KinSight™ Time-Dependent Inhibition Services

Inhibitor Characterization

We start by characterizing your inhibitor using dose response curves with and without pre-incubation of the target enzyme at various inhibitor doses. To recognize TDI, we perform both a qualitative assessment of the progress curve (signal vs time) and quantitative analysis of IC₅₀ (reaction rate as a function inhibitor concentration).

For the qualitative assessment, we visually inspect the linearity of the progress curve—compounds that demonstrate TDI will not appear linear; the slope of the progress curve decreases over time while those that do not demonstrate TDI will have a linear curve throughout.

For the quantitative analysis, we calculate the fold-change in IC₅₀ values with and without pre-incubation of the enzyme with compound. By pre-incubating the enzyme with the compound, the inhibitor can exert any time-dependent effects prior to activity determination, resulting in a more linear progress curve and a change in the IC₅₀.

If we determine there is no observable TDI, we report the IC₅₀ and K_i (if we use ATP K_m and binding is competitive with ATP) measurements that are correlated with drug potency. If we see a shift in IC₅₀ values, this is indicative of TDI but doesn’t tell you the whole story. Instead, the kinetic parameters that matter most for your compound’s potency assessment depend on whether it exhibits reversible or irreversible inhibition.

For inhibitors exhibiting TDI, we determine a “proxy IC₅₀”, an IC₅₀ inhibitor concentration derived from the pre-incubation dose response curve that is used as an optimal working inhibitor concentration for the downstream workflow.

Reversibility Characterization

Our next step is to identify whether your inhibitor is reversible or irreversible. To determine reversibility, we pre-incubate the enzyme with inhibitor at 10x the proxy IC₅₀ determined in the previous stage followed by a 100x jump dilution experiment. In this assay, the reaction rate of an enzyme bound to a reversible inhibitor would recover nearly to the uninhibited rate, because the inhibitor would quickly dissociate. On the other hand, an irreversible inhibitor would not dissociate, and there would be no recovery of the reaction rate.

Reversible inhibitors will advance to residence time determinations. Irreversible inhibitors advance to determination of k_inact/K_I, a rate constant describing the efficiency of irreversible bond formation.

TDI Characterization

For reversible inhibitors, we perform forward progress curve analysis and jump dilution analysis and report residence time (tau), k_off, and t_1/2.

For irreversible inhibitors, we perform forward progress curve analysis and report the inactivation efficiency, k_inact/K_I, and where possible each kinetic parameter separately: k_inact, the maximal rate of inactivation, and K_I, the kinetic binding constant (depending on whether the global data fit to a 1 or 2 step model, respectively).

These parameters, particularly the inactivation efficiency, are critical to interpret structure-activity relationships and can more effectively translate activity from biochemical assays to the cell than traditional IC₅₀ comparisons. The inactivation efficiency is the best and most-often used parameter to describe the effectiveness of an irreversible inhibitor. Journals are beginning to require kinetic characterization for TDIs and will not accept steady-state IC₅₀ reporting.