Active site-directed enzyme inhibitors

VI. Conformationally Restricted Active-Site-Directed Enzyme Inhibitors... 

VI. CONFORMATIONALLY RESTRICTED ACTIVE-SITE-DIRECTED ENZYME INHIBITORS... 

W-R Shieh. Part I. Studies on the active site-directed enzyme inhibitors of soybean lipoxygenase. Part n. Purification and studies on the [i-keto ester reductases from baker s yeast. Ph.D thesis. University of Wisconsin, Madison, WI, 1987. 

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. 

In order to give useful information about an enzyme, a conformationally restricted active-site-directed analog inhibitor need not bind to the enzyme irreversibly. In a study of the enzyme fructose 1,6-diphosphatase from rabbit liver, Benkovic et al, have investigated the question of the reactive form of the fructose 1,6-diphosphate in the enzymatic process (104,105). Three likely forms are shown in structures 50, 51 and 52. 

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). 

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,... 

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. 

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... 

Comprehensive reviews (Kl, Ul) of the active sites of cholinesterase both postulated the presence not only of an esteratic site for butyrylcholinesterase but also of an anionic site. Additionally, in the region of the anionic site, there are two hydrophobic areas, one directly surrounding the anionic group and the second located at some distance from it (Kl). The presence of hydrophobic areas has been established (B32, C3, H29, H45, MIO) by the use of fluorescent probes with spectral responses which reflect the environment of the probe. Such probes can be used to monitor changes in the conformations of enzymes and can be designed to be active-site-directed, competitive inhibitors (H30). Aspects of the spectroscopy of intrinsic and extrinsic fluorescent probes have been reported (C3). 

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. 

Baker has developed an approach to selective cytotoxicity based on active-site-directed irreversible inhibitors that combine reversibly with the target enzyme and then inactivate it by exo-alkylation. Using this approach, he has prepared compounds that in vitro selectively inactivate folate reductase from leukemia L1210, but not the enzyme from mouse liver, spleen, and intestine. to vivo, however, the only compound that was effective in prolonging the life of mice with L1210 leukemia did not show selectivity for the reductase from leukemic cells. Hansch has applied regression analysis to a series of pyrimidine and triazine inhibitors of folate reductase. ... 

In a broad sense an enzyme is specifically inhibited when its active site is blocked physically and/or chemically without significant alteration of the rest of the molecule. For this, many types of covalent inhibitors have been developed. The desired goal is chemical modification of an active site amino acid residue of the enzyme and subsequent loss of catalytic activity. The most common approach has been the synthesis of structurally and chemically reactive analogues of a substrate of the target enzyme. Such inhibitors have been referred to as active-site-directed irreversible inhibitors or affinity labels (312,313). Generally the affinity label has a reactive electrophilic substituent that can generate a stable covalent bond with an active site nucleophilic... 

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. 

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). 

If k2 > kj, the glycosyl-enzyme intermediate will accumulate, and may be trapped by the rapid denaturation of the enzyme in the presence of (saturating) amounts of substrate. With -glucoside Aj from Asp. wentii and 4-nitrophenyl [ C]-2-deoxy-) -D-irra />jo-hexopyranoside, it was possible to identify the intermediate as a glycosyl ester (acylal) of 2-deoxy-D-arabino-hexose bound to the same aspartate residue that had previously been labeled with the active-site-directed inhibitor conduritol B epoxide and with D-glucal." This constituted an important proof that the carboxylate reacting with the epoxide is directly involved in catalysis. 

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