Affinity of inhibitors

Successful lead optimization can drive the affinity of inhibitors for their target enzymes so high that the equilibrium assumptions used to derive the equations for calculating enzyme-inhibitor K, values no longer hold. 

Figure 2. TCP cycle for calculating binding affinities of inhibitors to HIV-1 protease...

While the effect of primary mutations on reduced binding affinities of inhibitors can be at least partially explained in view of the accumulated structural data, the function of secondary, or compensatory mutations in the resistant HIV PR is difficult to rationalize as yet. The predominant compensatory mutations observed in the resistant variants involve residues Leu63/63, 1 71/7 , Met46/46, ... 

Although it is not known whether the base is an essential feature of a substrate, the nucleoside moiety is necessary since bis- and tris-p-nitrophenyl phosphate esters are not hydrolyzed (61). The nature of the R substitution on the 3 -OH clearly affects the affinity of the substrate or inhibitor for the enzyme, even though it does not affect the maximal catalytic rate constant (Table I). The importance of the 5 - and 3 -phosphate groups in determining the affinity of inhibitors (3, 66) is consistent with the contribution of these groups to substrate affinity (61). These effects result from the phosphoryl groups themselves rather than... 

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

The IC50 value is that concentration of a drag that reduces the activity (or binding) of another drag to an enzyme by 50%. Under certain conditions it can used to express the affinity of the enzyme inhibitor. 

A general mechanism of resistance is reducing the affinity of the antiretroviral compound for its mutant target protein. Resistance mutations associated with reduced affinity are observed during treatment failure with a fusion inhibitor, nonnucleoside reverse transcriptase inhibitors (NNRTl), integrase inhibitor, and protease inhibitors as reviewed in Chaps. 3,4, 6, and 7 (Hazuda et al. 2007 Hsiou et al. 2001 King et al. 2002 Mink et al. 2005). 

As glycon affinity of glycosidases is generally low for monosaccharides (see Tables III and IV), problems with unspecific labeling may arise with glycon-derived inhibitors of high intrinsic reactivity which do not have additional features to provide enhanced affinity, for example, a suitably positioned... 

A competitive inhibitor and substrate exert reciprocal effects on the concentration of the EI and ES complexes. Since binding substrate removes free enzyme available to combine with inhibitor, increasing the [S] decreases the concentration of the EI complex and raises the reaction velocity. The extent to which [S] must be increased to completely overcome the inhibition depends upon the concentration of inhibitor present, its affinity for the enzyme K-, and the of the enzyme for its substrate. 

For simple noncompetitive inhibition, E and EI possess identical affinity for substrate, and the EIS complex generates product at a negligible rate (Figure 8-10). More complex noncompetitive inhibition occurs when binding of the inhibitor does affect the apparent affinity of the enzyme for substrate, causing the tines to intercept in either the third or fourth quadrants of a double reciprocal plot (not shown). 

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