Biased agonism
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A concise overview of biased agonism, mechanism, beta arrestin pathway, types, examples, GPCR, pros and cons of biased agonism, beta blockers and angiostensin receptor in biased agonism.
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Biased agonism
- 1. BIASED AGONISM DR. SIDDHARTHA DUTTA MAMC, NEW DELHI
- 2. • The ability of some ligands to selectively activate some signaling pathways while blocking others • The ability of a receptor to differentially activate downstream signaling pathways depending on binding of a “biased” agonist compared to a “balanced” agonist • GPCR was believed to function as a binary switch • Different ligands at the same GPCR were shown to selectively activate one downstream pathway versus another • Stabilize different subsets of receptor conformations, which would then be transmitted to biasedly coupling and activating a subset of intracellular effectors for a specific effect
- 3. GPCRs as a target • After agonist activation, most GPCRs quickly lose the ability to respond to a ligand(desensitization) • G protein-coupled receptor kinases (GRKs) and Arrestins, were found to mediate desensitization • GRK family(GRK5/6) & Arrestins- 4 types • Phosphorylated GPCRs have a high-affinity for arrestins • βArrestin serves as as a scaffolding protein that bind to the phosphorylated receptor and adaptor for receptor endocytosis involved in clathrin-mediated GPCR internalization
- 5. Mechanism • β-arrestins is not only a desensitizer of G protein signals, but also a signaling molecule • Most-arrestin biased agonists place the receptor into a conformation that is recognized by the GRK but not the G heterotrimer allowing for phosphorylation and -arrestin recruitment • Molecular dynamics predicts that when proteins such as receptors change conformation, different regions of the receptor change independently (i.e., the protein does not form uniform global conformation) • These structural changes increase the binding affinties of GPCRs for β-arrestins, which are known to block G protein signaling
- 7. Balanced vs. Biased Signaling • Classical GPCRs signaling - ligands elicit their effects through one mechanism • Although agonists differ in terms of efficacy, their elicited downstream effects are identical • Unbiased or “balanced” ligands activate downstream signaling more globally without selectivity • Biased ligands, could selectively activate a subset of the receptor’s downstream signaling cascade • Stabilize distinct conformations of the GPCR to promote interaction with specific transducers, leading to the selective activation of downstream pathways
- 8. Key Determinant of Biased Signaling • Biased signaling leads to conformational changes of the receptor which allows different phosphorylation patterns of the cytoplasmic loops and C-terminal tail • β-arrestin displays different responses, depending on which class of GRKs phosphorylates the receptor(AT1R) • Differences in phosphorylation pattern of the C terminal tail of βAR determines the signaling activity of the ligand bound receptor • Barcode hypothesis • States that different GRKs establish a distinct phosphorylation barcode that recruits β-arrestin and regulates its downstream biased effects
- 9. True ligand bias • Limited to the ligand-receptor-transducer complex • Results from the generation of a conformational state of the receptor that is stabilized by a given ligand or • Which promotes interaction with specific transducers such as G-protein or β-arrestin, to evoke selective cellular signaling responses
- 10. Allosterism • In addition to receptor conformations and phosphorylation profiles, biased signaling may be modulated by the actual site of ligand-receptor binding • Allosteric ligands can lead to biased agonism 1) They may act as biased ligands themselves and activate a specific subset of receptor-mediated pathways 2) When an allosteric and orthosteric ligand are bound at the same time, the allosteric ligand may interact with the orthostatic site ligand and promote a specific subset of downstream signaling activation
