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BACKGROUND: There is increasing interest in developing FPR2 agonists (compound 43, ACT-389949 and BMS-986235) as potential pro-resolving therapeutics, with ACT-389949 and BMS-986235 having entered phase I clinical development. FPR2 activation leads to diverse downstream outputs. ACT-389949 was observed to cause rapid tachyphylaxis, while BMS-986235 and compound 43 induced cardioprotective effects in preclinical models. We aim to characterise the differences in ligand-receptor engagement and downstream signalling and trafficking bias profile. EXPERIMENTAL APPROACH: Concentration-response curves to G protein dissociation, ß-arrestin recruitment, receptor trafficking and second messenger signalling were generated using FPR2 ligands (BMS-986235, ACT-389949, compound 43 and WKYMVm), in HEK293A cells. Log(τ/KA) was obtained from the operational model for bias analysis using WKYMVm as a reference ligand. Docking of FPR2 ligands into the active FPR2 cryoEM structure (PDBID: 7T6S) was performed using ICM pro software. KEY RESULTS: Bias analysis revealed that WKYMVm and ACT-389949 shared a very similar bias profile. In comparison, BMS-986235 and compound 43 displayed approximately 5- to 50-fold bias away from ß-arrestin recruitment and trafficking pathways, while being 35- to 60-fold biased towards cAMP inhibition and pERK1/2. Molecular docking predicted key amino acid interactions at the FPR2 shared between WKYMVm and ACT-389949, but not with BMS-986235 and compound 43. CONCLUSION AND IMPLICATIONS: In vitro characterisation demonstrated that WKYMVm and ACT-389949 differ from BMS-986235 and compound 43 in their signalling and protein coupling profile. This observation may be explained by differences in the ligand-receptor interactions. In vitro characterisation provided significant insights into identifying the desired bias profile for FPR2-based pharmacotherapy.
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Accumulating evidence suggests that G protein-coupled receptors (GPCRs) can exist and function in homodimer and heterodimer forms. The adenosine A1 receptor (A1R) has been shown to form both homodimers and heterodimers, but there is a lack of chemical tools to study these dimeric receptor populations. This work describes the synthesis and pharmacological evaluation of a novel class of bivalent GPCR chemical tools, where each ligand moiety of the bivalent compound contains a sulfonyl fluoride covalent warhead designed to be capable of simultaneously reacting with each A1R of an A1R homodimer. The novel compounds were characterised using radioligand binding assays, including washout assays, and functionally in cAMP assays. The bivalent dicovalent compounds were competitive A1R antagonists and showed evidence of covalent binding and simultaneous binding across an A1R homodimer. Greater selectivity for A1R over the adenosine A3 receptor was observed for bivalent dicovalent over the equivalent monovalent compounds, indicating subtype selectivity can be achieved with dual occupation by a bivalent dicovalent ligand.
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LINKED ARTICLES: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc.
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Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Humanos , AnimalesRESUMEN
G protein-coupled receptors (GPCRs) represent the target for approximately a third of FDA-approved small molecule drugs. The adenosine A1 receptor (A1R), one of four adenosine GPCR subtypes, has important (patho)physiological roles in humans. A1R has well-established roles in the regulation of the cardiovascular and nervous systems, where it has been identified as a potential therapeutic target for a number of conditions, including cardiac ischemia-reperfusion injury, cognition, epilepsy, and neuropathic pain. A1R small molecule drugs, typically orthosteric ligands, have undergone clinical trials. To date, none have progressed into the clinic, predominantly due to dose-limiting unwanted effects. The development of A1R allosteric modulators that target a topographically distinct binding site represent a promising approach to overcome current limitations. Pharmacological parameters of allosteric ligands, including affinity, efficacy and cooperativity, can be optimized to regulate A1R activity with high subtype, spatial and temporal selectivity. This review aims to offer insights into the A1R as a potential therapeutic target and highlight recent advances in the structural understanding of A1R allosteric modulation.
