Classical enzyme inhibition

Allenic amino acids belong to the classical suicide substrates for the irreversible mechanism-based inhibition of enzymes [5], Among the different types of allenic substrates used for enzyme inhibition [128, 129], the deactivation of vitamin B6 (pyr-idoxal phosphate)-dependent decarboxylases by a-allenic a-amino acids plays an important role (Scheme 18.45). In analogy with the corresponding activity of other /3,y-unsaturated amino acids [102,130], it is assumed that the allenic amino acid 139 reacts with the decarboxylase 138 to furnish the imine 140, which is transformed into a Michael acceptor of type 141 by decarboxylation or deprotonation. Subsequent attack of a suitable nucleophilic group of the active site then leads to inhibition of the decarboxylase by irreversible formation of the adduct 142 [131,132]. 

It was previously thought that 5-FU inhibits the enzyme by classical competitive inhibition. However, it was found that 5-FU is a transition-state substrate, and it forms a covalent complex with tetrahydrofolate and the enzyme in the same way that the natural substrate does. The reaction, however, will not go to completion, since the fluoro-uridine derived from the antimetabolite remains attached to the enzyme, and the latter becomes irreversibly deactivated. Recovery can occur only through the synthesis of new enzyme. Fluorouracil is used in the treatment of breast cancer and has found limited use in some intestinal carcinomas. Unfortunately, this drug has the side effects usually associated with antimetabolites. Its prodrug, fluorocytosine (8.35, which is also an antifungal agent) is better tolerated. 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

The classical QSAR methodology started 1964 with the publications of Hansch and Fujita (1964) and Free and Wilson (1964) and the statement of Hansch (1969) resulted from a proposal by Fujita. They proposed to combine several physiochemical parameters (tt, a), also called descriptors, in a quantitative model. This Hansch-type analysis is very flexible and describes many different kinds of biological activities, e.g. in vitro data such as enzyme inhibition (Kubinyi 2002) ... 

The effect of NMMA is attributable to its prevention of NO formation by NOS and its reversal by excess substrate (l-arginine) is a classic example of competitive enzyme inhibition (Figure 2). 

Measurement of the in vitro efficacy of compounds as substrates is usually deduced by comparison of their k JK ratios where is the first-order rate constant for product formation and is the Michaelis equilibrium constant [38]. For those compounds which are classical, reversible inhibitors, K, the dissociation (or inhibition) equilibrium constant, and (kassoc) the rate constant for enzyme inhibition, are the most commonly reported kinetic values. These values may be measured while using either a high-molecular-weight natural substrate or a low-molecular-weight synthetic substrate. For alternate-substrate inhibitors, that is, compounds which form a stable complex (an acyl-enzyme ) that dissociates to enzyme and intact inhibitor or to enzyme and an altered form of the inhibitor, the usually reported value is K, the apparent K. For compounds which irreversibly inactivate the enzyme, the kinetics are usually measured under conditions such that the initial enzyme concentration [E] is much lower than the inhibitor concentration [I] which in turn is much lower than the Ky Under these conditions the commonly reported value is obs/[I]> the apparent... 

Classical enzyme inhibitors such as bacitracin, bes-tatin, and amastatin have been found to be effective for improving the nasal absorption of various peptide drugs such as LHRH and calcitonin. These inhibitors having peptide like structures appear to exert their inhibitory effects by a competitive mechanism. In addition, camostat mesilate and nafamostat mesilate, which are clinically used as primary ingredients for pancreatic diseases, have been found to improve the nasal absorption of vasopressin, desmopressin, and calcitonin by inhibiting aminopeptidase and trypsin activity. 

Rosowski, A., Eorsch, R. A., Freisheim, J. H., Moran, R. G., Wick, M. Methotrexate analogues. 19. Replacement of the glutamate side chain in classical antifolates by L-homocysteic acid and L-cysteic acid effect on enzyme inhibition and antitumor activity. J. Med. Chem. 1984,27, 600-604. 

The classic type of competitive reversible enzyme inhibition can be expressed simply as ... 

Structure-function analysis of peptide synthetases includes classical enzyme studies with no link to structural data. These have been carried out with enzyme kinetics, substrate analogs, inhibition studies, and the isolation of intermediates and products. Engineering approaches have employed mostly fragment expression and site-directed mutagenesis. In addition, first experiments with chimeric structures have been performed-... 

Many of the early examples of maerocyeles in drug discovery relied on these classical reaction types for the formation of the ring and they remain in regular use. This was due, in part, to the peptidomimetic nature of many of these structures, which often were targeted at protease enzyme inhibition, and thus lent themselves readily to macrolactamization for amide bond formation or macrolactonization for cyclic depsipeptide-like compounds. Representative examples of these two general transformations are shown in Scheme 11.1 (BOP (benzotriazol-l-ylo5ytris(dimethylamino)-phosphonium hexafluorophosphate, Castro s reagent), EDC (l-ethyl-3-(3-dimethylamino-propyl)-carbodiimide), DMAP (4-dimethylamino-pyridine)) for the matrix metalloproteinase (MMP) inhibitor template 2 and the renin inhibitor scaffold 4. °... 

Another, more classical, control mechanism exists for the feedback regulation of key enzymes for the synthesis of Gm-6-P04 and for N-acetylmannosamine. Each of these is the first enzyme in the metabolic commitment to hexosamine and sialic acid synthesis. Komfeld et al, (1964) showed that UDP-GlcNAc is an efficient feedback inhibitor for L-glutamine-D-fructose-6-phosphate aminotransferase and that CMP-NAN also inhibits UDP-GlcNAc-2-epimerase, which is responsible for the synthesis of A/ -acetylmannosamine. This is an example of the by now familar endproduct inhibition of the first enzyme of a metabolic pathway (see Figure 6). They were also able to demonstrate that in vivo administration of puromycin to rats, which inhibits de novo protein synthesis and also depresses sialic acid and hexosamine utilization, does not lead to an accumulation of UDP-GlcNAc. Furthermore, the turnover of the UDP-hexosamine pool was shown to be slowed down. These data suggest that impairment of the utilization of UDP-hexosamine leads to decreased synthesis of UDP-hexosamines or their precursors (i.e., classical feedback inhibition). 

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