Suicide substrates inactivators

Nazari K, Mahmoudi A, Khosraneh M et al (2009) Kinetic analysis for suicide-substrate inactivation of microperoxidase-11 a modified model for bisubstrate enzymes in the presence of reversible inhibitors. J Mol Catal B Enzym 56 61-69... 

Tienilic acid is oxidized in the liver by the cytochrome P-450 monooxygenase to 5-hydroxytienilic acid, which is the major urinary metabolite (about 50% in humans). This oxidation occurs through an electrophilic intermediate capable of alkylating very specifically the cytochrome P-450." This suicide-substrate inactivation is also observed with many xenobiotics such as alkenes with terminal... 

The efficiency of inactivation by covalent bond formation vs release of the reactive species into solution has been described by its partition ratio. The most efficient inactivators have catalytic partition ratios of 0, in which case each inhibitor molecule leads to inactivation of the enzyme. To this date, many of these inhibitors have been designed, and alternative names like suicide substrate, Trojan Horse inactivator, enzyme induced inactivator, inhibitor, and latent inactivator have been used for this class of inhibitors. A number of comprehensive reviews are available (26—32). 

Suicide Substrates —Mechanism-Based Enzyme Inactivators... 

Inactivators of class A (3-lactamases (clavulanate, sulbactam, tazobactam) are themselves (3-lactams and act as suicide substrates. They can be used in... 

SCHEME 11.3 Postulated mechanisms for the inhibition of serine proteases by coumarin derivatives. NuH nucleophile. Pathway a suicide-type inactivation (suicide substrate). Pathway b transient inactivation by formation of a stable acyl-enzyme (alternate substrate-inhibitor). 

With a 3,3-heterodihalogeno substitution of the (3-lactam ring, a selective interaction of each enantiomer of the chiral azetidinone with the enzyme active site is expected. The enantiomer 3R of the 3F, 3Br derivative indeed has a more favorable kinetic parameter k-JK, than the enantiomer 3S.33 The partition ratio kCA /kt (=k3/k4, Eq. 11.1) for the inactivation is also higher. Therefore, enantiomer 3R is a better suicide substrate for HLE since a lower partition ratio corresponds to abetter suicide substrate.20... 

Walsh, C. T. Suicide substrates mechanism-based enzyme inactivators recent developments. Ann. Rev. Biochem. 1984, 53, 493-535. 

Reboud-Ravaux, M. Vilain, A. C. Boggetto, N. Maillard, J.-L. Favreau, C. Xie, J. Mazaleyrat, J.-P. Wakselman, M. A cyclopeptidic suicide substrate preferentially inactivates urokinase-type plasminogen activator. Biochem. Biophys. Res. Commun. 1991, 178, 352-359. 

The biological activity of a compound can often be affected dramatically by the presence of even a single fluorine substituent that is placed in a particular position within the molecule. There are diverse reasons for this, which have been discussed briefly in the preface and introduction of this book. A few illustrative examples of bioactive compounds containing a single fluorine substituent are given in Fig. 3.1. These include what is probably the first example of enhanced bioactivity due to fluorine substitution, that of the corticosteroid 3-1 below wherein Fried discovered, in 1954, that the enhanced acidity of the fluorohydrin enhanced the activity of the compound.1 Also pictured are the antibacterial (3-fluoro amino acid, FA (3-2), which acts as a suicide substrate enzyme inactivator, and the well-known anti-anthrax drug, CIPRO (3-3). 

However, an important problem arises during the peroxidative removal of phenols from aqueous solutions PX is inactivated by free radicals, as well as by oligomeric and polymeric products formed in the reaction, which attach themselves to the enzyme (Nazari and others 2007). This suicide peroxide inactivation has been shown to reduce the sensitivity and efficiency of PX. Several techniques have been introduced to reduce the extent of suicide inactivation and to improve the lifetime of the active enzyme, such as immobilization. Moreover, Nazari and others (2007) reported a mechanism to prevent and control the suicide peroxide inactivation of horseradish PX by means of the activation and stabilization effects of Ni2+ ion, which was found to be useful in processes such as phenol removal and peroxidative conversion of reducing substrates, in which a high concentration of hydrogen peroxide may lead to irreversible enzyme inactivation. 

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... 

