Enzyme suicide inhibitors

NH2). The acid crystallises readily when 4g in 50mL H2O is treated with abs EtOH at 4°/ 3hrs, and is collected washed with cold abs EtOH and Et20 and dried in vac. Also recrystallises from aqueous Me2CO, Rp on Si02 TLC plates with n-BuOH-H20-AcOH (4 1 1) is 0.26. The racemate has m 238-240°. [Leukart et al. Helv Chim Acta 59 2181 1976 Eberle and Zeller Helv Chim Acta 68 1880 1985 Jansen et al. Reel Trav Chim Pays-Bas 88 819 7969.] It is a suicide inhibitor of y-cystathionase and other enzymes [Washtier and Abeles Biochemistry 16 2485 7977 Shinozuka et al. Eur J Biochem 124 377 7982]. 

A wide variety of a-tnfluoromethyl a-amino acids are readily available from the reaction of 5-fluoro-4-tnfluoromethyl-l,3 azoles with allylic alcohols [138, 139] a-Tnfluoromethyl-subsumted a-amino acids show anubactenal and antihy pertensive activity Some are highly specific enzyme inhibitors (suicide inhibitors) and may be important as bioregulators [140] Furthermore, they are interesting candidates for peptide modification... 

CrATP, a suicide inhibitor of Ca -ATPase [178], that arrests the enzyme in a Ca occluded E[ P state, also produced E -type crystals very similar to those obtained with lanthanides [119]. These observations further support the assignment of the PI-type crystals to the Ei and E P conformation of the Ca -ATPase. 

The starting point for much of the work described in this article is the idea that quinone methides (QMs) are the electrophilic species that are generated from ortho-hydro-xybenzyl halides during the relatively selective modification of tryptophan residues in proteins. Therefore, a series of suicide substrates (a subtype of mechanism-based inhibitors) that produce quinone or quinonimine methides (QIMs) have been designed to inhibit enzymes. The concept of mechanism-based inhibitors was very appealing and has been widely applied. The present review will be focused on the inhibition of mammalian serine proteases and bacterial serine (3-lactamases by suicide inhibitors. These very different classes of enzymes have however an analogous step in their catalytic mechanism, the formation of an acyl-enzyme intermediate. Several studies have examined the possible use of quinone or quinonimine methides as the latent... 

Unlike catecholamines and indoleamines, histamine itself is not a direct inhibitor of its biosynthetic enzyme, but it exerts feedback control through the H3 autoreceptor. Perhaps the most powerful tool in the study of the histamine system is S-a-fluoromethylhistidine, a highly selective and potent suicide inhibitor of HDC [22]. This compound has been used successfully to study many of the functions of histamine in brain. 

Mechanistic investigations have shown that these compounds behave as suicide inhibitors (preferably called mechanism-based inactivators) in the sense that they are recognized by /3-lactamases as substrates, but the great stability of the acyl-enzyme intermediate blocks turnover of the enzyme [46] [47]. /3-Lactamase inhibitors can be divided into two classes, class I and class II class-I inhibitors (e.g., clavulanic acid (5.12)), in contrast to those of class II (e.g., olivanic acid (5.15)), have a heteroatom at position 1 that can lead to ring opening at C(5). The mechanistic consequences of this difference in structure are illustrated by the general scheme in Fig. 5.3. 

In the earlier scheme, I represents a product formed by metabolism of the inhibitor by the enzyme. This product may be released into bulk solvent, or may interact (often covalently) with a suitably reactive component of the enzyme within the active site. This irreversibly inactivated enzyme complex is shown as El". There are two kinetic constants that can be obtained from relatively straightforward experiments with a suicide inhibitor. The Ki value is an equilibrium constant for the initial reversible step, and all the rate constants from the above scheme contribute to its value. The rate of irreversible inactivation of enzyme at a saturating concentration of the suicide inhibitor is given by fcinact. to which only k2> h, and k contribute (Silverman, 1995). At infinitely high concentrations of the inhibitor, the half-Ufe for inactivation is equal to ln2/ l inact ... 

