Binding kinetics of ligands acting at GPCRs

Sykes, David A.; Stoddart, Leigh A.; Kilpatrick, Laura E.; Hill, Stephen J.

doi:10.1016/j.mce.2019.01.018

Cited by 96 publications

(92 citation statements)

References 103 publications

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“…The (un)binding kinetics have a major impact on both pharmacodynamics [55][56][57] [60][61][62][63] and small changes in either the structure of the ligand or the protein can alter the nature and the position of the transition states along the (un)binding routes 64 .…”

Section: Towards the Definition Of The Structure-binding/unbinding Pamentioning

confidence: 99%

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Deganutti

Barkan

Preti

et al. 2020

Preprint

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Despite being amongst the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A1R) was determined in the active, Gi protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A1R. Our results identify new residues involved in the association and dissociation pathway, suggest the binding mode of N6-cyclopentyladenosine (CPA) related ligands, and highlight the dramatic effect that chemical modifications can have on the overall binding mechanism.

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Section: Towards the Definition Of The Structure-binding/unbinding Pamentioning

confidence: 99%

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Deganutti

Barkan

Preti

et al. 2020

Preprint

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“…BRET techniques have been successfully used to determine the real time kinetics of ligand binding to GPCRs (Stoddart et al, 2018, Sykes et al, 2019. In BRET ligand-binding experiments, we investigated the ability of the selective A3R antagonist MRS 1220, K5, K17 or K18 to inhibit specific binding of the fluorescent A3R antagonist CA200645 to Nluc-A3R.…”

Section: Kinetics Of A3r Antagonists Determined Through Bretmentioning

confidence: 99%

Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists

Barkan

Lagarias

Vrontaki

et al. 2019

Preprint

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Author Contributions:KB, AK and GL conceived and designed the research; KB performed the mammalian assays; KK conducted radioligand binding experiments, PL, DS, EV and AK performed the molecular dynamic simulations; SH derived the equations for the 'Rapid competitor dissociation kinetics' model; KB, GL and AK analysed data; KB and GL wrote manuscript, AK revised and edited the manuscript. Summary Background and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective receptor antagonists is a challenging but critical task.Experimental approach 39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamics (MD) simulations and consistent calculated binding 2 free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies. Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 µM) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. Conclusions and ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Word count: 241

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“…The phospholipid bilayer can effectively increase the efficiency of ligand binding to an intrabilayer receptor site, even at low concentrations, by reducing the degrees of freedom of the drug and limiting it to a specific region of the bilayer. Although membrane interactions can result in improved binding kinetics (Sykes et al, 2019), excessive membrane accumulation of ligands can lead to toxicity through off-target interactions. Therefore, simply increasing ligand lipophilicity to gain efficacy for membraneassociated targets is often unfavorable in terms of the overall drug profile.…”

Section: Introductionmentioning

confidence: 99%

Does the Lipid Bilayer Orchestrate Access and Binding of Ligands to Transmembrane Orthosteric/Allosteric Sites of G Protein-Coupled Receptors?

2019

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The ligand-binding sites of many G protein-coupled receptors (GPCRs) are situated around and deeply embedded within the central pocket formed by their seven transmembrane-spanning a-helical domains. Generally, these binding sites are assumed accessible to endogenous ligands from the aqueous phase. Recent advances in the structural biology of GPCRs, along with biophysical and computational studies, suggest that amphiphilic and lipophilic molecules may gain access to these receptors by first partitioning into the membrane and then reaching the binding site via lateral diffusion through the lipid bilayer. In addition, several crystal structures of class A and class B GPCRs bound to their ligands offer unprecedented details on the existence of lipid-facing allosteric binding sites outside the transmembrane helices that can only be reached via lipid pathways. The highly organized structure of the lipid bilayer may direct lipophilic or amphiphilic drugs to a specific depth within the bilayer, changing local concentration of the drug near the binding site and affecting its binding kinetics. Additionally, the constraints of the lipid bilayer, including its composition and biophysical properties, may play a critical role in "pre-organizing" ligand molecules in an optimal orientation and conformation to facilitate receptor binding. Despite its clear involvement in molecular recognition processes, the critical role of the membrane in binding ligands to lipid-exposed transmembrane binding sites remains poorly understood and warrants comprehensive investigation. Understanding the mechanistic basis of the structure-membrane interaction relationship of drugs will not only provide useful insights about receptor binding kinetics but will also enhance our ability to take advantage of the apparent membrane contributions when designing drugs that target transmembrane proteins with improved efficacy and safety. In this minireview, we summarize recent structural and computational studies on membrane contributions to binding processes, elucidating both lipid pathways of ligand access and binding mechanisms for several orthosteric and allosteric ligands of class A and class B GPCRs.

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Binding kinetics of ligands acting at GPCRs

Cited by 96 publications

References 103 publications

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists

Does the Lipid Bilayer Orchestrate Access and Binding of Ligands to Transmembrane Orthosteric/Allosteric Sites of G Protein-Coupled Receptors?

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Binding kinetics of ligands acting at GPCRs

Cited by 96 publications

References 103 publications

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

Does the Lipid Bilayer Orchestrate Access and Binding of Ligands to Transmembrane Orthosteric/Allosteric Sites of G Protein-Coupled Receptors?

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Pharmacological Characterisation of Novel Adenosine Receptor A₃R Antagonists