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Cognición , Receptor de Adenosina A1 , Humanos , Adenosina , Sitios de Unión , Corazón , LigandosRESUMEN
The application of artificial intelligence (AI) approaches to drug discovery for G protein-coupled receptors (GPCRs) is a rapidly expanding area. Artificial intelligence can be used at multiple stages during the drug discovery process, from aiding our understanding of the fundamental actions of GPCRs to the discovery of new ligand-GPCR interactions or the prediction of clinical responses. Here, we provide an overview of the concepts behind artificial intelligence, including the subfields of machine learning and deep learning. We summarise the published applications of artificial intelligence to different stages of the GPCR drug discovery process. Finally, we reflect on the benefits and limitations of artificial intelligence and share our vision for the exciting potential for further development of applications to aid GPCR drug discovery. In addition to making the drug discovery process "faster, smarter and cheaper," we anticipate that the application of artificial intelligence will create exciting new opportunities for GPCR drug discovery.
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Epilepsy is one of the most serious and common chronic neurological conditions, characterised by recurrent hypersynchronous electrical activity in the brain that lead to seizures. Despite over 50 million people being affected worldwide, only ~70% of people with epilepsy have their seizures successfully controlled with current pharmacotherapy, and many experience significant psychiatric and physical comorbidities. Adenosine, a ubiquitous purine metabolite, is a potent endogenous anti-epileptic substance that can abolish seizure activity via the adenosine A1 G protein-coupled receptor. Activation of A1 receptors decreases seizure activity in animal models, including models of drug-resistant epilepsy. Recent advances have increased our understanding of epilepsy comorbidities, highlighting the potential for adenosine receptors to modulate epilepsy-associated comorbidities, including cardiovascular dysfunction, sleep and cognition. This review provides an accessible resource of the current advances in understanding the adenosine system as a therapeutic target for epilepsy and epilepsy-associated comorbidities.
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Heart failure remains a leading cause of morbidity and mortality worldwide. Current treatment for patients with heart failure include drugs targeting G protein-coupled receptors such as ß-adrenoceptor antagonists (ß-blockers) and angiotensin II type 1 receptor antagonists (or angiotensin II receptor blockers). However, many patients progress to advanced heart failure with persistent symptoms, despite treatment with available therapeutics that have been shown to reduce mortality and mortality. GPCR targets currently being explored for the development of novel heart failure therapeutics include adenosine receptor, formyl peptide receptor, relaxin/insulin-like family peptide receptor, vasopressin receptor, endothelin receptor and the glucagon-like peptide 1 receptor. Many GPCR drug candidates are limited by insufficient efficacy and/or dose-limiting unwanted effects. Understanding the current challenges hindering successful clinical translation and the potential to overcome existing limitations will facilitate the future development of novel heart failure therapeutics.
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G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.
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Regulación Alostérica , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G , Animales , Humanos , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico/efectos de los fármacos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Conformación Proteica/efectos de los fármacos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/clasificación , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/ultraestructuraRESUMEN
Cardiovascular disease (CVD) remains the leading cause of death worldwide. An ongoing challenge remains the development of novel pharmacotherapies to treat CVD, particularly atherosclerosis. Effective mechanism-informed development and translation of new drugs requires a deep understanding of the known and currently unknown biological mechanisms underpinning atherosclerosis, accompanied by optimization of traditional drug discovery approaches. Current animal models do not precisely recapitulate the pathobiology underpinning human CVD. Accordingly, a fundamental limitation in early-stage drug discovery has been the lack of consensus regarding an appropriate experimental in vivo model that can mimic human atherosclerosis. However, when coupled with a clear understanding of the specific advantages and limitations of the model employed, preclinical animal models remain a crucial component for evaluating pharmacological interventions. Within this perspective, we will provide an overview of the mechanisms and modalities of atherosclerotic drugs, including those in the preclinical and early clinical development stage. Additionally, we highlight recent preclinical models that have improved our understanding of atherosclerosis and associated clinical consequences and propose model adaptations to facilitate the development of new and effective treatments.
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Aterosclerosis , Enfermedades Cardiovasculares , Animales , Humanos , Aterosclerosis/tratamiento farmacológico , Descubrimiento de Drogas , Modelos AnimalesRESUMEN
Alzheimer's disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer's disease. The adenosine A1 and A2A receptors are expressed in the human brain and have a proposed involvement in the pathogenesis of dementia. Targeting these receptors preclinically can mitigate pathogenic ß-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer's disease and the therapeutic potential of A1 and A2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response.
