Kinetic radioligand binding assays analyze the rates at which radioligands and receptors bind to and dissociate from one another. Providing comprehensive radiolabeling services and supporting the development of RDCs with high-quality data, Alfa Cytology offers radioligand binding assay services, including kinetic radioligand binding assays. These advanced studies are critical for quantitatively characterizing the real-time binding interactions between a radiolabeled molecule and its biological target, delivering essential parameters for lead optimization and candidate selection in drug discovery and development.
Kinetic radioligand binding assays are used to calculate the rates of association (Kon) and dissociation (Koff) of a radioligand to its specific receptor, enzyme, or other macromolecular target. When compared to equilibrium binding assays, which provide a snapshot of affinity (Kd), kinetic assays examine the binding event over time. This time-resolved analysis is important. Indeed, the residence time of a drug on its target is becoming more acknowledged as a vital factor regarding the in vivo efficacy and duration of action, and is often a more viable correlate to the pharmacological activity than affinity alone.
Fig.1 Measured kinetics are analyzed using a dynamic simulation. (Bäck, T., et al., 2024)
Lead Optimization
Directing medicinal chemistry efforts through in-depth kinetic structure-activity relationships, enabling the selection of compounds with optimal target residence times alongside binding affinity.
Candidate Selection Profiling
Determining and predicting in vivo pharmacokinetic and pharmacodynamic profiles using kinetic parameters helps provide the essential information needed to choose promising candidates.
Mechanism of Action Studies
Identifying the distinct effects on binding and unbinding rates to differentiate competitive, allosteric, and irreversible binding mechanisms.
Target Engagement Validation
Confirming and quantifying the interaction of a novel compound with its intended biological target in a native or recombinant system.
Alfa Cytology offers full-service, customized kinetic radioligand binding assay services, providing solutions unique to each project's needs. Comprehensive solutions span from the development and validation of the assay to the performance of high-throughput screening and/or mechanistic studies. All studies are conducted in compliance with industry best practices, ensuring data integrity, precision, and regulatory readiness.
Recognizing the unique challenges presented by different ligand classes, we specialize in developing and optimizing kinetic radioligand binding assays for diverse radioligands.
Small Molecule-based Radioligands
Experience with a wide range of targets, including GPCRs, ion channels, kinases, and nuclear receptors, utilizing tritium (3H) labels, or carbon-14 (14C) where appropriate.
Specialized protocols for characterizing the binding kinetics of monoclonal antibodies, antibody fragments, and other protein scaffolds, often employing iodine-125 or other suitable isotopes.
Capabilities for assessing the kinetics of peptide-receptor interactions, crucial for oncology and metabolic disease research.
A kinetic radioligand binding assay was performed to characterize the binding properties of [3H]-small molecule to the adenosine A1 receptor. The assay consisted of association and dissociation phases. For association, membrane preparations were incubated with a fixed concentration of [3H]-labeled small molecule at 30°C, and specific binding was measured at regular intervals over 3 hours. For dissociation, membranes were first pre-incubated with the radioligand to reach equilibrium. Dissociation was then initiated by adding a vast excess of an unlabeled ligand, and the remaining bound radioligand was quantified periodically over 7 hours to ensure complete dissociation. Non-specific binding, determined in the presence of an excess of the unlabeled ligand, was subtracted from total binding to calculate specific binding in all experiments.
| Item | Group | Description |
|---|---|---|
| Association Assay | Total Binding | [3H]-ligand + Receptor Incubated together, and samples were taken at multiple time points to measure the increase in binding over time until equilibrium was reached. |
| Non-specific Binding | [3H]-ligand + Receptor + Excess unlabeled ligand Incubated together, and samples were taken at the same time points as the total binding tubes. |
|
| Dissociation Assay | Total Binding | Pre-equilibrated [3H]-ligand-Receptor complex + Excess unlabeled ligand (added at t=0) Dissociation was initiated at t=0 by adding an excess of unlabeled ligand. Samples were taken at multiple time points after this addition. |
| Non-specific Binding | Pre-equilibrated mixture of [3H]-ligand, Receptor, and Excess unlabeled ligand Excess unlabeled ligand was present throughout pre-equilibration and dissociation. Samples were taken at matched time points to measure the background signal. |
The kinetic binding profile of [3H]-small molecule at the human adenosine A1 receptor is shown in Fig. 2. In the association phase, specific binding increased time-dependently until equilibrium was established (Fig. 2A). In the dissociation experiment, the addition of an excess unlabeled ligand prompted a rapid and complete decline in specific binding (Fig. 2B). The observed data were fitted to determine the association rate constant (Kon) and dissociation rate constant (Koff).
Fig.2 The association of [3H]-ligand to the human adenosine A1 receptor (A). The dissociation of [3H]-ligand from the human adenosine A1 receptor (B).
A partnership is offered that combines scientific depth with operational excellence, assuring that essential kinetic data is produced with excellence and dependability.
Focused on innovative approaches and profound knowledge in kinetic radioligand binding, Alfa Cytology delivers the critical data to assess the intricacies of your drug-target interactions and enable strategic insights in your discovery and development programs. For detailed discussions on how our services can advance your specific project, please contact us.
Reference
For research use only. Not intended for any clinical use.
Alfa Cytology, a provider of RDC preclinical services, specializes in RDC design, conjugation, and analysis. Supported by a rich library of radionuclides, chelators, and linkers, we excel in targeting molecule design. We offer customized RDC services tailored to diverse requirements-your demand is our mission.
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