[Show abstract][Hide abstract] ABSTRACT: Scintillation proximity assay (SPA) is a radio-isotopic technology format used to measure a wide range of biological interactions, including drug-target binding affinity studies. The assay is homogeneous in nature, as it relies on a “mix and measure” format. It does not involve a filtration step to separate bound from free ligand as is the case in a traditional receptor-binding assay. For G protein-coupled receptors (GPCRs), it has been shown that optimal binding kinetics, next to a high affinity of a ligand, can result in more desirable pharmacological profiles. However, traditional techniques to assess kinetic parameters tend to be cumbersome and laborious. We thus aimed to evaluate whether SPA can be an alternative platform for real-time receptor-binding kinetic measurements on GPCRs. To do so, we first validated the SPA technology for equilibrium binding studies on a prototypic class A GPCR, the human adenosine A1 receptor (hA1R). Differently to classic kinetic studies, the SPA technology allowed us to study binding kinetic processes almost real time, which is impossible in the filtration assay. To demonstrate the reliability of this technology for kinetic purposes, we performed the so-called competition association experiments. The association and dissociation rate constants (k
on and k
off) of unlabeled hA1R ligands were reliably and quickly determined and agreed very well with the same parameters from a traditional filtration assay performed simultaneously. In conclusion, SPA is a very promising technique to determine the kinetic profile of the drug-target interaction. Its robustness and potential for high-throughput may render this technology a preferred choice for further kinetic studies.
Full-text · Article · Dec 2015 · Purinergic Signalling
[Show abstract][Hide abstract] ABSTRACT: The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can be roughly divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491 and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists, and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491 and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYX(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists.
No preview · Article · Jul 2014 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: We report the synthesis and evaluation of previously unreported 4-Amino-6-aryl-5-cyano-2-thiopyrimidines as selective human adenosine A1 receptor (hA1AR) agonists with tunable binding kinetics, this without affecting their nanomolar affinity for the target receptor. They show a very diverse range of kinetic profiles (from 1 minute (compound 52) up to 1 hour (compound 43)), and their Structure-Affinity Relationships (SAR) and Structure-Kinetics Relationships (SKR) were established. When put in perspective with the increasing importance of binding kinetics in drug discovery, these results bring new evidence of the consequences of affinity-only-driven selection of drug candidates, i.e. the potential elimination of slightly less active compounds that may display preferable binding kinetics.
No preview · Article · Mar 2014 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: Pre-clinical models of inflammatory diseases (e.g. neuropathic pain, rheumatoid arthritis and multiple sclerosis) have pointed to a critical role of the chemokine receptor CCR2 and its ligand, CCL2. However, one of the biggest problems of high affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high affinity and long residence time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of ligand's structure and its receptor residence time (i.e. SKR) next to traditional structure-affinity relationships (SAR). By applying combined knowledge of SAR and SKR we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity based optimization yielded compound 1 with good binding (Ki = 6.8 nM), but very short residence time (2.4 min). However, when the optimization was based also on residence time, the hit-to-lead process yielded compound 22a - a new high affinity CCR2 antagonist (3.6 nM) with a residence time of 135 min.
No preview · Article · Sep 2013 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: The chemokine receptor CCR2 is a G protein-coupled receptor that is primarily activated by the endogenous chemokine CCL2. Many different small molecule antagonists have been developed to inhibit this receptor, as it is involved in a variety of diseases characterized by chronic inflammation. Unfortunately, all of these antagonists lack clinical efficacy and therefore a better understanding of their mechanism of action is warranted. In this study we examined the pharmacological properties of small molecule CCR2 antagonists in radioligand binding and functional assays. Six structurally different antagonists were selected for this study, which all displaced the endogenous agonist (125)I-CCL2 from CCR2 with nanomolar affinity. Two of these antagonists, INCB3344 and CCR2-RA, were radiolabeled in order to study the binding site in more detail. We discovered that [(3)H]-INCB3344 and [(3)H]-CCR2-RA bind to distinct binding sites at CCR2, the latter being the first allosteric radioligand for CCR2. Besides the binding properties of the antagonists, we also examined CCR2 inhibition in multiple functional assays, including a novel label-free whole cell assay. INCB3344 was found to competitively inhibit CCL2-induced G protein activation, whereas CCR2-RA showed a non-competitive or allosteric mode of inhibition. These findings demonstrated that the CCR2 antagonists examined in this study can be classified in two groups with a different binding site and thereby a different mode of inhibition. We have provided further insights in CCR2 antagonism, which are important for the development of novel CCR2 inhibitors.
