Inhibitor affinity, quantitation

Characterization of inhibition modality, and from this quantitative determination of enzyme-inhibitor dissociation constants, constitutes the only rational, quantitative means of assessing relative compound affinity for a target enzyme. 

In this chapter we consider the situation where this assumption is no longer valid, because the affinity of the inhibitor for its target enzyme is so great that the value of K w approaches the total concentration of enzyme ( / T) in the assay system. This situation is referred to as tight binding inhibition, and it presents some unique challenges for quantitative assessment of inhibitor potency and for correct assessment of inhibitor SAR. 

The partitioning of the activated inhibitor between direct covalent inactivation of the enzyme and release into solution is an important issue for mechanism-based inactivators. The partition ratio is of value as a quantitative measure of inactivation efficiency, as described above. This value is also important in assessing the suitability of a compound as a drug for clinical use. If the partition ratio is high, this means that a significant proportion of the activated inhibitor molecules is not sequestered as a covalent adduct with the target enzyme but instead is released into solution. Once released, the compound can diffuse away to covalently modify other proteins within the cell, tissue, or systemic circulation. This could then lead to the same types of potential clinical liabilities that were discussed earlier in this chapter in the context of affinity labels, and would therefore erode the potential therapeutic index for such a compound. 

Dunn, B.M. and Chaiken, I.M., Quantitative affinity chromatography. Determination of binding constants by elution with competitive inhibitors, Proc. Natl. Acad. Set U.S.A., 71, 2382-2385, 1974. 

Figure 11.4 Illustration of the SPR experiment. The binding of BACE to the immobilized small peptidic reference compound is detected by a change in refractive index at the chip surface. Preincubation of BACE with a test compound blocks the interaction with the immobilized reference compound in a manner that is dependent on the concentration of the test compound and its affinity for BACE. In practice, we could rank affinities by comparison of the SPR response at a fixed concentration of the test compound and then quantitatively determine the affinity by analyzing the concentration response dependence. In this example, the inhibitor in the middle panel (Inhibitor 1) has weaker affinity than the inhibitor in the lower panel (Inhibitor 2) because, at equivalent concentrations, Inhibitor 2 more extensively blocks the interaction between BACE and the immobilized peptidic inhibitor.

The next step was made by Klebe et al. [50]. Two 3D-QSAR methods were applied to get three-dimensional quantitative structure-activity relationships using a training set of 72 inhibitors of the benzamidine type with respect to their binding affinities toward Factor Xa to yield statistically reliable models of good predictive power [51-54] the widely used CoMFA method (for steric and electrostatic properties) and the comparative molecular similarity index analysis (CoMSlA) method (for steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor properties). These methods allowed the consideration of various physicochemical properties, and the resulting contribution maps could be intuitively interpreted. 

Adsorption of the eel enzyme to the affinity column was quantitative and approx. 80% of the activity was recovered after elution with the inhibitor tensilon. Specific activity of the enzyme which migrated on disk electrophoresis as a single band was 16 000 units/mg. Results with the erythrocyte enzyme were equally good, with a 2500-fold purification achieved. 

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