New approaches for the reliable in vitro assessment of binding affinity based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics

Abstract

BackgroundResolving the kinetic mechanisms of biomolecular interactions have become increasingly important in early-phase drug development. Since traditional in vitro methods belong to dose-dependent assessments, binding kinetics is usually overlooked. The present study aimed at the establishment of two novel experimental approaches for the assessment of binding affinity of both, radiolabelled and non-labelled compounds targeting the A3R, based on high-resolution real-time data acquisition of radioligand-receptor binding kinetics. A novel time-resolved competition assay was developed and applied to determine the Ki of eight different A3R antagonists, using CHO-K1 cells stably expressing the hA3R. In addition, a new kinetic real-time cell-binding approach was established to quantify the rate constants k on and k off, as well as the dedicated K d of the A3R agonist [125I]-AB-MECA. Furthermore, lipophilicity measurements were conducted to control influences due to physicochemical properties of the used compounds.ResultsTwo novel real-time cell-binding approaches were successfully developed and established. Both experimental procedures were found to visualize the kinetic binding characteristics with high spatial and temporal resolution, resulting in reliable affinity values, which are in good agreement with values previously reported with traditional methods. Taking into account the lipophilicity of the A3R antagonists, no influences on the experimental performance and the resulting affinity were investigated.ConclusionsBoth kinetic binding approaches comprise tracer administration and subsequent binding to living cells, expressing the dedicated target protein. Therefore, the experiments resemble better the true in vivo physiological conditions and provide important markers of cellular feedback and biological response.

Structure–Affinity Relationships and Structure–Kinetics Relationships of Pyrido[2,1-f]purine-2,4-dione Derivatives as Human Adenosine A3 Receptor Antagonists

“…While the binding kinetics of some (labeled) hA 3 R agonists have been studied, 20 this parameter has not been part of medicinal chemistry efforts for antagonists, i.e., yielding structure–kinetics relationships (SKRs), next to SAFIRs. 21 Therefore, to provide the first SKR analysis on the hA 3 R, a highly potent and selective hA 3 R antagonist scaffold was chosen.…”

SNAPshots of the MCHR1: a Comparison Between the PET-Tracers [18F]FE@SNAP and [11C]SNAP-7941

“…This phenomenon contradicts previously elaborated findings on CHO-K1-hMCHR2 membranes [ 34 ] and might be explained by the difference in the biochemical approach (competition experiments with the unlabeled ligand vs. direct binding with the radiolabeled ligand) and the experimental setup (membranes vs. living cells). In this context, it has to be highlighted that experiments on living cells, as performed in the present study, enhance the understanding of the complex interplay between the radiotracer and the dedicated biological target [ 39 ]. Moreover, previous experiments revealed higher binding affinity for (±)-SNAP-7941 ( K i = 3.91 ± 0.74 nM) compared to FE@SNAP ( K i = 9.98 ± 1.12 nM) [ 25 ].…”

“…Association-time curves were monitored in real-time until the binding equilibrium was achieved. The observed rate constant of the association reaction ( k obs ) was determined using non-linear regression curve fitting algorithms implemented in GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA), as previously reported [ 39 , 40 ]. Cell survival was continuously examined using the perimeter trace (signal vs. dish position) of the LigandTracer® 1.0.1 software (Ridgeview Instruments AB, Uppsala, Sweden).…”

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [¹¹C]SNAP-7941

“…Precondition a SPE (solid phase extraction) cartridge with 10 mL of 96% ethanol and 20 mL of water and attach it between V11 and V12. 13 ) and finally cool down to below 40 °C. 14.…”