Affinity label

at concentrations of inactivator well below Kh a plot of kohs as a function of [/] will be linear, and the slope of the line will be equal to k JK This is exactly the case that we encountered above for nonspecific affinity labeling. 

for any irreversible enzyme inactivator, we can quantify the effectiveness of inactivation using the second-order rate constant kanJKi. This constant thus becomes the key metric that the medicinal chemist can use in exploring the SAR of enzyme inactivation by a series of compounds. In terms of individual rate constants, the definitions of both nact and A) depend on the details of the mechanisms of inactivation, as will be described below. 

An important point to realize here is that attempts to quantify the relative potency of irreversible enzyme inactivators by more traditional parameters, such as IC50 values, are entirely inappropriate because these values will vary with time, in different ways for different compounds. Hence the SAR derived from IC50 values, determined at a fixed time point in the reaction progress curve, is meaningless and can be misleading in terms of compound optimization. Unfortunately, the literature is rife with examples of this type of inappropriate quantitation of irreversible inactivator potency, making meaningful comparisons with literature data difficult, at best. 

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

This calculated rate acceleration is conservative since the ratio of 5 M presented in eq. (4.8) assumes no significant orientation effects. Rate ratios as high as 10 M have been found for a variety of intramolecular reactions. A careful study of a series of affinity labels for trypsin has revealed that the first order rate constants for reagents with comparable reactive groups vary by at least a factor of 5. The differences in these first order rate constants must reflect the difference in the 

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Affinity labeling Affinity labels Affinity values Affirm Aflas... 

Herbicidal Inhibition of Enzymes. The Hst of known en2yme inhibitors contains five principal categories group-specific reagents substrate or ground-state analogues, ie, rapidly reversible inhibitors affinity and photo-affinity labels suicide substrate, or inhibitors and transition-state, or reaction-intermediate, analogues, ie, slowly reversible inhibitors (106). 

Affinity Labels. Active site-directed, irreversible inhibitors or affinity labels are usually substrate analogues that contain a reactive electrophilic functional group. In the first step, they bind to the active site of the target enzyme in a reversible fashion. Subsequentiy, an active site nucleophile in close proximity reacts with the electrophilic group on the substrate to form a covalent bond between the enzyme and the inhibitor, typically via S 2 alkylation or acylation. Affinity labels do not require activation by the catalysis of the enzyme, as in the case of a mechanism-based inhibitor. 

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). 

A good example of an affinity label for creatine kinase has been presented (35). This enzyme catalyzes the reversible transfer of a phosphoryl group from adenosine triphosphate [56-65-5] (17) to creatine [57-00-1] (18), leading to adenosine diphosphate [7584-99-8] (19) and phosphocreatine [67-07-2]... 

A/-(2,3-Epoxypropyl)-A/-amidinoglycine [70363-44-9] (21) was shown to be an affinity label of creatine kinase. Its mechanism of covalent bond formation is outlined as follows ... 

Berg R. H., G. W. Erdos, M. Gritzali and R. D. Brown. (1988). Enzyme-gold affinity labeling of cellulose. Journal of Electron Microscopy Techniques 8 371-379. 

The third mechanism that results in slow binding behavior is covalent inactivation of the enzyme by affinity labeling or mechanism-based inhibition (Scheme... 

Figure 8.2 Mechanisms of irreversible enzyme inactivation. (A) Nonspecific affinity labeling, (B) quiescent affinity labeling, and (C) mechanism-based inactivation.

Depending on the mechanism of irreversible reaction, inactivation can appear to proceed through either a single-step or a two-step mechanism (Figure 8.2). In the case of nonspecific affinity labels (see Section 8.2) many amino acid residues on the enzyme molecule, and on other protein molecules in the sample, can be covalently modified by the affinity label. Not every modification event will lead to inactiva-... 

However, the conversion of omeprazole to the active sulphenamide does not result in formation of a reversible enzyme inhibitor, but rather results in in situ formation of a powerful affinity label. Hence we can consider omeprazole to be a unique example of quiescent affinity labeling in which selectivity results from the unique environment of the target enzyme. 

More recently attempts to generate highly selective quiescent affinity labels have been made for a number of protease and kinase targets. As examples, inhibitors of the Rhinovirus 3C protease (Mathews et al 1999) and of the epidermal growth factor receptors (Boschelli, 2002), both incorporating Michael acceptors to covalently inactivate cysteine residues in their target enzymes (Lowry and Richardson, 1981 Figure 8.6), have entered human clinical trials for the treatment of rhinovirus infection and cancer, respectively. 

The lack of target enzyme specificity is a critical liability for the use of affinity labels as drugs. The inherent chemical reactivity of these compounds almost ensures that... 

Table 8.1 Some examples of quiescent affinity labels of clinical interest...

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