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Enfermedad de Alzheimer , Adenosina/metabolismo , Anciano , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Humanos , Receptores Purinérgicos P1/metabolismoRESUMEN
We discuss the fascinating pharmacology of formylpeptide receptor 2 (FPR2; often referred to as FPR2/ALX since it binds lipoxin A4 ). Initially identified as a low-affinity 'relative' of FPR1, FPR2 presents complex and diverse biology. For instance, it is activated by several classes of agonists (from peptides to proteins and lipid mediators) and displays diverse expression patterns on myeloid cells as well as epithelial cells and endothelial cells, to name a few. Over the last decade, the pharmacology of FPR2 has progressed from being considered a weak chemotactic receptor to a master-regulator of the resolution of inflammation, the second phase of the acute inflammatory response. We propose that exploitation of the biology of FPR2 offers innovative ways to rectify chronic inflammatory states and represents a viable avenue to develop novel therapies. Recent elucidation of FPR2 structure will facilitate development of the anti-inflammatory and pro-resolving drugs of next decade.
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Lipoxinas , Receptores de Lipoxina , Células Endoteliales/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipoxinas/farmacología , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/metabolismoRESUMEN
The adenosine A1 receptor is a therapeutic target based on its ability to provide cardioprotection during episodes of myocardial ischemia and reperfusion injury. However, the clinical translation of A1R agonists has been hindered by dose-limiting adverse effects (bradycardia and hypotension). Previously, we demonstrated that the bitopic agonist VCP746 (1), consisting of an adenosine pharmacophore linked to an allosteric moiety, can stimulate cardioprotective A1R signaling effects in the absence of unwanted bradycardia. This study maps the structure-activity relationships of 1 through modifications to the linker moiety. Derivatives differing in the flexibility, length, and nature of the linker were assessed, which revealed that the linker is tolerant of several modifications including added rigidity. Ligands featuring 1,4-disubstituted 1,2,3-triazoles were the most biased of the novel analogues but also displayed sub-nanomolar potency in a cAMP accumulation assay at the A2BR. To our knowledge, 10 is the most potent A2BR agonist published to date.
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Bradicardia , Agonistas del Receptor Purinérgico P1 , Adenosina/farmacología , Agonistas del Receptor de Adenosina A1/farmacología , Humanos , Ligandos , Receptor de Adenosina A1 , Receptor de Adenosina A3 , Receptores Purinérgicos P1RESUMEN
The adenosine A1 receptor (A1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain1,2. However, development of analgesic orthosteric A1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A1R co-bound to adenosine, MIPS521 and a Gi2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts.
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Analgesia , Receptor de Adenosina A1/metabolismo , Adenosina/química , Adenosina/metabolismo , Regulación Alostérica/efectos de los fármacos , Analgesia/métodos , Animales , Sitios de Unión , Modelos Animales de Enfermedad , Femenino , Subunidad alfa de la Proteína de Unión al GTP Gi2/química , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Hiperalgesia/tratamiento farmacológico , Lípidos , Masculino , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , Estabilidad Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor de Adenosina A1/química , Transducción de Señal/efectos de los fármacosRESUMEN
Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A1 receptor (A1R) and adenosine A3 receptor (A3R) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the A1R and A3R. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A2A and A2B adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of A1R and A3R localization and trafficking in native cells and living systems.
