Kinases, including protein kinases, phosphatases, and phosphodiesterases, play vital roles in cellular processes by transferring phosphate groups through covalent modifications. Protein kinases, in particular, regulate the activity of proteins by transferring phosphate groups from high-energy donor molecules like adenosine triphosphate (ATP) to specific target molecules, a process known as phosphorylation. Dysregulated signaling pathways mediated by kinases are implicated in various human pathologies, such as cancer, cardiovascular diseases, neurodegenerative disorders, and metabolic imbalances. Consequently, kinases have emerged as crucial targets for drug development and screening.
Protein kinase active site showing structural interactions associated with catalytic and EPK-ELK component residues (Oruganty et al., 2013).
A kinase binding assay is a laboratory technique used to measure the binding affinity and potency of a compound to a specific kinase target. It provides valuable insights into the interaction between the compound and the kinase, helping researchers evaluate its potential as a therapeutic agent.
Kinase binding assays serve as invaluable tools for evaluating the interaction between potential drug candidates and kinase targets. These assays provide insights into the compound's ability to inhibit or modulate kinase activity, thereby guiding the selection and optimization of potential therapeutic agents. By assessing the affinity and specificity of small molecules or biologics towards kinases, researchers can identify lead compounds with the potential to interfere with aberrant signaling pathways.
Compound binding assays can be used to identify compounds that bind to kinases and modulate their activity. These assays involve screening a library of small molecules or biologics to identify potential lead compounds. Creative Proteomics offers techniques such as bio-layer interferometry (BLI), microscale thermophoresis (MST), surface plasmon resonance (SPR), isothermal titration calorimetry ( ITC) and other techniques that can measure the binding affinity and kinetics between compounds and kinases. By understanding the interactions between compounds and kinases, researchers can optimize lead compounds and design more effective therapeutic agents.
SPR is a label-free technique widely utilized in compound binding assays. By immobilizing one binding partner, typically the kinase target, on a sensor surface, researchers can monitor real-time binding events. As compounds flow over the immobilized target, changes in the refractive index near the sensor surface are detected, providing valuable information on binding affinity, kinetics, and thermodynamics. SPR enables the determination of crucial parameters such as association and dissociation rates, equilibrium constants, and stoichiometry, aiding in the characterization and optimization of lead compounds.
Binding interaction studies by Surface Plasmon Resonance (SPR) (Manschwetus et al., 2019)
Isothermal Titration Calorimetry is a powerful technique employed to measure the heat released or absorbed during a binding event. In this assay, a solution containing the kinase target is titrated with small amounts of a compound of interest. The heat generated or consumed during each injection is precisely measured, allowing for the determination of binding constants, enthalpy changes, stoichiometry, and binding kinetics. ITC provides a comprehensive understanding of the thermodynamics driving compound binding to kinases, offering insights into the nature of the interaction and guiding the selection of promising candidates for further development.
Bio-Layer Interferometry is a label-free technique that measures the interference patterns generated when a biosensor tip, coated with the kinase target, interacts with a compound of interest. BLI detects changes in the optical thickness of the biomolecular layer surrounding the tip as the compound binds to the kinase. This real-time analysis provides valuable information on binding kinetics, affinity, and specificity. BLI enables the determination of crucial parameters such as association and dissociation rates, equilibrium constants, and stoichiometry, aiding in the characterization and optimization of lead compounds.
Binding curves determined by biolayer interferometry for mAbs targeting antigenic site IV (Mousa et al., 2018).
Microscale Thermophoresis is a powerful technique employed to measure the movement of molecules in a temperature gradient. In this assay, a fluorescently labeled compound is titrated with the kinase target, and the thermophoretic movement of the labeled molecule is measured. The magnitude of the thermophoretic effect is directly related to the binding affinity between the compound and the kinase. MST allows for the determination of binding constants, affinity measurements, and the analysis of binding thermodynamics. It provides valuable insights into the strength and nature of the compound-kinase interaction, aiding in the selection and optimization of potential drug candidates.
Understanding protein-protein interactions is essential for deciphering the complex signaling networks involving kinases. Techniques such as co-immunoprecipitation, yeast two-hybrid assays, and fluorescence resonance energy transfer (FRET) enable researchers to identify interacting partners of kinases. These interactions provide insights into the regulation and modulation of kinase activity within the cellular context. Additionally, protein-protein interaction analysis can aid in the identification of potential biomarkers or targets for therapeutic interventions.
Combining these advanced technologies into the drug discovery process enables researchers to make informed decisions regarding the selection, optimization and further development of lead compounds. With its expertise in compound binding assays, Creative Proteomics is committed to providing comprehensive solutions to accelerate drug discovery efforts and advance precision medicine.