Affinity labeling, selection

To obtain affinity labels selective for a single receptor type, Portoghese incorporated less reactive electrophiles at the 6j3 position leading to the preparation of P-FNA (186) (604). This compound illustrates how the second step in the mechanism of affinity labels can enhance selectivity whereas jS-FNA interacts reversibly with k receptors, where it is an agonist, it is an irreversible antagonist at fji... 

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. 

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. 

Table 8.3 Examples of amino acid selective affinity labels...

Bergstrom, M.. Fasth, K.J., Kilpatrick, G., et al . Brain uptake and receptor binding of two [11C] labeled selective high affinity NKl-antagonists, GR203040 and GR205171 - PET studies in rhesus monkey. Neuropharmacology 39(4), 664-670, 2000. 

Kettner C, Shaw E. d-PHE-PRO-ARGCH2C1-1 selective affinity label for thrombin. Thrombosis Research 1979 14 969-973. 

The photoaffinity labels have been useful in mapping out residues at the active sites of enzymes and the binding sites of proteins such as antibodies. The normal affinity labels are more useful for kinetic work, since they are selective for the basic and nucleophilic groups that are so prevalent in catalysis, and since they also give information on the pATfl s of the groups that are modified. 

Publications on the synthesis of aryl azides as photoactivatable reagents selected for their experimental procedures are Fleet et al. (1972) Galardy et al. (1974) Katzenellenbogen et al. (1973) Schwyzer and Calviezel (1971) Bridges and Knowles (1974) Huang and Richards (1977). The reader should also consult the detailed description of Smith and Boyer (1963) and the Methods in Enzymology volume on Affinity Labeling... 

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