A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A(3) receptor

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A3 receptor (A3AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A3AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A3AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A3AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A3AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([3H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [3H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.Medicinal Chemistr

Kinetics for Drug Discovery: an industry-driven effort to target drug residence time

A considerable number of approved drugs show non-equilibrium binding characteristics, emphasizing the potential role of drug residence times for in vivo efficacy. Therefore, a detailed understanding of the kinetics of association and dissociation of a target-ligand complex might provide crucial insight into the molecular mechanism-of-action of a compound. This deeper understanding will help to improve decision making in drug discovery, thus leading to a better selection of interesting compounds to be profiled further. In this review, we highlight the contributions of the Kinetics for Drug Discovery (K4DD) Consortium, which targets major open questions related to binding kinetics in an industry-driven public-private partnership.PharmacologyMedicinal Chemistr

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362

Constitutive activity of GPR40/FFA1 intrinsic or assay dependent?

Free fatty acid receptor 1 (FFA1; previously designated GPR40) is a potential therapeutic target for the treatment of diabetes and related metabolic disorders. Agonist-independent or constitutive activity is a feature associated with essentially all G protein-coupled receptors but the extent of this varies substantially between family members. In many situations, detection of such activity can be both assay- and context-dependent and may reflect the presence in the assay of an endogenous agonist. In studies on FFA1, experiments employing cell membrane preparations and the binding of [(35)S]guanosine 5'-O-[γ-thio]triphosphate to G proteins produce data consistent with a high-level constitutive activity of this receptor. Herein, we detail these assays and discuss approaches to determine if this is a measure of intrinsic receptor constitutive activity or if such results reflect the presence of an endogenous agonist. FFA1 is coupled predominantly to G proteins of the Gα(q) subfamily. Activation of the receptor results, therefore, in the transient elevation of intracellular [Ca(2+)]. We also detail assays to measure such signals and consider whether they are appropriate to detect receptor constitutive activity

Functional homomers and heteromers of dopamine D_2L and D₃ receptors co-exist at the cell surface

Human dopamine D&lt;sub&gt;2long&lt;/sub&gt; and D&lt;sub&gt;3&lt;/sub&gt; receptors were modified by N-terminal addition of SNAP or CLIP forms of C&lt;sup&gt;6&lt;/sup&gt;-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D&lt;sub&gt;2long&lt;/sub&gt; and D&lt;sub&gt;3&lt;/sub&gt; receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D&lt;sub&gt;2long&lt;/sub&gt;-D&lt;sub&gt;3&lt;/sub&gt; heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D&lt;sub&gt;2long&lt;/sub&gt;–D&lt;sub&gt;3&lt;/sub&gt; heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D&lt;sub&gt;2long&lt;/sub&gt; and D&lt;sub&gt;3&lt;/sub&gt; receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D&lt;sub&gt;2long&lt;/sub&gt; and D&lt;sub&gt;3&lt;/sub&gt; receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D&lt;sub&gt;2long&lt;/sub&gt; and D&lt;sub&gt;3&lt;/sub&gt; receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites