The Importance of Continuous Assays for Time-Dependent Inhibition
PhosphoSens® continuous assay format enables easy identification and analysis of time-dependent kinase inhibitors.
Interest in time-dependent inhibition from today's drug discovery research has led to a greater need for a continuous assay format to replace its end-point counterpart. While the end-point format has been used historically to determine inhibitor potency, this format has significant disadvantages in studies involving time-dependent inhibition. In particular, an end-point analysis assumes that the initial reaction rate is maintained throughout the forward progress curve. Thus, lags at the beginning and/or saturation towards the end of the curve are not captured.
Following this understanding, scientists are turning to the continuous assay format of PhosphoSens®. This technology is powered by chelation-enhanced fluorescence (ChEF) using the sulfonamido-oxine (Sox) chromophore and a proprietary peptide substrate resulting in an assay that is continuous, direct, catalytic and homogenous, yielding a progress curve in every well.
Time-Dependent Inhibition Workflow at AssayQuant Technologies
In the presentation, we walk you through our TDI (time-dependent inhibition) workflow, which includes:
- Determination of the presence/absence of time-dependent inhibition
- If time-dependent, determine reversibility/irreversibility
- Quantitative characterization of the reversible or irreversible inhibition
And we'll show you how this workflow yields the kinetic parameters that are of greatest interest to drug discovery.
Presentation Includes:
- PhosphoSens Technology overview enabling a continuous assay format
- Comparison of continuous and end-point assays
- Introduction to time-dependent inhibition (TDI)
- PhosphoSens workflow for TDI
- Determination of reversible/irreversible inhibitor binding
- Reversible inhibitor characterization:
- koff (off-rate)
- t1/2 (half-life)
- τ (tau, residence time)
- Irreversible inhibitor characterization:
- kinact (max rate of inactivation)
- KI (kinetic binding constant)
- kinact/KI (inactivation efficiency)
- When to report IC50, and when to report kinact/KI
Earl May, Ph.D., Senior Director of Discovery Technologies
Earl has 25+ years of expertise in drug discovery and enzymology from GSK, OSI and several other biotechnology companies. He has used our Sox sensor peptide technology at all companies he had kinase inhibitor programs, with both reversible and covalent compounds.Additional Download
Access a PDF version of the presentation above.
♦ IC50, EC50 and Ki for catalytic inhibitor/activator potency ♦ Kinome Profiling for compound selectivity ♦ Progress Curve Analysis for time-dependent inhibition ♦ Jump Dilution for reversible vs irreversible determinations ♦ Residence Time for reversible inhibition studies ♦ kinact/KI for irreversible inhibition studies ♦ MOI/MOA for inhibitor-substrate competition studies ♦ Add Eu3+ for red-shifted endpoint HTS ♦
Find Your Next Kinetic Assay Kit
PhosphoSens continuous assays deliver kinetic answers to kinetic questions, with a simple format and superior substrates to streamline workflows and enable new discoveries. With hundreds of assays developed and a robust custom assay development process, AssayQuant is a leader in next-generation continuous assays.