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Antagonistas del Receptor de Adenosina A1/farmacología , Antagonistas del Receptor de Adenosina A3/farmacología , Sondas Moleculares/farmacología , Receptor de Adenosina A1/metabolismo , Receptor de Adenosina A3/metabolismo , Xantinas/farmacología , Antagonistas del Receptor de Adenosina A1/síntesis química , Antagonistas del Receptor de Adenosina A3/síntesis química , Alquinos/química , Animales , Azidas/química , Células CHO , Química Clic , Cricetulus , Diseño de Fármacos , Colorantes Fluorescentes/química , Humanos , Ligandos , Sondas Moleculares/síntesis química , Receptor de Adenosina A1/química , Receptor de Adenosina A3/química , Xantinas/síntesis químicaRESUMEN
Idiopathic pulmonary fibrosis is a chronic and progressive fibrotic lung disease, and current treatments are limited by their side effects. Proliferation of human lung fibroblasts in the pulmonary interstitial tissue is a hallmark of this disease and is driven by prolonged ERK signalling in the nucleus in response to growth factors such as platelet-derived growth factor (PDGF). Agents that increase cAMP have been suggested as alternative therapies, as this second messenger can inhibit the ERK cascade. We previously examined a panel of eight Gαs-cAMP-coupled G protein-coupled receptors (GPCRs) endogenously expressed in human lung fibroblasts. Although the cAMP response was important for the anti-fibrotic effects of GPCR agonists, the magnitude of the acute cAMP response was not predictive of anti-fibrotic efficacy. Here we examined the reason for this apparent disconnect by stimulating the Gαs-coupled prostacyclin receptor and measuring downstream signalling at a sub-cellular level. MRE-269 and treprostinil caused sustained cAMP signalling in the nucleus and complete inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. In contrast, iloprost caused a transient increase in nuclear cAMP, there was no effect of iloprost on PDGF-induced ERK in the nucleus, and this agonist was much less effective at reversing PDGF-induced proliferation. This suggests that sustained elevation of cAMP in the nucleus is necessary for efficient inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. This is an important first step towards understanding of the signalling events that drive GPCR inhibition of fibrosis.
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The adenosine A1 receptor (A1AR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of A1AR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying A1AR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of A1AR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel A1AR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of A1AR molecular pharmacology and signaling in living cells.
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Antagonistas del Receptor de Adenosina A1/síntesis química , Colorantes Fluorescentes/química , Receptor de Adenosina A1/química , Antagonistas del Receptor de Adenosina A1/metabolismo , Compuestos Bicíclicos con Puentes/química , Diseño de Fármacos , Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Cinética , Ligandos , Octanos/química , Receptor de Adenosina A1/metabolismo , Relación Estructura-Actividad , Xantina/química , Xantina/metabolismoRESUMEN
Adenosine A1 receptors (A1R) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for A1R-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the A1R agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the A1R biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca2+ influx relative to NECA and the cAMP pathway at the A1R, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the A1R. In contrast to VCP746, which displays more 'adenosine-like' signaling at the A2BR, neladenoson was a highly selective A1R agonist, with biased, weak agonism at the A2BR. Together these results show that unwanted hemodynamic effects of A1R agonists can be avoided by compounds biased away from Ca2+ influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that A1R-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.
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Adenosine modulates many aspects of human physiology and pathophysiology through binding to the adenosine family of G protein-coupled receptors, which are comprised of four subtypes, the A1R, A2AR, A2BR and A3R. Modulation of adenosine receptor function by exogenous agonists, antagonists and allosteric modulators can be beneficial for a number of conditions including cardiovascular disease, Parkinson's disease, and cancer. Unfortunately, many preclinical drug candidates targeting adenosine receptors have failed in clinical trials due to limited efficacy and/or severe on-target undesired effects. To overcome the key barriers typically encountered when transitioning adenosine receptor ligands into the clinic, research efforts have focussed on exploiting the phenomenon of biased agonism. Biased agonism provides the opportunity to develop ligands that favour therapeutic signalling pathways, whilst avoiding signalling associated with on-target undesired effects. Recent studies have begun to define the structure-function relationships that underpin adenosine receptor biased agonism and establish how this phenomenon can be harnessed therapeutically. In this review we describe the recent advancements made towards achieving therapeutically relevant biased agonism at adenosine receptors.
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Enfermedades Cardiovasculares/metabolismo , Neoplasias/metabolismo , Enfermedad de Parkinson/metabolismo , Receptores Purinérgicos P1/metabolismo , Descubrimiento de Drogas , Humanos , Ligandos , Relación Estructura-ActividadRESUMEN
Herein we describe the development of a focused series of functionalized pyridazin-3(2 H)-one-based formyl peptide receptor (FPR) agonists that demonstrate high potency and biased agonism. The compounds described demonstrated biased activation of prosurvival signaling, ERK1/2 phosphorylation, through diminution of the detrimental FPR1/2-mediated intracellular calcium (Cai2+) mobilization. Compound 50 showed an EC50 of 0.083 µM for phosphorylation of ERK1/2 and an approximate 20-fold bias away from Cai2+ mobilization at the hFPR1.