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Exploring the kinetic selectivity of drugs targeting the β₁ -adrenoceptor.

ABSTRACT: In this study, we report the β₁ -adrenoceptor binding kinetics of several clinically relevant β_1/2 -adrenoceptor (β_1/2 AR) agonists and antagonists. [³ H]-DHA was used to label CHO-β₁ AR for binding studies. The kinetics of ligand binding was assessed using a competition association binding method. Ligand physicochemical properties, including logD_7.4 and the immobilized artificial membrane partition coefficient (K_IAM ), were assessed using column-based methods. Protein Data Bank (PDB) structures and hydrophobic and electrostatic surface maps were constructed in PyMOL. We demonstrate that the hydrophobic properties of a molecule directly affect its kinetic association rate (k_on ) and affinity for the β₁ AR. In contrast to our findings at the β₂ -adrenoceptor, K_IAM , reflecting both hydrophobic and electrostatic interactions of the drug with the charged surface of biological membranes, was no better predictor than simple hydrophobicity measurements such as clogP or logD_7.4 , at predicting association rate. Bisoprolol proved kinetically selective for the β₁ AR subtype, dissociating 50 times slower and partly explaining its higher measured affinity for the β₁ AR. We speculate that the association of positively charged ligands at the β₁ AR is curtailed somewhat by its predominantly neutral/positive charged extracellular surface. Consequently, hydrophobic interactions in the ligand-binding pocket dominate the kinetics of ligand binding. In comparison at the β₂ AR, a combination of hydrophobicity and negative charge attracts basic, positively charged ligands to the receptor's surface promoting the kinetics of ligand binding. Additionally, we reveal the potential role kinetics plays in the on-target and off-target pharmacology of clinically used β-blockers.

SUBMITTER: Sykes DA

PROVIDER: S-EPMC9237807 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background and purposeStress-related catecholamines have a role in cancer and β-adrenoceptors; specifically, β2 -adrenoceptors have been identified as new targets in treating melanoma. Recently, β3 -adrenoceptors have shown a pleiotropic effect on melanoma micro-environment leading to cancer progression. However, the mechanisms by which β3 -adrenoceptors promote this progression remain poorly understood. Catecholamines affect the immune system by modulating several factors that can alter immune cell sub-population homeostasis. Understanding the mechanisms of cancer immune-tolerance is one of the most intriguing challenges in modern research. This study investigates the potential role of β3 -adrenoceptors in immune-tolerance regulation.Experimental approachA mouse model of melanoma in which syngeneic B16-F10 cells were injected in C57BL-6 mice was used to evaluate the effect of β-adrenoceptor blockade on the number and activity of immune cell sub-populations (Treg, NK, CD8, MDSC, macrophages, and neutrophils). Pharmacological and molecular approaches with β-blockers (propranolol and SR59230A) and specific β-adrenoceptor siRNAs targeting β2 - or β3 -adrenoceptors were used.Key resultsOnly β3 -, but not β2 -adrenoceptors, were up-regulated under hypoxia in peripheral blood mononuclear cells and selectively expressed in immune cell sub-populations including Treg, MDSC, and NK. SR59230A and β3 -adrenoceptor siRNAs increased NK and CD8 number and cytotoxicity, while they attenuated Treg and MDSC sub-populations in the tumour mass, blood, and spleen. SR59230A and β3 -adrenoceptor siRNAs increased the ratio of M1/M2 macrophages and N1 granulocytes.Conclusions and implicationsOur data suggest that β3 -adrenoceptors are involved in immune-tolerance, which opens the way for new strategic therapies to overcome melanoma growth.Linked articlesThis article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.

Project description:Objective3',5'-Cyclic adenosine monophosphate (cAMP) is a central second messenger governing brown adipocyte differentiation and function. β-adrenergic receptors (β-ARs) stimulate adenylate cyclases which produce cAMP. Moreover, cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDEs), which can generate subcellular microdomains of cAMP. Since the spatio-temporal organisation of the cAMP signalling pathway in adipocytes is still unclear, we sought to monitor real-time cAMP dynamics by live cell imaging in pre-mature and mature brown adipocytes.MethodsWe measured the real-time dynamics of cAMP in murine pre-mature and mature brown adipocytes during stimulation of individual β-AR subtypes, as well as its regulation by PDEs using a Förster Resonance Energy Transfer based biosensor and pharmacological tools. We also correlated these data with β-AR stimulated lipolysis and analysed the expression of β-ARs and PDEs in brown adipocytes using qPCR and immunoblotting. Furthermore, subcellular distribution of PDEs was studied using cell fractionation and immunoblots.ResultsUsing pre-mature and mature brown adipocytes isolated from transgenic mice expressing a highly sensitive cytosolic biosensor Epac1-camps, we established real-time measurements of cAMP responses. PDE4 turned out to be the major PDE regulating cytosolic cAMP in brown preadipocytes. Upon maturation, PDE3 gets upregulated and contributes with PDE4 to control β1-AR-induced cAMP. Unexpectedly, β3-AR initiated cAMP is resistant to increased PDE3 protein levels and simultaneously, the control of this microdomain by PDE4 is reduced upon brown adipocyte maturation. Therefore we postulate the existence of distinct cAMP pools in brown adipocytes. One cAMP pool is formed by β1-AR associated with PDE3 and PDE4, while another pool is centred around β3-AR and is much less controlled by these PDEs. Functionally, lower control of β3-AR initiated cAMP by PDE3 and PDE4 facilitates brown adipocyte lipolysis, while lipolysis activated by β1-AR and is under tight control of PDE3 and PDE4.ConclusionsWe have established a real-time live cell imaging approach to analyse brown adipocyte cAMP dynamics in real-time using a cAMP biosensor. We showed that during the differentiation from pre-mature to mature murine brown adipocytes, there was a change in PDE-dependent compartmentation of β1-and β3-AR-initiated cAMP responses by PDE3 and PDE4 regulating lipolysis.

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Exploring the kinetic selectivity of drugs targeting the β₁ -adrenoceptor.

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Exploring the kinetic selectivity of drugs targeting the β1 -adrenoceptor.

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OmicsDI is part of the ELIXIR infrastructure

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Exploring the kinetic selectivity of drugs targeting the β₁ -adrenoceptor.