- 12. • Small molecules acts as allosteric modulators to stabilize specific conformations of GPCR • A class of antibodies isolated from the Camelid family, called nanobodies, was found to function as allosteric modulators of the β2AR, stabilizing a specific agonist-activated conformation • Nanobodies transiently expressed intracellularly, or “intrabodies,” could bind the intracellular domain and stabilize specific receptor conformations • Intrabodies show a variety of effects on cAMP accumulation and β-arrestin recruitment and can inhibit β2AR signaling
- 13. Membrane stretch • Membrane stretch by mechanical stimuli- A form of allosteric modulation • AT1R activation by mechanical stretch of the receptor • Endogenous ligand, angiotensin II, and G protein recruitment may not be required • Mechanical stretch induces a unique β-arrestin- conformational state of the AT1R that is distinct from the G- protein bound AT1R • Recruitment of β-arrestin appears to be necessary to activate downstream signaling making mechanical stretch a β- arrestin biased phenomenon for the AT1R
- 14. Angiotensin II Type 1 Receptor (AT1R) • AT1R, which is stimulated by Ang II, an important regulator in the cardiovascular system • S II- β-arrestin-2-dependent biased ligand • (ERK) Extracellular Signal-regulated Kinase-1 and 2 • TRV120027, a β-arrestin biased ligand, can block vasoconstriction reduces arterial pressure induced by G protein activation and increase cardiac performance/ contractility via β- arrestin biased signaling • TRV120027 also suppresses angiotensin II-induced cardiac hypertrophy
- 16. Biased agonism and cvs diseases • β-adrenergic receptors (βARs) play a pivotal role in heart failure therapeutics • βARs can exert a cardioprotective effect transactivating Epidermal growth factor receptor (EGFR) through βarrestin • β-blockers carvedilol and alprenolol • Induces β-arrestin dependent EGFR-transactivation-a possible mechanism for the cardioprotective effect of β-arrestin
- 17. Carvedilol • β-arrestin-biased ligand for β1-AR and β2-AR • GRK-5 and -6 have been shown to play a pivotal role in β1AR biased agonism • Activates ERK1/2 via β-arrestin in the absence of G protein activation, and transactivate EGFR • EGFR activated NO production • Transactivations show that β-blocker, which acts as a β-arrestin biased ligand, has cardio protective effects in-vitro and in-vivo systems
- 18. PACAP (pituitary cyclase-activating polypeptide type 1) • Peptides PACAP1-27 and PACAP1-38 • Activate receptors to elevate cyclic AMP and increase production of IP3
- 19. Biased agonism examples • Urotensin Receptor (UT)—lead to a vasodilatory effect ora cardiostimulant effect • Adenosine A1 Receptor-VCP746, a hybrid adenosine linked to an allosteric modulator • Proteinase-Activated Receptor 1herapeutically used to • alter the effects of thrombin on the endothelium vs. increased bleeding
- 20. Biased agonism an offender?
- 21. NERVOUS SYSTEM • μ Opioid Receptor-Morphine • β-arrestin-2 KO mice, morphine leads to enhanced analgesia with reduced desensitization • TRV130, an IV G protein-biased ligand, which shows potent analgesic effects while causing less GI dysfunction and respiratory suppression compared to morphine in mice • Significant reduction in pain intensity after moderate to severe postoperative pain • TRV734 has also been developed as an oral agent
- 22. • Delta Opioid Receptor—targets for treatment of migraine Parkinson’s disease and neuropathic pain • Delta opioid receptor activation-No addiction but does lead to seizures • Lowered seizure threshold is a result of β-arrestin-mediated signaling • TRV250 -treatment of migraines without the associated lowered seizure threshold • Kappa Opioid Receptor— balanced agonist leads to analgesia with low abuse potential, but with severe side effects including anhedonia, hallucinations, and dysphoria • p38 MAP kinases through mediation by β-arrestin-2 • RB-64 induces analgesia without having an effect on sedation, anhedonia, or motor coordination
- 23. Platelet-Activating Factor Receptor (PAFR) • PAF, which binds to PAF receptor (GPCR ), was found to enhance MMP-2 expression levels • MMP-2 is considered to make an important contribution to atherosclerotic plaque instability • PAF-enhanced MMP-2 production has been shown to occur via activation of a β-arrestin-dependent ERK pathway • SIRT1(silent mating type information regulation 2 homolog 1), is a key regulator down-regulates PAFR via β-arrestin mediated internalization • Deacetylation (SIRT1), might be involved in GPCR regulation