A number of methods can assist in identifying and characterizing enol intermediates (as well as eneamine and carbanion intermediates) in enzyme-catalyzed reactions. These include (1) proton isotope exchange (2) oxidation of the intermediate (3) coupled elimination (4) spectrophotometric methods (5) use of transition-state inhibitors (6) use of suicide inhibitors (7) isolation of the enol and (8) destructive analysis. 

The synthesis of suicide inhibitors and the kinetics of enzyme inactivation have greatly advanced the study of enzyme mechanisms as well as the design of drugs and antimetabolites. 

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. 

Efforts to overcome the actions of the p-lactamases have led to the development of such p-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam. They are called suicide inhibitors because they permanently bind when they inactivate p-lactamases. Among the p-lactamase inhibitors, only clavulanic acid is available for oral use. Chemical inhibition of p-lactamases, however, is not a permanent solution to antibiotic resistance, since some p-lactamases are resistant to clavulanic acid, tazobactam, or sulbactam. Enzymes resistant to clavulanic acid include the cephalosporinases produced by Citrobacter spp., Enterobacter spp., and Pseudomonas aeruginosa. 

Linear furanocoumarins (psoralens) inhibit P450s as mechanism-based inactivators (suicide inhibitors). Thus, species that produce psoralens may have evolved C4H enzymes with enhanced tolerance to these compounds. Recombinant C4H from the psoralen-producing species R. graveolens showed markedly slower inhibition kinetics with psoralens, and possibly biologically significant tolerance, compared to C4H from a species that does not produce the compounds (H. tuberosus) ... 

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. 

Furans can also undergo covalent binding to macromolecules [20]. Furans, such as 8-methoxypsoralen, are epoxidized by Cyt P450 to yield a highly electrophilic species, which is so reactive that it covalently modifies an amino acid residue of CYP itself and thus inactivates the enzyme irreversibly (hence it is also in the class of suicide inhibitors) [21]. 

Reactive metabolites of xenobiotics may differ in reactivity, and therefore have varying impact on enzymatic activities in terms of proximity to their origin. For example, some intermediates are highly reactive and directly inhibit the enzyme that leads to their formation. These substances are commonly referred to as suicide inhibitors, for obvious reasons. Some suicide inhibitors, such as piperonyl butoxide (PBO), a pesticide synergist) are common inhibitors of certain CYP isozymes. PBO amplifies the toxicity of certain insecticides by inhibiting the insect s CYP enzymes that are involved in its degradation. It is metabolized to a highly reactive carbene, which forms an inhibitor complex with the heme iron of CYP, as shown in Scheme 3.6. 

Metabolites that are less reactive than suicide inhibitors may impact more distant enzymes, within the same cell, adjacent cells, or even in other tissues and organs, far removed from the original site of primary metabolism. For example, organopho-sphates (OPs), an ingredient in many pesticides, are metabolized by hepatic CYPs to intermediates, which, when transported to the nervous system, inhibit esterases that are critical for neural function. Acetylcholinesterase (AChE) catalyzes the hydrolysis of the ester bond in the neurotransmitter, acetylcholine, allowing choline to be recycled by the presynaptic neurons. If AChE is not effectively hydrolyzed by AChE in this manner, it builds up in the synapse and causes hyperexcitation of the postsynaptic receptors. The metabolites of certain insecticides, such as the phos-phorothionates (e.g., parathion and malathion) inhibit AChE-mediated hydrolysis. Phosphorothionates contain a sulfur atom that is double-bonded to the central phosphorus. However, in a CYP-catalyzed desulfuration reaction, the S atom is... 

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. 

True enough, treatment of PAP with FMPP resulted in a time-dependent inactivation of the enzyme. Competitive inhibitors of PAP protected against inactivation. The authors suggest that FMPP represents a useful basic structure which can be incorporated into the design of more specific phosphatase inhibitors for example, the modified tyrosine 77 could be incorporated into a particular peptide to give a suicide substrate that is selective for a protein phosphatase which preferentially hydrolyses that peptide. 