Scheme 2. Inactivation of (3-hydroxydecanoyl thioester dehydrase by the suicide substrate 3-decynoyl-A/-...

Silverman has pointed out that several criteria must be met to demonstrate that a compound is a true suicide substrate 1101 (1) Loss of enzyme activity must be time-dependent, and it must be first-order in [inactivator] at low concentrations and zero-order at higher concentrations (saturation kinetics), (2) substrate must protect the enzyme from inactivation (by blocking the active site), (3) the enzyme must be irreversibly inactivated and be shown to have a 11 stoichiometry of suicide substrate active site (dialysis of enzyme previously treated with radiolabeled suicide substrate must not release radiolabel into the buffer), (4) the enzyme must unmask the suicide substrate s potent electrophile via a catalytic step,1121 and (5) the enzyme must not be covalently labeled with the activated form of the suicide substrate following its escape from the active site (the presence of bulky scavenging thiol nucleophiles in the buffer must not decrease the observed rate of inactivation). 

Figure 7. Two examples of irreversible inactivators that are not suicide substrates a) TPCK, a classic" affinity label of the serine protease chymotrypsin, b) ZFK-CH2-mesitoate, a quiescent" affinity label of the cysteine protease cathepsin B, and c) the kinetic scheme for both forms of affinity label-inactivation.

These inactivators typically have negligible reactivity toward cellular nucleophiles, in contrast to the classic affinity labels and the activated (escaped) form of suicide substrates (I ). However, all classes of irreversible inactivators - even in the ideal case of covalently labeling only their target enzymes - suffer from the possibility of eliciting an undesired immune response against the inactivator-derivatized protein following protein denaturation and degradation.1171... 

An interesting dinically useful prodrug is 5-fluorouracil, which is converted in vivo to 5-fluoro-2 -deoxyuridine 5 -monophosphate, a potent irreversible inactivator of thymidylate synthase It is sometimes charaderized as a dead end inactivator rather than a suicide substrate since no electrophile is unmasked during attempted catalytic turnover. Rathei since a fluorine atom replaces the proton found on the normal substrate enzyme-catalyzed deprotonation at the 5 -position of uracil cannot occur. The enzyme-inactivator covalent addud (analogous to the normal enzyme-substrate covalent intermediate) therefore cannot break down and has reached a dead end (R. R. Rando, Mechanism-Based Enzyme Inadivators , Pharm. Rev. 1984,36,111-142). 

Funaki, T., Takanohashi, Y., Fukazawa, H. and Kuruma, I. (1991) Estimation of kinetic parameters in the inactivation of an enzyme by a suicide substrate. Biochimica et Biophysica Acta, 1078 (1), 43-46. 

Walsh, C.T. (1984) Suicide Substrates, Mechanism Based Enzyme Inactivators -Recent Developments. Annual Review of Biochemistry, 53, 493-535. 

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. 

Mechanism-based inhibitors or suicide substrates seem to be particularly prevalent with CYP3A4. Such compounds are substrates for the enzyme, but metabolism is believed to form products that deactivate the enzyme. Several macrolide antibiotics, generally involving a tertiary amine function, are able to inhibit CYP3A4 in this manner (147,148). Erythromycin is one of the most widely used examples of this type of interaction, although there are other commonly prescribed agents that inactivate CYP3A4 (149-151), and a consideration of this phenomenon partially explains a number of interactions that are not readily explained by the conventional in vitro data (152). 

The compound 5-fluorouridine targets thymidylate synthase. After a nucleoside kinase phosphorylates it, resembles the natural substrate for the enzyme, except that it contains a fluorine where dUMP has a hydrogen. The fluorine isn t removed from the ring by thymidylate synthase, and this causes the ring to remain covalently bound to the enzyme, which means that the enzyme is irreversibly inactivated. The 5-fluorouridine monophosphate is an example of a suicide substrate —a compound whose reaction with an enzyme causes the enzyme to no longer function. 

One successful approach to suicide inhibitors for serine proteases is outlined in Figure 8. The dihydrocoumarin reacts with chymotrypsin to form an acyl enzyme and uncover a p-hydroxybenzyl chloride functional group. This is an extremely reactive alkylating agent due to formation of the quinone methide and the enzyme is rapidly inactivated (44). It is likely that suicide substrates will be applied to other proteases in the future. 

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