Indacaterol is a novel beta(2)-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human beta(2)-adrenoceptor and in human primary airway smooth muscle (ASM) cells.Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human beta(2)-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3',5'-cyclic-monophosphate production was used as an outcome measure.In both the low- and high-expression recombinant GEVT 'wild type' cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the beta(2)-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3',5'-cyclic-monophosphate in response to beta(2)-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol.These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of beta(2)-adrenoceptors.
An increased appreciation of the importance of optimizing drug-binding kinetics has lead to the development of various techniques for measuring the kinetics of unlabeled compounds. One approach is the competition-association kinetic binding method first described in the 1980s. The kinetic characteristics of the tracer employed greatly affects the reliability of estimated kinetic parameters, a barrier to successfully introducing these kinetic assays earlier in the drug discovery process. Using a modeling and Monte Carlo simulation approach, we identify the optimal tracer characteristics for determining the kinetics of the range of unlabeled ligands typically encountered during the different stages of a drug discovery program (i.e., rapidly dissociating, e.g., koff = 10 minute−1 low-affinity "hits" through to slowly dissociating e.g., koff = 0.01 minute−1 high-affinity "candidates"). For more rapidly dissociating ligands (e.g., koff = 10 minute−1), the key to obtaining accurate kinetic parameters was to employ a tracer with a relatively fast off-rate (e.g., koff = 1 minute−1) or, alternatively, to increase the tracer concentration. Reductions in assay start-time ≤1second and read frequency ≤5 seconds significantly improved the reliability of curve fitting. Timing constraints are largely dictated by the method of detection, its inherent sensitivity (e.g., TR-FRET versus radiometric detection), and the ability to inject samples online. Furthermore, we include data from TR-FRET experiments that validate this simulation approach, confirming its practical utility. These insights into the optimal experimental parameters for development of competition-association assays provide a framework for identifying and testing novel tracers necessary for profiling unlabeled competitors, particularly rapidly dissociating low-affinity competitors.
Abstract Adenosine is elevated in the tumour microenvironment and plays a critical role in suppressing T cell function through high affinity interaction with the A2a receptor. Genetic deficiency of A2a in mice is associated with enhanced cytotoxic responses and reduced tumour burden in syngeneic models. These effects are mimicked by small molecule A2a antagonists and some of these compounds are currently being evaluated in clinical trials for the treatment of solid tumours, particularly in combination with checkpoint inhibitors. However, the high levels of adenosine in the tumour microenvironment can dramatically reduce the effectiveness of competitive A2a antagonists. The challenge therefore is to identify highly potent and selective A2a antagonists which retain potency in the presence of high concentrations of adenosine and therefore have the potential to nullify the adenosinergic pathway within the tumour microenvironment ARX001822 binds A2a with high affinity (Ki=0.9 nM) and with greater than 660-fold selectivity over A1, A2b and A3. In a functional assay utilising CHO cells expressing recombinant A2a ARX001822 inhibited cAMP production in response to the selective A2a agonist CGS21680 in a competitive manner (KB= 0.3nM). Activation of A2a leads to the suppression of T cell-derived cytokine production and ARX001822 prevented this suppression even in the presence of high concentration of the adenosine receptor ligand NECA (IC50=38 nM). ARX001822 was also active in human whole blood, preventing NECA-mediated elevation of pCREB in CD8+ T cells and restoring production of interferon-γ with a potency 5-20 times higher than that of competitor molecules undergoing clinical evaluation in cancer. ARX001822 was orally bioavailable in rats and mice and was effective in inhibiting elevation of pCREB in mouse CD8+ T cells in an ex vivo pharmacodynamic assay. ARX001822 is a highly potent and selective A2a antagonist which is effective in preventing adenosinergic mediated suppression of cytokine production in the presence of high concentrations of adenosine receptor ligands and a full complement of plasma proteins. Knowledge of whole blood potency on both pCREB and interferon-γ modulation combined with the exposure required for activity in the pharmacodynamic model is helpful in estimating the clinical exposure required for A2a receptor blockade and downstream events related to modulating T cell function. Citation Format: Peter M. Finan, Roy Pettipher, Jonathan White, Viral Patel, Ben Moulton, Soraya Porres, Karolina Gherbi, Elizabeth M. Rosethorne, Steven J. Charlton, Clive McCarthy. Profile of ARX001822, a highly potent, selective and orally bioavailable A2a antagonist effective in preventing adenosinergic suppression of T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5014.
Overdose deaths from fentanyl have reached epidemic proportions in the USA and are increasing worldwide. Fentanyl is a potent opioid agonist that is less well reversed by naloxone than morphine. Due to fentanyl's high lipophilicity and elongated structure we hypothesised that its unusual pharmacology may be explained by its interactions with the lipid membrane on route to binding to the μ-opioid receptor (MOPr). Through coarse-grained molecular dynamics simulations, electrophysiological recordings and cell signalling assays, we determined how fentanyl and morphine access the orthosteric pocket of MOPr. Morphine accesses MOPr via the aqueous pathway; first binding to an extracellular vestibule, then diffusing into the orthosteric pocket. In contrast, fentanyl may take a novel route; first partitioning into the membrane, before accessing the orthosteric site by diffusing through a ligand-induced gap between the transmembrane helices. In electrophysiological recordings fentanyl-induced currents returned after washout, suggesting fentanyl deposits in the lipid membrane. However, mutation of residues forming the potential MOPr transmembrane access site did not alter fentanyl's pharmacological profile in vitro. A high local concentration of fentanyl in the lipid membrane, possibly in combination with a novel lipophilic binding route, may explain the high potency and lower susceptibility of fentanyl to reversal by naloxone.
Integral membrane proteins (IMPs) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand-target interactions, robust, reliable, high-throughput, and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimetry (DSF) screening method using the thiol-reactive BODIPY FL-cystine dye to monitor thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2. To validate this method, the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners.
G protein-coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β
We give an evaluation for the stuffle-regularised $t^{\ast,V}(\{2\}^a,1,\{2\}^b)$ as a polynomial in single-zeta values, $\log(2)$ and $V$. We then apply this to establish some linear independence results of certain sets of motivic multiple $t$ values. In particular, we prove the elements of Saha's conjectural basis are linearly independent, on the motivic level, and that the (suitably regularised) elements $t^\mathfrak{m}(\{1,2\}^\times)$ form a basis for both the (extended) motivic MtV's and the alternating MZV's.
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