Mechanism-based Covalent Inhibitors

Can we, nevertheless, use mechanism-based inhibitors to study the molecular interactions in active sites Unfortunately the simple answer appears to be negative on- and off-rates and structural rearrangements are difficult to interpret in terms of the energetics of specific interactions. Although detailed spectroscopic studies have begun to shed light on these complex mechanisms (e.g., [14, 15]), much more work will be required before all the enthalpic and entropic effects can be unscrambled. 

Knowledge of nothing more than the catalytic mechanism and the Pl residue can indeed be used directly to design thrombin inhibitors. All that is required is an arginine analogue with an electrophilic center in the correct position. The simplest of these is APPA (Fig. 7.3). 

The seminal work of Bode and Huber produced crystal structures of both ben-zamidine and APPA bound to trypsin [3ptb, Itpp] [16]. Thrombin has a very similar primary sequence to trypsin, with amino acid identities (similarities) of about 40% (55%), depending on species. The structures of the enzymes are also very similar, with 200 Ca positions superimposing with about 0.75 A rms deviation. 

The remaining 60 thrombin residues are in surface loops which are much shorter in trypsin, where there are only 30 corresponding residues. 

Given this similarity, it is no surprise that both benzamidine and APPA show lithe selectivity among thrombin, trypsin, and the large number of closely related enzymes. 

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Another nucleoside-derived mechanism-based enzyme inhibitor is Fluoronepla-nocin A [79]. This compound is of interest as a broad-spectrum antiviral drug which acts by irreversible inhibition of S-adenosylhomocystein hydrolase (SAH). In a first enzymatic reaction step the 3 -hydroxy group of the inhibitor is oxidized to the corresponding ketone (Scheme 4.34). This leads to depletion of the biochemical oxidizer nicotinamide adenine dinudeotide (NAD ). In the next step a nucleophilic residue of the enzyme undergoes Michael addition to the /i-fluoro a,/>-unsatu-rated ketone moiety. This is followed by fluoride elimination and thus the inhibitor stays covalently trapped in the active site and disables the enzyme permanently. 

Some of the more reactive irreversible inhibitors have been variously called suicide substrates, koa inhibitors, and mechanism-based inhibitors. These compounds are relatively innocuous substrate analogs until converted by the enzyme to highly reactive products capable of covalent attachment at the active site. Since the enzyme mechanism is involved, the covalently conjugated amino acid is often directly involved in catalysis. There has recently been increased interest in mechanism-based glycosidase inhibitors because of their value in studying the reaction mechanism (4, 48) and in their potential therapeutic application 47). 

One class of mechanism-based MAO inhibitors includes the unsaturated alkylamines (propargylamine analogs) (Table II). Although the kinetics of enzyme inactivation for these compounds are consistent with a mechanism-based inhibitor, in only a few cases has the chemical mechanism and site of protein modification been determined. Pargyline (iV-benzyl-N-methyl-2-propynylamine) is a classic example. Pargyline reacts stoichiometrically and irreversibly with the MAO of bovine kidney, with protection from inactivation afforded by substrate benzylamine (91). Furthermore, the reaction involves bleaching of the FAD cofactor at 455 nm and the formation of a new absorbing species at 410 nm and a covalent adduct of inactivator with flavin cofactor (92). 

Because the sulfone looks like the original antibiotic, penicillinase accepts it as a substrate, forming an ester, as it does with penicillin. If the ester were then hydrolyzed, penicillinase would be liberated and, therefore, would be free to react with penicillin. However, the electron-withdrawing sulfone provides an alternative pathway to hydrolysis that forms a stable imine. Because imines are susceptible to nucleophilic attack, an amino group at the active site of penicillinase reacts with the imine, forming a second covalent bond between the enzyme and the inhibitor. The covalently attacked group inactivates penicillinase, thereby wiping out the resistance to penicillin. The sulfone is another example of a mechanism-based suicide inhibitor (Section 25.8). 

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 significant difference between pseudoirreversible inhibitors and mechanism-based inactivators is the reversibiUty of the inactivation. A complete evaluation of the mechanism involved would require evidence not only for the covalent enzyme-inhibitor complex, but also for its decomposition products and its rate of reactivation. It is often difficult to identify the active site amino acid residue covalently linked to the inhibitor because of the instabiUty of the complex. 

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

Mechanism-based inactivation results in formation of a covalent adduct between the active inhibitor and the enzyme, or between the active inhibitor and a substrate or cofactor molecule. If the mechanism involves covalent modification of the enzyme, then one should not be able to demonstrate a recovery of enzymatic activity after dialysis, gel filtration, ultrafiltration, or large dilution, as described in Chapters 5 to 7. Additionally, if the inactivation is covalent, denaturation of the enzyme should fail to release the inhibitory molecule into solution. If a radiolabeled version of the inactivator is available, one should be able to demonstrate irreversible association of radioactivity with the enzyme molecule even after denaturation and separation by gel filtration, and so on. In favorable cases one should likewise be able to demonstrate covalent association of the inhibitor with the enzyme by a combination of tryptic digestion and LC/MS methods. 

