Protease inhibitors affinity labels

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 diazomethyl ketone functional group was first observed to be an affinity label by Buchanan and co-workers who showed that the antibiotic azaserine, an O-diazoacetyl derivative, 9 inhibited an enzyme in the biosynthesis of purine by alkylation of a cysteine residue. 10 The acid protease pepsin was then observed to be inhibited by peptidyl diazomethyl ketones in the presence of copper ions with the resulting esterification of an aspartate residue. 11 Two peptidyl diazomethyl ketones, Z-Phe-CHN2 and Z-Phe-Phe-CHN2, were found to irreversibly inactivate papain, a cysteine protease. 12 Since these reports, many peptidyl diazomethyl ketones have been prepared primarily as inhibitors of various cysteine proteases. 7 Peptidyl diazomethyl ketones are also synthetic intermediates and have been used to prepare chloromethyl ketones (Section 15.1.3), 13 bromomethyl ketones (Section 15.1.3), acyloxymethyl ketones, 14 and (i-peptides. 15 A few peptidyl diazoalkyl ketones have been reported. 16,17 ... 

Serine Protease Inhibitors. The two major catalytic residues of serine proteases are a serine residue and a histidine residue. Affinity labels have been developed that modify both of these residues. 

Reviews. A number of reviews are available on the general topic of affinity labeling or on specific types of inhibitors (30,42,43,46,49,50, 51). These should be consulted for a more complete discussion of irreversible inhibitors for proteases. 

Figure 4 Chemical tools for the study of y-secretase. Transition-state anaiog inhibitors inciude hydroxyi-containing moieties that interact with the catalytic aspartates of aspartyl proteases. Helical peptides mimic the APR transmembrane domain and interact with the substrate docking site on the protease. These potent inhibitors were converted into affinity labeling reagents that contain a chemicaiiy reactive bromoacetyi or photoreactive benzophenone for covalent attachment to the protein target and a biotin moiety to allow isolation and detection of the labeled protein. Both types of chemical probes interacted with the two presenilin subunits but at distinct locations, which suggests that both the active site and the docking site of y-secretase lie at the interface between these subunits.

The most interesting class of protease inhibitors in viral studies has been the chloromethyl ketone derivatives of amino acids (54j 55) These were designed as affinity labels of serine-type proteases, and react irreversibly with histidine and serine residues in the active sites of proteases. There is a basis for selectivity of the chloromethyl ketones phenylalanyl and lysyl derivatives were synthesized, which had specificity for chymotrypsin and trypsin, respectively (54) This specificity led to studies on inhibition of poliovirus protein cleavage, with positive results (25, 26). 

The principle of transition state affinity requires the development during enzyme catalysis of very powerful forces of attraction between the enzyme and the altered form of the substrate that is present in the transition state. > This principle has served as a basis for the synthesis of some very strong reversible enzyme inhibitors/" and may also in principle be useful in the design of affinity labeling reagents. The latter possibility remains to be explored, but seems already to be partly substantiated by a few serendipitous examples of inhibitors of glycosidases and proteases. 

Representative examples of halomethyl ketones which have been used as affinity labels for serine proteases are listed in the table. Several thiol proteases (papain, clostripain, cathepsin B, and cocoonase) are also listed since the mechanism of the inhibition is probably similar for the two classes of enzymes. The inhibitors chosen for the table were either the most readily available or among the more reactive for a particular enzyme. For more information the reader is referred to a recent comprehensive review of all the enzymes that have been studied with halomethyl ketone inhibitors. ... 

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