In the preceding section, four diagnostic tests of affinity labeling were listed (inactivation inhibited by substrates, pH dependence of inactivation similar to that of catalysis, labeled inhibitor covalently bound in 1 1 stoichiometry, and saturation kinetics obeyed). The same criteria may be used to diagnose suicide inhibition. In addition, tests must be made to detect any diffusion of the activated intermediate I into solution. For example, the addition of —SH reagents that rapidly react with electrophiles and hence scavenge them should not slow down the rate of reaction. The suicide inhibitor should not, in any case, react with the thiol at an appreciable rate in the absence of enzyme. 

Most suicide inhibitors are based on the generation of an intermediate that has conjugated double bonds and that is susceptible to a Michael addition reaction. A nucleophilic group on the enzyme may then be alkylated by the intermediate (equation 9.7). The conjugated intermediate is usually generated by proton-abstraction by a basic group on the enzyme. 

Often, the basic group that is responsible for the proton abstraction is also the nucleophilic group in the Michael addition. Thus, most of the suicide inhibitors made so far have been aimed at enzymes that catalyze the formation of carban-ions or carbanion-like intermediates. Suicide inhibitors are typically based on acetylenic compounds (as in equation 9.8), /3, y-unsaturated compounds (as in equation 9.9), or /3-halo compounds (as in equation 9.10). (The a protons in such compounds are acidic because the negative charge in the carbanion is delocalized by the conjugation with X.)... 

It should be noted that there is a kinetic isotope effect on the normal reaction (9.11) when the a-deuterated compound is used as the substrate. A similar effect is found when the deuterated suicide inhibitor is used. Thus, both reactions involve a proton transfer in the rate-determining step of the reaction. It has also been shown that a sample of the allenic intermediate that is prepared chemically does in fact irreversibly inhibit the enzyme.18... 

One drug that is used to treat both Parkinson s disease and depression is (-)deprenyl. It, too, is an acetylenic suicide inhibitor that inhibits the enzyme by binding covalently to the flavin.27... 

Active-Site-Directed and Enzyme-Activated Irreversible Inhibitors Affinity Labels and Suicide Inhibitors ... 

These reactions, which have provided a means of inhibiting the flavin-linked monoamine oxidases, enable us to end on a clinical note. The monoamine oxidases are responsible for the deamination of monoamines such as adrenaline, noradrenaline, dopamine, and serotonin, which act as neurotransmitters. Imbalances in the levels of monoamines cause various psychiatric and neurological disorders Parkinson s disease is associated with lowered levels of dopamine, and low levels of other monoamines are associated with depression. Inhibitors of monoamine oxidases may consequently be used to treat Parkinson s disease and depression. The flavin moiety is covalently bound to the enzyme by the thiol group of a cysteine residue (equation 9.17). The acetylenic suicide inhibitor N,N-dimethyl-propargylamine inactivates monoamine oxidases by alkylating the flavin on N-5.25 A likely mechanism for the reaction is the Michael addition of the N-5 of the reduced flavin to the acetylenic carbon 2... 

The "suicide inhibitor." This enzyme inhibitor binds irreversibly to the enzyme protein, permanently inhibiting the enzyme. 

FIGURE 2—47. Some drugs are inhibitors of enzymes. Shown here is an irreversible inhibitor of an enzyme, depicted as binding to the enzyme with chains. The binding is locked so permanently that such irreversible enzyme inhibition is sometimes called the work of a suicide inhibitor, since the enzyme essentially commits suicide by binding to the irreversible inhibitor. Enzyme activity cannot be restored unless another molecule of enzyme is synthesized by the cell s DNA. The enzyme molecule that has bound the irreversible inhibitor is permanently incapable of further enzymatic activity and therefore is essentially dead. ... 

However, when an irreversible inhibitor binds to the enzyme, it cannot be displaced by the substrate and thus binds irreversibly (Fig. 2—51). The irreversible type of enzyme inhibitor is sometimes called a suicide inhibitor because it covalently and irreversibly binds to the enzyme protein, permanently inhibiting it and therefore essentially killing the enzyme by making it nonfunctional forever (Fig. 2—51). Enzyme activity in this case is only restored when new enzyme molecules are synthesized. 

The suicide inhibitor cannot be moved off of the enzyme by a competing substrate. 

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