Partial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1... Show morePartial agonists for G protein-coupled receptors (GPCRs) provide opportunities for novel pharmacotherapies with enhanced on-target safety compared to full agonists. For the human adenosine A1 receptor (hA1AR) this has led to the discovery of capadenoson, which has been in phase IIa clinical trials for heart failure. Accordingly, the design and profiling of novel hA1AR partial agonists has become an important research focus. In this study, we report on LUF7746, a capadenoson derivative bearing an electrophilic fluorosulfonyl moiety, as an irreversibly binding hA1AR modulator. Meanwhile, a nonreactive ligand bearing a methylsulfonyl moiety, LUF7747, was designed as a control probe in our study.In a radioligand binding assay, LUF7746’s apparent affinity increased to nanomolar range with longer pre-incubation time, suggesting an increasing level of covalent binding over time. Moreover, compared to the reference full agonist CPA, LUF7746 was a partial agonist in a hA1AR-mediated G protein activation assay and resistant to blockade with an antagonist/inverse agonist. An in silico structure-based docking study combined with site-directed mutagenesis of the hA1AR demonstrated that amino acid Y2717.36 was the primary anchor point for the covalent interaction. Additionally, a label-free whole-cell assay was set up to identify LUF7746’s irreversible activation of an A1 receptor-mediated cell morphological response.These results led us to conclude that LUF7746 is a novel covalent hA1AR partial agonist and a valuable chemical probe for further mapping the receptor activation process. It may also serve as a prototype for a therapeutic approach in which a covalent partial agonist may cause less on-target side effects, conferring enhanced safety compared to a full agonist. Show less
While equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In... Show moreWhile equilibrium binding affinities and in vitro functional antagonism of CB1 receptor antagonists have been studied in detail, little is known on the kinetics of their receptor interaction. In this study, we therefore conducted kinetic assays for nine 1-(4,5-diarylthiophene-2-carbonyl)-4-phenylpiperidine-4-carboxamide derivatives and included the CB1 antagonist rimonabant as a comparison. For this we newly developed a dual-point competition association assay with [3H]CP55940 as the radioligand. This assay yielded Kinetic Rate Index (KRI) values from which structure-kinetics relationships (SKR) of hCB1 receptor antagonists could be established. The fast dissociating antagonist 6 had a similar receptor residence time (RT) as rimonabant, i.e. 19 and 14 min, respectively, while the slowest dissociating antagonist (9) had a very long RT of 2222 min, i.e. pseudo-irreversible dissociation kinetics. In functional assays, 9 displayed insurmountable antagonism, while the effects of the shortest RT antagonist 6 and rimonabant were surmountable. Taken together, this study shows that hCB1 receptor antagonists can have very divergent RTs, which are not correlated to their equilibrium affinities. Furthermore, their RTs appear to define their mode of functional antagonism, i.e. surmountable vs. insurmountable. Finally, based on the recently resolved hCB1 receptor crystal structure, we propose that the differences in RT can be explained by a different binding mode of antagonist 9 from short RT antagonists that is able to displace unfavorable water molecules. Taken together, these findings are of importance for future design and evaluation of potent and safe hCB1 receptor antagonists. Show less
The structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served... Show moreThe structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA(2A) receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A(2A)-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A(2A) receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine. Show less
A covalent antagonist for the human adenosine A2A receptor Xue Yang, Guo Dong, Thomas J.M. Michiels, Eelke B. Lenselink, Laura Heitman, Julien Louvel, Ad P. IJzerman Abstract The structure of the... Show moreA covalent antagonist for the human adenosine A2A receptor Xue Yang, Guo Dong, Thomas J.M. Michiels, Eelke B. Lenselink, Laura Heitman, Julien Louvel, Ad P. IJzerman Abstract The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine. Show less
The structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served... Show moreThe structure of the human A(2A) adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA(2A) receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A(2A)-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A(2A) receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine. Show less
The structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as... Show moreThe structure of the human A2A adenosine receptor has been elucidated by X-ray crystallography with a high affinity non-xanthine antagonist, ZM241385, bound to it. This template molecule served as a starting point for the incorporation of reactive moieties that cause the ligand to covalently bind to the receptor. In particular, we incorporated a fluorosulfonyl moiety onto ZM241385, which yielded LUF7445 (4-((3-((7-amino-2-(furan-2-yl)-[1, 2, 4]triazolo[1,5-a][1, 3, 5]triazin-5-yl)amino)propyl)carbamoyl)benzene sulfonyl fluoride). In a radioligand binding assay, LUF7445 acted as a potent antagonist, with an apparent affinity for the hA2A receptor in the nanomolar range. Its apparent affinity increased with longer incubation time, suggesting an increasing level of covalent binding over time. An in silico A2A-structure-based docking model was used to study the binding mode of LUF7445. This led us to perform site-directed mutagenesis of the A2A receptor to probe and validate the target lysine amino acid K153 for covalent binding. Meanwhile, a functional assay combined with wash-out experiments was set up to investigate the efficacy of covalent binding of LUF7445. All these experiments led us to conclude LUF7445 is a valuable molecular tool for further investigating covalent interactions at this receptor. It may also serve as a prototype for a therapeutic approach in which a covalent antagonist may be needed to counteract prolonged and persistent presence of the endogenous ligand adenosine.KEYWORDS: A2A adenosine receptor; Adenosine; Covalent antagonist; G protein-coupled receptors; Radioligand binding Show less
Kv11.1-induced cardiotoxicity has emerged as an unanticipated adverse effect of many pharmacological agents and has become a major obstacle in drug development over the past decades. In this thesis... Show moreKv11.1-induced cardiotoxicity has emerged as an unanticipated adverse effect of many pharmacological agents and has become a major obstacle in drug development over the past decades. In this thesis, allosteric modulation of the Kv11.1 channel has been extensively explored, and negative allosteric modulators were shown to relieve the proarrhythmic effects of structurally and therapeutically diverse Kv11.1 blockers. The most potent modulators may be developed as a new class of antiarrhythmic medications in the future. On the other hand, kinetic binding parameters of a wide range of Kv11.1 blockers at the channel have been thoroughly investigated in this thesis. Association and dissociation rates or residence times are strongly suggested to be integrated with equilibrium affinity values into the future paradigms for a better and more comprehensive evaluation of Kv11.1 liability of drug candidates. The __kon-koff-KD__ kinetic map provides a first and promising classification of Kv11.1 blockers, which could be beneficial and indicative for drug researchers to design compounds with less Kv11.1-mediated cardiac side effects in the early stage of drug development. Hopefully, all findings in this thesis have brought new insights into Kv11.1-induced cardiac arrhythmias, and will offer opportunities for restoring or preventing this kind of arrhythmias in the near future. Show less