The partitioning of the activated inhibitor between direct covalent inactivation of the enzyme and release into solution is an important issue for mechanism-based inactivators. The partition ratio is of value as a quantitative measure of inactivation efficiency, as described above. This value is also important in assessing the suitability of a compound as a drug for clinical use. If the partition ratio is high, this means that a significant proportion of the activated inhibitor molecules is not sequestered as a covalent adduct with the target enzyme but instead is released into solution. Once released, the compound can diffuse away to covalently modify other proteins within the cell, tissue, or systemic circulation. This could then lead to the same types of potential clinical liabilities that were discussed earlier in this chapter in the context of affinity labels, and would therefore erode the potential therapeutic index for such a compound. 

Our second example of drugs that function as mechanism-based inactivators is the steroid 5a-reductase inhibitors finasteride and dutasteride. The mechanism of inactivation by these compounds is an interesting departure from the typical target enzyme covalent modification seen with most mechanism-based inactivators. 

Two laboratories have independently disclosed an interesting series of mechanism-based inhibitors. The dihydropyrrole 31, which appeared in a patent application [61], was reported to inhibit rat lung SSAO/VAP-1 with an IC50 = 500 nM. Recently, the Sayre team extended earlier work [74] and showed that these inhibitors, exemplified by 32, covalently bound to the enzyme with the cofactor in the reduced form [75]. Presumably, aromatization of the dihydropyrrole moiety accounts for the observed potencies. 

Suicide substrates and quiescent affinity labels, unlike the other types of inhibitors discussed in this chapter, form covalent bonds with active site nucleophiles and thereby irreversibly inactivate their target enzymes. A suicide substrate,191 also described by Silverman in a comprehensive review1101 as a mechanism-based inactivator, is a molecule that resembles its target enzyme s true substrate but contains a latent (relatively unreactive) electrophile. When the target enzyme attempts to turn over the... 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

Sometimes CYPs can also produce reactive metabolite species that, instead of undergoing the normal detoxification pathway, can act as irreversible CYP inhibitors, thus causing toxicity. Such reactive metabolites that cause CYP inactivation are called MBI and are described in Chapter 9. Mechanism-based enzyme inhibition is associated with irreversible or quasi-irreversible loss of enzyme function, requiring synthesis of new enzymes before activity is restored. The consequences of MBI could be auto-inhibition of the clearance of the inactivator itself or prolonged inhibition of the clearance of other drugs that are cleared by the same isozyme. There may also be serious immunotoxicological consequences if a reactive intermediate is covalently bound to the enzyme. Therefore, screening of new compounds for MBI is now a standard practice within the pharmaceutical industry. 

Aromatase inhibitors may be classified into two types. Type 1 aromatase inhibitors bind to the aromatase enzyme irreversibly, so they are called inactivators. In some cases they are dubbed mechanism-based or suicide inhibitors when they are metabolized by the enzyme into reactive intermediates that bind covalently to the active site. Type 1 aromatase inhibitors are usually steroidal in structure as represented by exemestane (1), formestane (13), and atamestane (14). Formestane (13) was launched by Ciba-Geigy in 1992. As formestane (13) is readily and extensively metabohzed when administered orally, it is used as a depot formulation for injection. 

Irreversible inhibitors combine or destroy a functional group on the enzyme so that it is no longer active. They often act by covalently modifying the enzyme. Thus a new enzyme needs to be synthesized. Examples of irreversible inhibitors include acetylsal-icyclic acid, which irreversibly inhibits cyclooxygenase in prostaglandin synthesis. Organophosphates (e.g., malathion, 8.10) irreversibly inhibit acetylcholinesterase. Suicide inhibitors (mechanism-based inactivators) are a special class of irreversible inhibitors. They are relatively unreactive until they bind to the active site of the enzyme, and then they inactivate the enzyme. 

Mechanism-based inhibitors (also known as suicide inhibitors or as kcat inhibitors) are actually substrates for their target enzymes. A reactive group is only revealed by enzyme action it is therefore not subject to hydrolysis until it has been revealed in the vicinity of the enzyme. The ability of the inhibitor then to inactivate the enzyme will depend upon relative rates of (a) covalent bond formation with the enzyme, (b) diffusion of the reactive entity away from the enzyme, and (c) hydrolysis. 

Irreversible inhibitors may be classified for convenience as active site directed inhibitors and suicide or irreversible mechanism based inhibitors (IMBIs). They bind to the enzyme by either strong non-covalent or strong covalent bonds. Inhibitors bound by strong non-covalent bonds will slowly dissociate, releasing the enzyme to carry out its normal function. However, whatever the type of binding, the enzyme will resume its normal function once the organism has synthesized a sufficient number of additional enzyme molecules to overcome the effect of the inhibitor. 

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