No preview · Article · Jul 2013 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: The four subtypes of adenosine receptors form relevant drug targets in the treatment of, e.g., diabetes and Parkinson's disease. In the present study, we aimed at finding novel small molecule ligands for these receptors using virtual screening approaches based on proteochemometric (PCM) modeling. We combined bioactivity data from all human and rat receptors in order to widen available chemical space. After training and validating a proteochemometric model on this combined data set (Q(2) of 0.73, RMSE of 0.61), we virtually screened a vendor database of 100910 compounds. Of 54 compounds purchased, six novel high affinity adenosine receptor ligands were confirmed experimentally, one of which displayed an affinity of 7 nM on the human adenosine A(1) receptor. We conclude that the combination of rat and human data performs better than human data only. Furthermore, we conclude that proteochemometric modeling is an efficient method to quickly screen for novel bioactive compounds.
No preview · Article · Jul 2012 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: The four subtypes of adenosine receptors form relevant drug targets in the treatment of e.g., diabetes and Parkinson’s disease. In the present study we aimed at finding novel small molecule ligands for these receptors using virtual screening approaches based on proteochemometric (PCM) modeling. We combined bioactivity data from all human and rat receptors in order to widen available chemical space. After training and validating a proteochemometric model on this combined dataset (Q2 of 0.73, RMSE of 0.61) we virtually screened a vendor database of 100,910 compounds. Of 54 compounds purchased, six novel high affinity adenosine receptor ligands were confirmed experimentally, one of which displayed an affinity of 7 nM on the human adenosine A1 receptor. We conclude that the combination of rat and human data performs better than human data only. Furthermore, we conclude that proteochemometric modeling is an efficient method to quickly screen for novel bioactive compounds.
No preview · Article · Jul 2012 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: The A(2B) adenosine receptor (A(2B)R) is highly expressed in macrophages and vascular smooth muscle cells and has been established as an important regulator of inflammation and vascular adhesion. Recently, it has been demonstrated that A(2B)R deficiency enhances neointimal lesion formation after vascular injury. Therefore, we hypothesize that A(2B)R agonism protects against injury-induced intimal hyperplasia.
Apolipoprotein E-deficient mice were fed a Western-type diet for 1 week, after which the left common carotid artery was denuded. Mice were treated with the A(2B) receptor agonist BAY60-6583 or vehicle control for 18 days. Interestingly, lumen stenosis as defined by the neointima/lumen ratio was inhibited by treatment with the A(2B) receptor agonist, caused by reduced smooth muscle cell proliferation. Collagen content was significantly increased in the BAY60-6583-treated mice, whereas macrophage content remained unchanged. In vitro, vascular smooth muscle cell proliferation decreased dose dependently whereas collagen content of cultured smooth muscle cells was increased by BAY60-6583.
Our data show that activation of the adenosine A(2B) receptor protects against vascular injury, while it also enhances plaque stability as indicated by increased collagen content. These outcomes thus point to A(2B) receptor agonism as a new therapeutic approach in the prevention of restenosis.
Preview · Article · Jun 2012 · Arteriosclerosis Thrombosis and Vascular Biology
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND PURPOSE Niacin can effectively treat dyslipidaemic disorders. However, its clinical use is limited due to the cutaneous flushing mediated by the nicotinic acid receptor HCA(2) . In the current study, we evaluated two partial agonists for HCA(2) , LUF6281 and LUF6283, with respect to their anti-dyslipidaemic potential and cutaneous flushing effect. EXPERIMENTAL APPROACH In vitro potency and efficacy studies with niacin and the two HCA(2) partial agonists were performed using HEK293T cells stably expressing human HCA(2) . Normolipidaemic C57BL/6 mice received either niacin or the HCA(2) partial agonists (400 mg·kg(-1) ·day(-1) ) once a day for 4 weeks for evaluation of their effects in vivo. KEY RESULTS Radioligand competitive binding assay showed K(i) values for LUF6281 and LUF6283 of 3 and 0.55 µM. [(35) S]-GTPγS binding revealed the rank order of their potency as niacin > LUF6283 > LUF6281. All three compounds reduced plasma VLDL-triglyceride concentrations similarly, while LUF6281 and LUF6283, in contrast to niacin, did not also exhibit the unwanted flushing side effect in C57BL/6 mice. Niacin reduced the expression of lipolytic genes HSL and ATGL in adipose tissue by 50%, whereas LUF6281 and LUF6283 unexpectedly did not. In contrast, the decrease in VLDL-triglyceride concentration induced by LUF6281 and LUF6283 was associated with a parallel >40% reduced expression of APOB within the liver. CONCLUSIONS AND IMPLICATIONS The current study identifies LUF6281 and LUF6283, two HCA(2) partial agonists of the pyrazole class, as promising drug candidates to achieve the beneficial lipid lowering effect of niacin without producing the unwanted flushing side effect.
