Enzyme inhibitors affinity labels

Active-Site-Directed and Enzyme-Activated Irreversible Inhibitors Affinity Labels and Suicide Inhibitors ... 

Although the organic modifiers are usually not specific for a given enzyme, the second group, the affinity labels, have a degree of specificity built in. Sometimes described as active-site directed, irreversible inhibitors, affinity labels are usually substrate or product analogs that contain an additional chemically reactive moiety. They first bind to the en-... 

L Irreversible inactivation. Inactivation by affinity labels leads to irreversible covalent bond formation between the enzyme and the inhibitor. Unlike the complex between and enzyme and a rapid, reversible inhibitor, the covalent enzyme-inhibitor complex is no longer in equilibrium with free enzyme and inhibitor. Therefore, exhaustive dialysis or gel filtration of the covalent enzyme-inhibitor complex cannot lead to the recovery of free, active enzyme. However, such experiments do not allow distinction among tight-binding, noncovalent inhibitors, affinity labels, and mechanism-based inactivators. 

During irreversible inhibition, after initial binding of the inhibitor to the enzyme, covalent bonds are formed between a functional group on the enzyme and the inhibitor. This is the case, for example, for the active-site-directed inhibitors (affinity labelling). 

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). 

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 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. 

Suicide substrates and quiescent affinity labels, unlike the other types of inhibitors discussed in this chapter, form covalent bonds with active site nucleophiles and thereby irreversibly inactivate their target enzymes. A suicide substrate,191 also described by Silverman in a comprehensive review1101 as a mechanism-based inactivator, is a molecule that resembles its target enzyme s true substrate but contains a latent (relatively unreactive) electrophile. When the target enzyme attempts to turn over the... 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

More specific evidence came from affinity labeling with molecules which could react with specific amino acid group sat or adjacent to the substrate site. These labels were substrate analogues and competitive inhibitors. Substituted aryl alkyl ketones were used. TV-p-toluene-sulphonyl-L-phenylalanine chloromethyl ketone (TPCK) blocked the activity of chymotrypsin. Subsequent sequence analysis identified histidine 57 as its site of binding (see Hess, 1971, p 213, The Enzymes, 3rd ed.). Trypsin, with its preference for basic rather than aromatic residues adjacent to the peptide bond, was not blocked by TPCK but was susceptible to iV-p-toluenesulphonyl-L-lysine chloromethyl ketone (TLCK) (Keil, ibid, p249). 

General aspects of enzymatic reactions cateuLyzed by kinases are briefly mentioned. Many alternate substrates, competitive inhibitors and affinity labels based either on the structure of ATP or on the structure of the non-ATP kinase substrates are described. Several examples are presented that should be of particular interest to the medicinal chemist. Finally, the design of an affinity label for creatine kinase is reviewed as an example of how such information can be used in the search for agents directed at an enzyme s active site. 

Designing specific enzyme inhibitors on a rational basis when one does not have a detailed three-dimensional crystal structure to which to relate is a rather sophisticated challenge. Some viable approaches to such a challenge are discussed in a review chapter by Santi and Kenyon (91) This discussion will focus on our rationale for the design of an affinity label for creatine kinase, namely N-(2,3-epoxypropyl)-N-amidinoglycine (epoxycreatine ) ... 

Evidence that Epoxycreatine is an Affinity Label. All the available evidence is consistent with the hypothesis that epoxycreatine behaves like an affinity label for the enzyme and that it is attacked by a carboxylate group of the enzyme. That is, it inactivated the enzyme rapidly at 0°C. Inactivation was complete and activity did not return upon exhaustive dialysis. Creatine was shown to give protection against the inactivation in the expected manner. Most importantly, though, the irreversible binding of the Inhibitor was shown to be stoichiometric using [ Cj-epoxycrea-tine that is, one and only one inhibitor molecule becomes bound per active site, even in the presence of excess inhibitor. 

In consideration of the close resemblance of the inhibitor to a natural substrate for the enzyme, and of the enzymic process of inhibition, the reaction of the enzyme with TPCK has been described as affinity labelling. (Schoellman and Shaw, 1962, 1963). Many other examples of affinity labelling have subsequently been discovered. 

In the preceding section, four diagnostic tests of affinity labeling were listed (inactivation inhibited by substrates, pH dependence of inactivation similar to that of catalysis, labeled inhibitor covalently bound in 1 1 stoichiometry, and saturation kinetics obeyed). The same criteria may be used to diagnose suicide inhibition. In addition, tests must be made to detect any diffusion of the activated intermediate I into solution. For example, the addition of —SH reagents that rapidly react with electrophiles and hence scavenge them should not slow down the rate of reaction. The suicide inhibitor should not, in any case, react with the thiol at an appreciable rate in the absence of enzyme. 

An affinity label, or active-site-directed irreversible inhibitor, is a chemically reactive compound that is designed to resemble a substrate of an enzyme, so that it binds specifically to the active site and forms covalent bonds with the protein residues.1-3 Affinity labels are very useful for identifying catalytically important residues and determining their pKa values from the pH dependence of the rate of modification. 

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