How Carvedilol activates β2-adrenoceptors

Abstract

Carvedilol is among the most effective β-blockers for improving survival after myocardial infarction. Yet the mechanisms by which carvedilol achieves this superior clinical profile are still unclear. Beyond blockade of β1-adrenoceptors, arrestin-biased signalling via β2-adrenoceptors is a molecular mechanism proposed to explain the survival benefits. Here, we offer an alternative mechanism to rationalize carvedilol’s cellular signalling. Using primary and immortalized cells genome-edited by CRISPR/Cas9 to lack either G proteins or arrestins; and combining biological, biochemical, and signalling assays with molecular dynamics simulations, we demonstrate that G proteins drive all detectable carvedilol signalling through β2ARs. Because a clear understanding of how drugs act is imperative to data interpretation in basic and clinical research, to the stratification of clinical trials or to the monitoring of drug effects on the target pathway, the mechanistic insight gained here provides a foundation for the rational development of signalling prototypes that target the β-adrenoceptor system.

General Metadynamics Protocol To Simulate Activation/Deactivation of Class A GPCRs: Proof of Principle for the Serotonin Receptor

“…In a separate study, we have used the protocol for an extensive study of the β 2adrenergic receptor with similar success to that reported here for 5-HT 1A , and very recently, a paper was published reporting A 100 free-energy profiles to support experimental data. 34 Given that A 100 is rapidly gaining acceptance as a general activation measure, 33 and considering the reliability of the optimized protocol described here, we are confident to have described a metadynamics simulation technique that is superior to other activation CVs, for instance, the TM3−TM6 distance, as described above.…”

“…19 The A 100 index was initially introduced as an analysis tool, and its use as a CV has been very recently reported to support experimental data rationalizing carvedilol's cellular signaling in the β 2 -adrenergic receptor. 34 Here, we define the computational protocol exactly and describe its application to the serotonin 5-HT 1A receptor in its apo-form (ligand-free) and in the presence of two known ligands: the full agonist serotonin and the partial agonist aripiprazole. Apart from its simple definition and general applicability, further advantages of using A 100 with multiple walkers metadynamics are that no a priori knowledge of either the active or the inactive state is required, nor an initial path connecting the two states.…”

“…Here, we report a metadynamics-based multiple-walkers computational protocol that uses the general activation index A 100 as a single activation CV. The A 100 index is defined as a linear combination of 5 distances between α-carbons of 10 residues located on different helices and can thus be used within the PLUMED interface. , Given the general applicability of the activation index A 100 , ,, the computational protocol introduced here can potentially be applied to all class A, rhodopsin-like GPCRs that exhibit the residues that occur in the A 100 definition. As shown below, A 100 , which was specifically designed to indicate the activation/deactivation state of the receptor, is better suited than microswitches such as the TM3–TM6 distance, which can be ambiguous with respect to activation …”

“…The A 100 index was initially introduced as an analysis tool, and its use as a CV has been very recently reported to support experimental data rationalizing carvedilol’s cellular signaling in the β 2 -adrenergic receptor . Here, we define the computational protocol exactly and describe its application to the serotonin 5-HT 1A receptor in its apo -form (ligand-free) and in the presence of two known ligands: the full agonist serotonin and the partial agonist aripiprazole.…”

Allosteric Regulation of G-Protein-Coupled Receptors: From Diversity of Molecular Mechanisms to Multiple Allosteric Sites and Their Ligands

“…This compound interfered with the effects of the β-AR blocker carvedilol. Cmpd-6, on the one hand, increased the affinity of carvedilol for β 2 -AR and thereby enhanced its inhibitory effect on β 2 -agonist-induced stimulation of G s -proteins and cAMP signaling and, on the other hand, enhanced the stimulatory effects of carvedilol on ERK1/2 activity, endocytosis of β 2 -Ars, and their trafficking into lysosomes [ 426 ], which, as was shown later, is also due to the activation of G s -proteins [ 427 ].…”