A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor

There is a growing interest in understanding the binding kinetics of compounds that bind to G proteincoupled 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 10oC 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

Agonists for the adenosine A(1) receptor with tunable residence time: a case for nonribose 4-Amino-6-aryl-5-cyano-2-thiopyrimidines

Medicinal Chemistr

Data belonging to the thesis: Corpora Non Agunt Nisi Fixata: Ligand Receptor Binding Kinetics in G Protein-Coupled Receptors.

The present thesis focuses on the pharmacological concept of drug-target interaction, which dates back to the beginning of modern pharmacology. A traditional equilibrium metrics-based rationale (i.e. optimization of drug affinity leads to better efficacy and safety) is unable to prevent current high attrition rates in the early phase of drug discovery. In the past decade drug-target binding kinetics (i.e. association and dissociation rate constants, residence time) has been gaining more and more attention, which constitutes a paradigm shift to better predict parameters of drug efficacy and safety. We decided to investigate binding kinetics of G protein-coupled receptors (GPCR), since GPCR are involved in various critical physiological and pharmacological functions, being the target of about 30% of all drugs on the market. Both the human cannabinoid receptor 1 and the human adenosine A1 and A3 receptors were chosen as prototypical GPCR as well as potential drug targets. The binding kinetics investigations described in this thesis provide a better and multi-faceted understanding of drug-target interactions and offer suggestions for the design of better ligands with an appropriate kinetic profile, new technologies for rapid kinetic assessment, and ultimately suitable evaluation schemes for a better translation towards effective and safe drugs

Fabrication of Chitosan/Hydroxyethyl Cellulose/TiO2 Incorporated Mulberry Anthocyanin 3D-Printed Bilayer Films for Quality of Litchis

In this study, a bilayer antibacterial chromogenic material was prepared using chitosan (CS) and hydroxyethyl cellulose (HEC) as inner substrate, mulberry anthocyanins (MA) as a natural tracer, and titanium dioxide nanoparticles (nano-TiO2)/CS:HEC as a bacteriostatic agent for the outer layer. By investigating their apparent viscosity and suitability for 3D printing links, the optimal ratio of the substrates was determined to be CS:HEC = 3:3. Viscosity of the CH was moderate. The printing process was consistent and exhibited no breakage or clogging. The printed image was highly stable and not susceptible to collapse and diffusion. Scanning electron microscopy and infrared spectroscopy indicated that intermolecular binding between the substances exhibited good compatibility. Titanium dioxide nanoparticles (nano-TiO2) were evenly distributed in the CH and no agglomeration was observed. The inner film fill rates affected the overall performance of the chromogenic material, with strong inhibitory effects against Escherichia coli and Staphylococcus aureus at different temperatures, as well as strong color stability. The experimental results indicated that the double-layer antibacterial chromogenic material can, to a certain extent, extend the shelf life of litchi fruit and determine the extent of its freshness. Therefore, from this study, we can infer that the research and development of active materials have a certain reference value

Identification of driving factors for green building development in China

Green building (GB) has been actively promoted in many countries, but it has not become the mainstream in Chinese construction industry due to various reasons. This paper aims to investigate the major driving factors for the development of GB with reference of the Chinese construction market. Twenty-one factors influencing the development of GB were identified through a literature review, questionnaire survey, and face-to-face interview with professionals in the construction industry. Structural equation model was established to identify the critical driving path and three critical factors hierarchies. The result of model analysis also verifies the theoretical hypotheses that government body is the biggest motivation for the development of GB, and the path coefficient is high. The results demonstrate the necessity for the formulation of incentive policies and power of GB propaganda. We identify distinct government and market effects and then induce a government-led GB development path. These findings provide a valuable reference for government body aiming at promoting GB in the construction industry to put forward relevant policies and incentives and for the market body to understand the major driving factors and path when making decisions.</p

Identification of driving factors for green building development in China

Green building (GB) has been actively promoted in many countries, but it has not become the mainstream in Chinese construction industry due to various reasons. This paper aims to investigate the major driving factors for the development of GB with reference of the Chinese construction market. Twenty-one factors influencing the development of GB were identified through a literature review, questionnaire survey, and face-to-face interview with professionals in the construction industry. Structural equation model was established to identify the critical driving path and three critical factors hierarchies. The result of model analysis also verifies the theoretical hypotheses that government body is the biggest motivation for the development of GB, and the path coefficient is high. The results demonstrate the necessity for the formulation of incentive policies and power of GB propaganda. We identify distinct government and market effects and then induce a government-led GB development path. These findings provide a valuable reference for government body aiming at promoting GB in the construction industry to put forward relevant policies and incentives and for the market body to understand the major driving factors and path when making decisions.Integral Design and Managemen

Cellular Assay to Study β-Arrestin Recruitment by the Cannabinoid Receptors 1 and 2

Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of β-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit β-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a β-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here