Full-text · Article · May 2012 · British Journal of Pharmacology
[Show abstract][Hide abstract] ABSTRACT: We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the β-2 adrenoreceptor (ADRB2), the adenosine A(2A) receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts.
No preview · Article · May 2012 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N6-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A3 receptor (hA3R) ligands with affinities in the high nanomolar range (Ki values of 159 and 649 nM, respectively). These values were comparable to the observed Ki value of adenosine on hA3R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A3R. In a functional assay in Chinese hamster ovary cells transfected with hA3R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A3R antagonist VUF5574. Both IPA and reference A3R agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A3R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A3R-independent mechanism, as was previously reported for other A3R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A3R.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-011-9244-9) contains supplementary material, which is available to authorized users.
[Show abstract][Hide abstract] ABSTRACT: Little is known about the distribution of diagnoses that account for fatigue in patients in primary care. We evaluated the diagnoses established within 1 year after presentation with fatigue in primary care that were possibly associated with the fatigue.
We conducted a prospective observational cohort study with 1-year follow-up. We included adult patients who presented with a new episode of fatigue between June 2004 and January 2006. We extracted data on diagnoses during the follow-up period from the patients' medical records as well as data on pre-existing chronic diseases.
Of the 571 patients for whom diagnostic data were available, 268 (46.9%) had received one or more diagnoses that could be associated with fatigue. The diagnoses were diverse and mostly included symptom diagnoses, with main categories being musculoskeletal (19.4%) and psychological problems (16.5%). Clear somatic pathology was diagnosed in 47 (8.2%) of the patients. Most diagnoses were not made during the consultation when fatigue was presented.
Only a minority of patients were diagnosed with serious pathology. Half of the patients did not receive any diagnosis that could explain their fatigue. Nevertheless, because of the wide range of conditions and symptoms that may explain or co-occur with the fatigue, fatigue is a complex problem that deserves attention not only as a symptom of underlying specific disease.
Full-text · Article · Nov 2009 · Canadian Medical Association Journal
[Show abstract][Hide abstract] ABSTRACT: In this study we followed a new approach to analyze molecular substructures required for hERG channel blockade. We designed and synthesized 40 analogues of dofetilide (1), a potent hERG potassium channel blocker, and established structure-activity relationships (SAR) for their interaction with this important cardiotoxicity-related off-target. Structural modifications to dofetilide were made by diversifying the substituents on the phenyl rings and the protonated nitrogen and by varying the carbon chain length. The analogues were evaluated in a radioligand binding assay and SAR data were derived with the aim to specify structural features that give rise to hERG toxicity.
[Show abstract][Hide abstract] ABSTRACT: The luteinizing hormone (LH) receptor plays an important role in fertility and certain cancers. The endogenous ligands human chorionic gonadotropin (hCG) and LH bind to the large N terminal domain of the receptor. We recently reported on the first radiolabeled low molecular weight (LMW) agonist for this receptor, [(3)H]Org 43553, which was now used to screen for new LMW ligands. We identified a terphenyl derivative that inhibited [(3)H]Org 43553 binding to the receptor, which led us to synthesize a number of derivatives. The most potent compound of this terphenyl series, 24 (LUF5771), was able to increase the dissociation rate of [(3)H]Org 43553 by 3.3-fold (at 10 muM). In a functional assay, the presence of 24 resulted in a 2- to 3-fold lower potency of both Org 43553 and LH. Thus, the compounds presented in this paper are the first LMW ligands that allosterically inhibit the LH receptor.
No preview · Article · May 2009 · Journal of Medicinal Chemistry
[Show abstract][Hide abstract] ABSTRACT: The adenosine receptor subfamily consists of the adenosine A(1), A(2A), A(2B), and A(3) receptors, which are localized in a variety of tissues throughout the human body. It is, therefore, a challenge to develop receptor specific ligands with improved tissue selectivity. Allosteric modulators could have these therapeutic advantages over orthosteric ligands. In the present study, a series of 2,4-disubstituted quinolines were synthesized on the basis of the structure of LUF6000 (34). Compound 27 (LUF6096) was able to allosterically enhance agonist binding to a similar extent as 34. In addition, this new compound showed low, if any, orthosteric affinity for any of the adenosine receptors. In a functional assay, compound 27 showed improved activity in comparison to 34, as it increased both the intrinsic efficacy and the potency of the reference agonist Cl-IB-MECA at the human adenosine A(3) receptor.
No preview · Article · Feb 2009 · Journal of Medicinal Chemistry