, either by control of VEGF165b expression by regulation of splicing, overexpression of VEGF165b, or therapeutic delivery of VEGF165b to tumours.

Growth of new blood vessels (angiogenesis), required for all tumor growth, is stimulated by the expression of vascular endothelial growth factor (VEGF). VEGF is up-regulated in all known solid tumors but also in atherosclerosis, diabetic retinopathy, arthritis, and many other conditions. Conventional VEGF isoforms have been universally described as proangiogenic cytokines. Here, we show that an endogenous splice variant, VEGF165b, is expressed as protein in normal cells and tissues and is circulating in human plasma. We also present evidence for a sister family of presumably inhibitory splice variants. Moreover, these isoforms are down-regulated in prostate cancer. We also show that VEGF165b binds VEGF receptor 2 with the same affinity as VEGF165 but does not activate it or stimulate downstream signaling pathways. Moreover, it prevents VEGF165-mediated VEGF receptor 2 phosphorylation and signaling in cultured cells. Furthermore, we show, with two different in vivo angiogenesis models, that VEGF165b is not angiogenic and that it inhibits VEGF165-mediated angiogenesis in rabbit cornea and rat mesentery. Finally, we show that VEGF165b expressing tumors grow significantly more slowly than VEGF165-expressing tumors, indicating that a switch in splicing from VEGF165 to VEGF165b can inhibit tumor growth. These results suggest that regulation of VEGF splicing may be a critical switch from an antiangiogenic to a proangiogenic phenotype.

The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by Gs -protein-coupled adenosine receptors (A2A and A2B ), or inhibit AC activity, in the case of Gi/o -coupled adenosine receptors (A1 and A3 ). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A1 and A3 receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A1 - and A3 -receptor allosteric modulators on in vivo pharmacology.

The polyketide natural product borrelidin 1 is a potent inhibitor of angiogenesis and spontaneous metastasis. Affinity biopanning of a phage display library of colon tumour cell cDNAs identified the tandem WW domains of spliceosome-associated protein formin binding protein 21 (FBP21) as a novel molecular target of borrelidin, suggesting that borrelidin may act as a modulator of alternative splicing. In support of this idea, 1, and its more selective analog 2, bound to purified recombinant WW domains of FBP21. They also altered the ratio of vascular endothelial growth factor (VEGF) isoforms in retinal pigmented endothelial (RPE) cells in favour of anti-angiogenic isoforms. Transfection of RPE cells with FBP21 altered the ratio in favour of pro-angiogenic VEGF isoforms, an effect inhibited by 2. These data implicate FBP21 in the regulation of alternative splicing and suggest the potential of borrelidin analogs as tools to deconvolute key steps of spliceosome function. © The Royal Society of Chemistry 2011.

Objectives Angiotensin-converting enzyme inhibition (ACEI) in adult spontaneously hypertensive rats (SHRs) produces reductions in mean arterial pressure (MAP) and vascular structure that persist after treatment cessation. This study used an intermittent treatment strategy to determine the time course of changes in MAP, vascular resistance properties, and the tissue levels of endothelin. Methods Adult SHRs were treated with enalapril and low sodium diet for three 2-week treatment cycles, each separated by 2-week washout periods. MAP was measured via radiotelemetry. Hindlimb structurally based vascular resistance properties were assessed after two treatment cycles. Endothelin was measured in mesenteric vessels, renal cortex and medulla in untreated SHR (Con), and at day 10 of the first and third treatment cycles. Results Treatment produced a persistent reduction in MAP; however, the magnitude of change in the ‘off-treatment’ level decreased following successive treatments (cycle 1: −15 ± 1.7%, cycle 2: −8 ± 1.9%, and cycle 3: −1 ± 1.7%). Reduction in hindlimb vascular structure after two cycles of treatment was not different from that previously observed after one cycle. Endothelin levels were significantly elevated during the third cycle in renal medulla (Con: 797 ± 102 pg/g tissue, cycle 1: 767 ± 81 pg/g tissue, cycle 3: 1097 ± 205 pg/g tissue) and mesenteric vessels (Con: 711 ± 226 pg/g tissue, cycle 1: 696 ± 231 pg/g tissue, cycle 3: 1063 ± 741 pg/g tissue). Concomitant treatment with an endothelin antagonist did not impact arterial pressure. Conclusion These findings demonstrate that during ACEI treatment, most of the changes that confer persistent changes in MAP and vascular structure occur within the first 2 weeks. Elevation in endothelin levels is likely unrelated to arterial pressure.

Vascular endothelial growth factor A (VEGFA; hereafter referred to as VEGF) is a key regulator of physiological and pathological angiogenesis. Two families of VEGF isoforms are generated by alternate splice-site selection in the terminal exon. Proximal splice-site selection (PSS) in exon 8 results in pro-angiogenic VEGFxxx isoforms (xxx is the number of amino acids), whereas distal splice-site selection (DSS) results in anti-angiogenic VEGFxxxb isoforms. To investigate control of PSS and DSS, we investigated the regulation of isoform expression by extracellular growth factor administration and intracellular splicing factors. In primary epithelial cells VEGFxxxb formed the majority of VEGF isoforms (74%). IGF1, and TNFα treatment favoured PSS (increasing VEGFxxx) whereas TGFβ1 favoured DSS, increasing VEGFxxxb levels. TGFβ1 induced DSS selection was prevented by inhibition of p38 MAPK and the Clk/sty (CDC-like kinase, CLK1) splicing factor kinase family, but not ERK1/2. Clk phosphorylates SR protein splicing factors ASF/SF2, SRp40 and SRp55. To determine whether SR splicing factors alter VEGF splicing, they were overexpressed in epithelial cells, and VEGF isoform production assessed. ASF/SF2, and SRp40 both favoured PSS, whereas SRp55 upregulated VEGFxxxb (DSS) isoforms relative to VEGFxxx. SRp55 knockdown reduced expression of VEGF165b. Moreover, SRp55 bound to a 35 nucleotide region of the 3′UTR immediately downstream of the stop codon in exon 8b. These results identify regulation of splicing by growth and splice factors as a key event in determining the relative pro-versus anti-angiogenic expression of VEGF isoforms, and suggest that p38 MAPK-Clk/sty kinases are responsible for the TGFβ1-induced DSS selection, and identify SRp55 as a key regulatory splice factor.