Tight Binding Inhibitors

The catalytic subunit of cAPK contains two domains connected by a peptide linker. ATP binds in a deep cleft between the two domains. Presently, crystal structures showed cAPK in three different conformations, (1) in a closed conformation in the ternary complex with ATP or other tight-binding ligands and a peptide inhibitor PKI(5-24), (2) in an intermediate conformation in the binary complex with adenosine, and (3) in an open conformation in the binary complex of mammalian cAPK with PKI(5-24). Fig.l shows a superposition of the three protein kinase configurations to visualize the type of conformational movement. 

Slow, tight-binding inhibition occurs when slow-binding inhibition takes place at inhibitor concentrations comparable to that of the enzyme, in which case the previous two mechanisms can still apply. Comprehensive review articles on the subject of tight, slow, and slow, tight-binding inhibitors ate available in the literature (12,14). 

This class of inhibitors usually acts irreversibly by permanently blocking the active site of an enzyme upon covalent bond formation with an amino acid residue. Very tight-binding, noncovalent inhibitors often also act in an irreversible fashion with half-Hves of the enzyme-inhibitor complex on the order of days or weeks. At these limits, distinction between covalent and noncovalent becomes functionally irrelevant. The mode of inactivation of this class of inhibitors can be divided into two phases the inhibitors first bind to the enzyme in a noncovalent fashion, and then undergo subsequent covalent bond formation. 

If the inhibitor potency is such that the concentration of inhibitor required to affect significant, time-dependent inhibition is similar to the concentration of enzyme, then one must account for the tight binding nature of the inhibition (discussed further in Chapter 7). In this case Equation (6.1) is modified as follows ... 

Often high-affinity, or tight binding, interactions with enzymes is the result of a very slow dissociation rate of the enzyme-inhibitor binary complex. 

In this chapter we consider the situation where this assumption is no longer valid, because the affinity of the inhibitor for its target enzyme is so great that the value of K w approaches the total concentration of enzyme ( / T) in the assay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. 

Thus plots of IC50 as a function of [ TPP under conditions of Strauss and Goldstein s zone B allow one to simultaneously determine the values of Kfpp and [E T using Equations (7.13) and (7.15). Later in this chapter we will see other methods by which tight binding inhibitors can be used to provide accurate determinations of the total concentration of catalytically active enzyme in a sample. 

MORRISON S QUADRATIC EQUATION FOR FITTING CONCENTRATION-RESPONSE DATA FOR TIGHT BINDING INHIBITORS... 

A better method for analyzing concentration-response data for tight binding inhibitors was developed by Morrison and coworkers (Morrison, 1969 Williams and Morrison, 1979). This treatment is based on defining the Kt value of an inhibitor, or... 

Figure 7.4 Concentration-response plots for the data presented in Figure 7.1 fitted to Morrison s quadratic equation for tight binding inhibitors. The left panel shows the concentration-response behavior on a semilog scale, while the right panel shows the same data when the inhibitor concentration is plotted on a linear scale.

Murphy (2004) has reported an in-depth analysis of simulations for various assay conditions using Morrison s equation for tight binding inhibitors. From these studies several recommendations emerge for optimizing conditions for the determination... 

Figure 7.5 Concentration-response plot for a tight binding enzyme inhibitor, highlighting the three regions of the curve described by Murphy (2004).

Use of a Cubic Equation When Both Substrate and Inhibitor Are Tight Binding... 

DETERMINING MODALITY FOR TIGHT BINDING ENZYME INHIBITORS... 

Figure 7.6 Double reciprocal plot for a tight binding competitive enzyme inhibitor, demonstrating the curvature of such plots. The dashed lines represent an attempt to fit the data at lower substrate concentrations to linear equations. This highlights how double reciprocal plots for tight binding inhibitors can be misleading, especially when data are collected only over a limited range of substrate concentrations.

Determining Modality for Tight Binding Enzyme Inhibitors... 

Figure 7.7 Plot of IC50 as a function of substrate concentration (plotted as the ratio [S]/ATM on the x-axis) for tight binding competitive (closed circles) and tight binding uncompetitive (open circles) enzyme inhibitors.

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