Kinact / Ki Assay for Irreversible Covalent Compounds

Covalent inhibition is dependent on both the affinity of the compound for the binding pocket, as well as the rate of covalent bond formation by the warhead. Since this process is time dependent, the preferred measure of potency for irreversible covalent compounds is the second-order rate constant K_inact/K_i, rather than the IC₅₀ value. Generation of K_inact/K_i data allows us to ascertain a quantitative potency metric which can be used to drive chemical optimisation during hit-to-lead  and lead optimisation campaigns.

For reversible covalent inhibitors see our reversible covalent inhibitor binding assay.

Domainex’s Standard Experimental Procedure:

The target protein is incubated with the test compound at a range of concentrations at 37 °C over a period of 7 hours. The binding percentage of the compound to the protein is analysed in real time using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS). The resulting binding percentages at each time point for each concentration are plotted to generate K_obs, then these K_obs values and the corresponding compound concentrations are plotted to calculate K_inact/K_i. Example data is shown in Figure 1. 

Figure 1: K_obs and K_inact/K_i data generated for a fragment from Domainex’s internal covalent fragment library against a client’s target protein. The binding percentage for the fragment at different concentrations was measured over a period of 7 hours

Data analysis

The increase of binding percentage over time for a compound concentration is used to calculate the K_obs value using the following equation:

The relationship between K_obs and compound concentration is used to calculate the K_inact/K_i value as illustrated in the equation below: 

Deliverables

The results are reported in Excel file format including the K_inact/K_i value, as well as compound solubility and stability in PBS at 37 °C.