Enzyme inhibitors active-site-directed irreversible

Muehlbacher M, Poulter CD. Isopentenyl-diphosphate isomerase Inactivation of the enzyme with active-site-directed irreversible inhibitors and transition-state analogues. Biochemistry-USA, 1988 27 7315-7328. 

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. 

Information relevant to the mechanism of an enzyme-catalyzed reaction can, in general, only be obtained from irreversible inhibitors which react specifically at the active site and thereby inactivate the enzyme. As active-site-directed inhibition is treated in detail in Ref. 142 general aspects will be discussed here only briefly. In order to be suitable as an active-site-directed inhibitor, a compound must fulfil the following requirements. 

B. R. Baker, Design of Active-Site-Directed Irreversible Enzyme Inhibitors-, The Organic Chemistry of the Enzymic Active Site, John Wiley, Inc. New York, 1967... 

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... 

Active-site-directed irreversible enzyme inhibitor,... 

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). 

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,... 

B R Baker, Design of Active-Site-Directed Irreversible Enzyme Inhibitors, Wiley, New York (1967), L A Cohen, Ann Rev Biochem 37, 695, (1968), J F Moran and D J Tnggle, in Fundamental Concepts in Drug-Receptor Interactions (J F Danielli, J F Moran, and D J Tnggle, eds ), Academic Press, New York (1970)... 

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 17.28. Usinga substrate to protect an enzyme from inactivation 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. Design of Active-Site-Directed Irreversible Enzyme Inhibitors Wiley New York, 1967. 

Baker, B. R. Design of active-site-directed irreversible enzyme inhibitors. New... 

Active-Site-Directed Irreversible Enzyme 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... 

B. R. Baker, "Design of Active-Site Directed Irreversible Enzyme Inhibitors", Wiley,... 

Irreversible antagonists or inhibitors active site-directed alkylating agents, suicide enzyme inhibitors Types of proteases (4) transition state (or mechanism-based) inhibitors. 

Similar strategies have been used to develop inhibitors that target the C-18 hydroxylation involved in the biosynthesis of aldosterone . Thus, aldosterone analogs with C-18 iodomethyl, chloromethyl, allyl, propargyl, vinyl, and methyl-thiomethyl functionalities have been synthesized and some have been found to irreversibly inactivate the enzyme. An active site-directed 18-acetylenic deoxycorticosterone [21 -hydroxy-13(-2-)propy-... 

As previously described, irreversible enzyme inhibition is defined as time-dependent inactivation of the enzyme, which implies that the enzyme has, in some way or form, been permanently modified, because it can no longer carry out its function. This modification is the result of a covalent bond being formed with the inhibitor and some amino acid residue in the protein. Furthermore, this bond is extremely stable and, for all practical purposes, is not hydrolyzed fo give back the enzyme in its original state or structure. In most examples of irreversible inhibition, a new enzyme must be generated through gene transcription and translation for the enzyme to continue its normal catalytic action. Basically, there are two types of irreversible enzyme inhibitors, the affinity labels or active site-directed irreversible inhibitors and the mechanism-based irreversible enzyme inactivators. 

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