Active-site-directed irreversible inhibitors

Associated with the problem of active-site titration is the question of the location of the active site in the three-dimensional structure of the protein. As a prelude to this investigation, a study is needed to indicate which amino acid residues in the overall peptide sequence are in the active site. The active site is defined as the location of the enzyme catalysis thus, the substrate complexes at the active site prior to the catalytic process. Addition of a substrate will, therefore, protect the enzyme against reagents, such as inhibitors, which react at the active site. Of course, the active site may include amino acid residues from distant parts of the peptide chain for example, both serine-195 and histidine-57 are in the active site of a-chymo trypsin. 

Active-site directed inhibitors have reactivity with the enzyme greatly enhanced over that of non-specific inhibitors thus phenacyl iodide inhibits papain 50-fold faster than iodoacetamide whereas the active-site directed inhibitor 4-toluenesulphonylamidomethyl chloromethyl ketone reacts some 650-fold faster. The enhanced rate is due to complexation of the inhibitor with the enzyme, and indicates that the inhibitor must be reacting at the active site. 

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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. 

A class of DUBs only identified since 2002 is the OTU (ovarian tumor protein) DUB class. The OTU domain was originally identified in an ovarian tumor protein from Drosophila mdanogaster, and over 100 proteins from organisms ranging from bacteria to humans are annotated as having an OTU domain. The members of this protein superfamily were annotated as cysteine proteases, but no specific function had been demonstrated for any of these proteins. The first hint of a role for OTU proteins in the ubiquitin pathway was afforded by the observation that an OTU-domain-containing protein, HSPC263, reacted with ubiquitin vinyl sulfone (an active-site-directed irreversible inhibitor of DUBs) [41]. 

Active-site-directed Irreversible Inhibitors and Substrates... 

The most promising tools developed for this sort of analysis are active-site-directed irreversible inhibitors of DUBs. These inhibitors are ubiquitin or ubiquitin-like proteins chemically modified at the C-terminus by an electrophilic moiety such as a Michael acceptor or alkyl halide. The modified ubiquitin can be incubated with a purified DUB or a cell lysate containing DUB activity. Ubiquitin vinyl sul-fone (UbVS) is one such irreversible inhibitor because the vinyl sulfone moiety reacts with the active-site cysteine of the DUB, forming a thioether linkage. The covalent adduct is stable and can be detected in a variety of ways. Labeling of DUBs is specific, as only a DUB active-site cysteine will efficiently react with the vinyl sulfone moiety. 

The weight (or more correctly, the mass) of a protein expressed in grams per mole of active sites. Not all oligomeric proteins, even some with identical subunits, have a number of active sites equal to the number of subunits. Enzyme normality (Le., the concentration of enzyme active sites) is typically determined by active site titration with an active-site-directed irreversible inhibitor. This is... 

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. 

Since guanine aminohydrolase catalyzes the deamination of thioguanine and 8-azaguanine thereby destroying their anti-neoplastic effects, Baker and his colleagues have prepared a series of active site directed irreversible inhibitors to block the enzyme in tumor tissue (193). The most effective inhibitor, 9-(4-methoxy phenyl)guanine, effected a 50 inhibition at 0.38 nM in the presence of 13.3 juM substrate (194). 

Holmes, M. A., D. E. Tronrud, and B. W. Matthews, Structural analysis of the inhibition of thermolysin by an active-site-directed irreversible inhibitor. Biochem. 22 236, 1983. 

Active site directed inhibitors are compounds that bind at or near to the active site of the enzyme. These inhibitors usually form strong covalent bonds with either the functional groups that are found at the active site or close to that site. Since these groups are usually nucleophiles, the incorporation of electrophilic groups in the structure of a substrate can be used to develop new inhibitors (Table 7.3). This approach may also be used to enhance the action of a known inhibitor. Most of the active site directed irreversible inhibitors in clinical use were not developed from a substrate. They were obtained or developed by other routes and only later was their mode of action discovered. For example, aspirin,... 

One of the first interesting structures with cholinergic properties isolated from a marine source is the chemical onchidal. This compound was first isolated from the mollusc Onchidella binneyi and has an acetate ester similar to acetylcholine. Upon isolation, onchidal was discovered to be an active site-directed irreversible inhibitor of AChE (Abramson et ah, 1989). 

The natural toxins onchidal and fasciculins behave as anti-ChE agents. Onchidal is an active site-directed irreversible inhibitor of AChE, and fasciculins are proteinic AChE inhibitors which bind to a peripheral regulatory anionic site of AChE in a noncompetitive and irreversible manner. 

Covalent bonds are not as important in drug-receptor binding as noncovalent interactions. Alkylating agents in chemotherapy tend to react and form an immonium ion, which then alkylates proteins, preventing their normal participation in cell divisions. Baker s concept of active site directed irreversible inhibitors was well established by covalent formation of Baker s antifolate and dihydrofolate reductase (46). 

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-... 

Figure 17.26. Pseudo first-order inactivation kinetics of an active-site directed irreversible inhibitor.

The above results were confirmed subsequently by Holmes and Stevenson by determining the amino acid sequence and establishing the position of the C-labelled histidine in the peptide B obtained from E3. A reliable amino acid sequence 162 has been obtained for peptide B up to residue 30 which was in agreement with the amino acid sequence determined from the gene sequence of E3. The authors demonstrated that a histidine residue is selectively alkylated within the active site of E. coli E3 when the enzyme is a component of the PD complex. The reagent 161 functions as a unique form of an active-site-directed irreversible inhibitor. 

Baker, B. R. Factors in the design of active-site-directed irreversible inhibitors. 

Finally, it may be worthwhile to point out that one of the most successful chemotherapeutic drugs provided by nature may be considered as a non-classical active-site-directed irreversible inhibitor This drug is penicillin (70). As a non-classical antimetabolite of D-alanyl-D-alanine, it first forms a reversible complex with the enzyme peptidoglycan transpeptidase, and then by a ringopening reaction of its p-lactam moiety, it forms a covalently linked penicil-loyl-enzyme complex97. However, the reaction involves the acylation of a sulfhydryl group in the active site of the enzyme in this respect, 70 resembles the classical-type endoalkylating antimetabolites, azaserine and DON (8 and 9, see Section 2.2.). Some of the more recently discovered antibiotics and natural products from plants with antitumor activity (e.g., camptothecin) are... 

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