- 24. Hydroxyl Carboxylic Acid Receptor 2 (HCA2) • Lower TG and raises HDL levels • G protein mediated signaling that leads to lowering of serum free fatty acids • NIACIN • Limitation- cutaneous flushing • Recruitment of β–arrestin to the receptor and generation of arachidonate, leading to the undesired response of flushing
- 25. • Famotidine • Inverse agonist for Gs protein dependent activation of adenylyl cyclase • But act as an agonist for Gs protein independent effects leading to up-regulation of the expression of histidine decarboxylase • leading to rebound acid hypersecretion after withdrawal
- 26. Conclusion • Biased ligands can be used to selectively to achieve greater benefits or even negate unwanted results of GPCR activation, like side effects • Smarter drugs- selectively targets the therapeutically significant signaling pathway
- 27. THANK YOU
Editor's Notes
- refers to the ability of a ligand to activate a subset of a receptor’s signaling cascade. almost 40% of approved drugs such as α- and β-blockers, opiates, β-agonist and angiotensin-converting enzyme inhibitors that could be activated by agonist binding or inhibited by antagonist blockade of agonist binding
- Multiple signaling cascade Once activated it continuously activates the intracellular signaling molecules like a chain reaction—amplification of the response Once desired effect is obtained the cell stops the signaling by the process of desensitization To limit the side effects GRK family(GRK 1-7) is a class of seryl-threonyl kinases, which phosphorylate the cytoplasmic tail of agonist-occupied receptors β-Arrestin competes with G proteins for receptor binding sites and therefore leads to receptor desensitization desensitization process via GPCR phosphorylation by the GRKs with the involvement of β-arrestin either the -arrestin-mediated signaling event or G and G events but not both pathways simultaneously as would occur with a traditional agonist
- The specific conformation change is recognized by the intracellular effectors leading to a different subset of response with the Phosphorylated GPCRs have a high-affinity for arrestins, which exist as four isoforms: two expressed mostly in photoreceptors and so called “visual” arrestins (arrestin-1 and arrestin-4) and two ubiquitous arrestins (β-arrestin-1 and βarrestin-2). -There is the possibility that a GPCR can interact directly with a -arrestin independent of phosphorylation to recruit and activate cytoplasmic signaling proteins and pathways
- Normal endogenous ligand--sufficient to determine the character of a ligand, i.e., full, partial, inverse agonist or neutral antagonist agonists differ qualitatively (e.g., one agonist selectively activates one signal whereas another predominantly stimulates a diffrent signal via the same receptor) and quantitatively (e.g. partial verses full) Functional Selectivity
- GRK-5 and -6 have been shown to play a pivotal role in β1AR EGFr transactivation HEK293 cells- phosphorylation by targeted GRK-2 or -3---- no activation of biased pathway(egfr) AT1R, following agonist stimulation or mechanical stretch, GRK-5 and -6 seem to be responsible for β-arrestin dependent ERK activation Unique phophorylation changes behaves as a barcode to be recognized by the intracellular signalling molecules used mass spectrometry proteomics to show
- Most ligands bind a receptor where its endogenous ligand binds, known as the orthosteric site. However, some ligands bind at a site different from the orthosteric site, called an allosteric site . Allosteric binding causes a conformational change of the receptor, which alters its function.
- single-domain Intrabodies could represent a novel and interesting tool to understand GPCR biology
- as ligand binding at the orthosteric site is not required T1R-β-arrestin chimeric receptors it was demonstrated that
- Most studied AT1R antagonism --effective approach-tx htn since 1985 launch of losartan, firstARBs) However, in addition to blocking adverse effects of angiotensin II, such as hypertension and cardiac remodeling, ARBs also prevent beneficial effects of angiotensin II, including the AT1Rmediated inotropy which supports cardiac function. Consequently, reduced cardiac output was long thought to be an inseparable side effect associated with AT1R antagonism and ARBs in general.
