Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the... Show moreEvaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1. Show less
Aims Neladenoson bialanate is a partial adenosine A1 receptor agonist with demonstrated beneficial effects on cardiac function in animal models. We aimed to assess the dose-response effect of... Show moreAims Neladenoson bialanate is a partial adenosine A1 receptor agonist with demonstrated beneficial effects on cardiac function in animal models. We aimed to assess the dose-response effect of neladenoson bialanate on cardiac structure and function, clinical outcome, and safety in patients with heart failure (HF) with reduced ejection fraction (HFrEF).Methods and results PANTHEON was a dose-finding, phase IIb, randomized, double-blind, placebo-controlled trial conducted in 92 centres in 11 countries including 462 patients with chronic HFrEF, randomized to once daily oral dose of neladenoson bialanate (5, 10, 20, 30, and 40mg) or placebo. The primary endpoints were change from baseline to 20weeks in left ventricular ejection fraction (LVEF) (echocardiography) and in N-terminal pro-B-type natriuretic peptide (NT-proBNP). Mean age of the patients was 67 years, 17% were female, mean LVEF was 28%, mean NT-proBNP was 2085 ng/L. After 20weeks of treatment, there was no dose-effect of neladenoson bialanate on changes in NT-proBNP or LVEF (primary endpoints). No effect of neladenoson bialanate was found on left ventricular volumes, high-sensitivity troponin T, or cardiovascular mortality, HF hospitalization, and urgent visits for HF (secondary endpoints). There was a dose-dependent increase in creatinine and cystatin C, and a dose-dependent decrease in estimated glomerular filtration rate and heart rate.Conclusions In patients with chronic HFrEF, treatment with neladenoson bialanate was not associated with dose-dependent favourable effects on cardiac structure and function, cardiac risk markers, or clinical outcome but was associated with a dose-dependent decrease in renal function.Clinical Trial Registration: ClinicalTrials.gov Identifier NCT02992288. Show less
Aims Neladenoson bialanate is a partial adenosine A1 receptor agonist with demonstrated beneficial effects on cardiac function in animal models. We aimed to assess the dose-response effect of... Show moreAims Neladenoson bialanate is a partial adenosine A1 receptor agonist with demonstrated beneficial effects on cardiac function in animal models. We aimed to assess the dose-response effect of neladenoson bialanate on cardiac structure and function, clinical outcome, and safety in patients with heart failure (HF) with reduced ejection fraction (HFrEF). Methods and results PANTHEON was a dose-finding, phase IIb, randomized, double-blind, placebo-controlled trial conducted in 92 centres in 11 countries including 462 patients with chronic HFrEF, randomized to once daily oral dose of neladenoson bialanate (5, 10, 20, 30, and 40 mg) or placebo. The primary endpoints were change from baseline to 20 weeks in left ventricular ejection fraction (LVEF) (echocardiography) and in N-terminal pro-B-type natriuretic peptide (NT-proBNP). Mean age of the patients was 67 years, 17% were female, mean LVEF was 28%, mean NT-proBNP was 2085 ng/L. After 20 weeks of treatment, there was no dose-effect of neladenoson bialanate on changes in NT-proBNP or LVEF (primary endpoints). No effect of neladenoson bialanate was found on left ventricular volumes, high-sensitivity troponin T, or cardiovascular mortality, HF hospitalization, and urgent visits for HF (secondary endpoints). There was a dose-dependent increase in creatinine and cystatin C, and a dose-dependent decrease in estimated glomerular filtration rate and heart rate. Conclusions In patients with chronic HFrEF, treatment with neladenoson bialanate was not associated with dose-dependent favourable effects on cardiac structure and function, cardiac risk markers, or clinical outcome but was associated with a dose-dependent decrease in renal function. Clinical Trial Registration: identifier NCT02992288. 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
The main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As... Show moreThe main theme of this thesis, allosteric modulation effectuated through the sodium ion site of GPCRs, is inspired by the important role that this site appears to play in GPCR signaling. As sodium ions are abundant under physiological conditions they may affect GPCR signaling considerably. Receptor activation causes a substantial rearrangement of the sodium ion site, suggesting an important role in this process. Chapter 2 reviews the current knowledge on allosteric modulation of amiloride and its derivatives binding to the sodium ion site of Class A GPCRs. Chapters 3 to 5 follow-up on the recent crystal structure of the adenosine A2A receptor with a sodium ion bound. Chapters 3 and 4 complement the crystal structure with additional results from combined biochemistry, biophysical, molecular dynamics, and mutational studies. Chapter 5 describes the synthesis of novel amiloride derivatives that bind in the sodium ion site but also protrude into the orthosteric binding site. In Chapters 3 to 5, radio-labeled ligands were used to quantify ligand binding to the receptor, and Chapter 6 describes an alternative approach towards ligand binding assays. Instead of using a radio-label, mass spectrometry was used to quantify binding of an unlabeled ligand to adenosine A1 and A2A receptors. Show less
In this thesis, several orthosteric and allosteric agonists are presented for the newly discovered hydroxy-carboxylic acid (HCA) receptor 2, and their in vivo activity or in vitro structure... Show moreIn this thesis, several orthosteric and allosteric agonists are presented for the newly discovered hydroxy-carboxylic acid (HCA) receptor 2, and their in vivo activity or in vitro structure-activity relationships are described. The literature on HCA receptors was also thoroughly reviewed, providing some insight into the future of this receptor family as drug targets. The anti-cancer drug N6-(2-isopentenyl)adenosine (IPA) was shown to be a specific ligand for the adenosine A3 receptor, and its antiproliferative effect seems to be mediated by this receptor at low concentrations. A ligand discovery screen for orphan receptor GPR88 was performed, in which over 4000 compounds were tested. Show less