- Agonists β-adrenergic used as positive inotropes in tx of AHF with depressed ejection function. β-Blockers, antagonists of these receptors, are one of the mainstays for the treatment of CHF, as they mediate cardioprotection β1AR makes up 70% of all cardiac βAR. sustained β1-AR activation is cardiotoxic( increasing apoptosis, heart rate, blood pressure β-arrestin-dependent cardioprotective effct Although drugs used in CVDs bind same receptor, the pharmacologic effcts of them are dependent on ligands muscarinic receptors, the α2 adrenergic receptor, and the adenylyl cyclase-activating polypeptide receptor potential benefit of developing β-arrestin-biased ligands as novel therapeutic agents.
- however, it is also involved in regulation of nitric oxide(NO) production Consequently, it EGFR is infamous for its role in cancer----NO production activation of the EGFR by carvedilol is a greater good due to the role of NO in the cardiovascular system than the hypothetical risk of inducing a cancer.
- it plays a role in paracrine and autocrine regulation of certain types of cells. binds to vasoactive intestinal peptide receptor and to the PACAP receptor. post-traumatic stress disorder (PTSD) in women relative efficacy of PACAP1-27 for cyclic AMP elevation is higher than that of PACAP1-38 but lower for elevation of IP3 clearly indicate that agonist activation of multiple signaling mechanisms is not uniform but rather is often ‘biased’ toward some but not all signaling pathways
- biased agonism at the μ opioid receptor led to the development of TRV130, an intravenous G protein-biased ligand, which shows potent analgesic effects while causing less GI dysfunction and respiratory suppression compared to morphine in mice(phase 2) TRV734, oral agent that acts similar to TRV130 at the μ opioid receptor. A phase 1 study was completed, which showed good analgesic effects with mild to moderate adverse effects.
- biased agonism may also been implicated in undesirable effects
- morphine is very effective, its use is often limited by GI side effects including nausea, vomiting, and constipation. morphine can lead to respiratory suppression, sedation, tolerance, and addiction β-arrestin-mediated signaling leads to distinct mechanisms that increase the side effect profile of a balanced agonist at the μ receptor. Both drugs are in phase 2
- Limiting the use of balanced agonists has been identified as a G proteinbiased agonist at the delta receptor, with the goal of exploiting activation at the delta opioid receptor
- PAFR a plays a critical role in platelet aggregation Arachidonic acid,potent aggregation factor, induced platelet aggregation and thrombosis formation through PAFR SIRT1 down-regulates PAFR in platelets via proteasomal and/or lysosomal pathway SIRT1 activation suppresses platelet activation ex-vivo and pulmonary thromboembolism in-vivo G protein-related signaling pathway of PAFR is important to develop new cardiovascular drugs targeting PAFR
- 7TMR that couples through Gαi/Gαo endogenous ligand is 3-hydroxybutyrate
- ,
- Activation of signalling pathways depends on the conformational change induced by the agonist receptor complex and it is highly selective Not all agonist ll induce the same conformational change and hence lead to differential activation of downstream signalling pathways 3..Taking natural agonist for the receptor as useful point of reference ; this willhave a natural signaling bias can be used as astandard with which other agonists can be compared
- To make ideal drugs which are safer, more efficious , studies should focus on revealing the signals in detail and binding of ligand-receptor. Biased agonism is relatively novel concept, and despite the examples described above, comparatively few studies have been undertaken to elucidate the natures of GPCR pathway signals. Identifiation of receptor structures will support the structure based drug design. Thse approaches will undoubtedly lead to the discovery of developing ideal biased ligands for therapeutics.