Metabolism/mechanism-based inhibitor

It should be emphasized that the organic cosolvent concentration should be kept to a minimum as it may alter the basal enzyme activity. Also, a compound could be a weak direct inhibitor but a potent metabolism/mechanism-based inhibitor. Therefore, it is important to evaluate both types of inhibition [52],... 

AO is also effective in metabolizing a wide range of nitrogen-containing heterocycles such as purines, pyrimidines, pteridines, quinolines, and diazanaphthalenes (95). For example, phthalazine is rapidly converted to 1-phthalazinone by AO and the prodrug, 5-ethynyl-2-(l//)-pyrimidone, is oxidized to the dihydropyrimidine dehydrogenase mechanism-based inhibitor, 5-ethynyluracil, by AO (Fig. 4.40) (96). 

Fontana, E., Dansette, P.M. and Poli, S.M. (2005) Cytochrome P450 enzymes mechanism based inhibitors common sub-structures and reactivity. Current Drug Metabolism, 6, 413 -54. 

The CYPs as Antitarget Enzymes 278 The UGTs as Antitarget Enzymes 279 The MetaSite Technology 282 Mechanism-Based Inhibitors 285 Phase II Metabolism by UGTs 287 The Flowchart of the Overall Method 287 Conclusions 289 Software Package 290 References 290... 

In addition to the assessment of reversible inhibition, the role played by mechanism-based inhibitors (irreversible inhibitors) provides a focus during lead development, as it can result in a more profound and prolonged effect than that suggested by the therapeutic dose or exposure. Mechanism-based inhibition (MBI) occurs as a result of the CYP generating reactive intermediates that bind to the enzyme causing irreversible loss of activity. Oxidative metabolism via that CYP is only restored upon re-synthesis of that enzyme. Three mechanisms have been reported showing how intermediate species act as mechanism-based inhibitors ... 

Interactions with metabolic enzymes fluorinated amino acids are peptidomi-metic units or reactive entities used to design either reversible enzyme inhibitors (analogues of substrates) or irreversible enzyme inhibitors (mechanism-based inhibitors). 

Among the numerous enzymes that utilize pyridoxal phosphate (PLP) as cofactor, the amino acid racemases, amino acid decarboxylases (e.g., aromatic amino acids, ornithine, glutamic acid), aminotransferases (y-aminobutyrate transaminase), and a-oxamine synthases, have been the main targets in the search for fluorinated mechanism-based inhibitors. Pharmaceutical companies have played a very active role in this promising research (control of the metabolism of amino acids and neuroamines is very important at the physiological level). 

We often rationalize drug interactions as reflecting the reversible competition of two substrates for an active site. However, it is becoming increasingly clear that other mechanisms of inhibition are operational in vivo. For example, some mechanism-based inhibitors are activated during metabolism and form a complex with the heme of CYP3A, known as a metabolite... 

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). 

Chemical inhibition, which involves an evaluation of the effects of known CYP enzyme inhibitors on the metabolism of a drug candidate by human liver microsomes. Chemical inhibitors of CYP must be used cautiously because most of them can inhibit more than one CYP enzyme and some chemicals can inhibit one enzyme but activate another. Some chemical inhibitors are mechanism-based inhibitors that require biotransformation to a metabolite that inhibits or inactivates CYP. 

Studies in recent years have revealed a number of remarkable drug interactions with irreversible or mechanism-based inhibitors of CYP3A, many of which can be attributed to inhibition of sequential intestinal and hepatic first-pass metabolism. Mechanism-based inhibition involves the metabolism of an inhibitor to a reactive metabolite, which either forms a slowly reversible metabolic-intermediate (MI) complex with the heme moiety or inactivates the enzyme irreversibly via covalent binding to the enzyme catalyzing the last step in the bioactivation sequence. As a result, mechanism-based inhibition is both... 

A similar phenomenon has been observed with the well-established mechanism-based inhibitor, diltiazem (108). Diltiazem (Cardizem SR , 120 mg b.i.d. for 7 days) caused a decrease in small bowel CYP3A activity of 62% with no corresponding change in intestinal CYP3A mRNA or protein expression. Many clinical studies have shown that diltiazem, a calcium channel blocker, inhibits the metabolism of CYP3A substrates, such as triazolam (109), midazolam (110), and... 

Many drugs are mechanism-based inhibitors of CYP. This property could affect a drug s own metabolism or the metabolism of coadministered drugs, which could lead to serious drug interactions. Even though in vitro Ki values have been determined for a number of drugs and have been used to predict an in vivo interaction, the effect of mechanism-based inhibitors can be observed at in vivo concentrations below these Ki values. This effect can be predicted if in vitro estimates of kinetic constants (e.g., Kt and /cin ic,) for mechanism-based inhibitors are known. A theoretical basis and application have been presented that applies in vitro estimates of mechanism-based inhibitors to accurately predict in vivo drug interactions. 

In contrast, decreases in theophylline metabolism by selective inhibitors of CYP1A2, such as fluvoxamine and some quinolone antibiotics, or by selective and potent inhibitors of CYP3A4, such as the macrolide antibiotics, have resulted in serious theophylline toxicity (22). It is postulated that taken over time, the macrolide antibiotics act as mechanism-based inhibitors of CYP isoforms other than just CYP3A4. Some nonselective inhibitors of P450s, such as cimetidine, some p-blockers and calcium channel blockers, and others (19,22), also appear to inhibit the metabolism of theophylline enough to cause toxicity. 

Currently, efforts are underway to obtain more structural information on desaturases to address the mechanistic issues that have been raised through substrate-based studies. The need for more detailed 3-D active-site information is acute, particularly in the case of membrane-bound desaturases for which only hypothetical models currently are available. Design of mechanism-based inhibitors of medically relevant desaturases such as stearoyl CoA desaturase (SCD) (metabolic syndrome) (32), DesA3 (tuberculosis) (33), and dihydroceramide desaturase (apoptosis) (34, 35) also will be aided by new structural data. 

It should also be noted that the activation of a mechanism-based inhibitor by its target enzyme is, formally, an example of metabolic activation. However, there is a clear distinction between the activation of a mechanism-based inhibitor described above and the metabolic activation of a prodrug. In the latter case, an inactive precursor is metabolized in the body (either chemically or enzymatically) to metabolites that possess the desired activity. For example. Acyclovir (3a) must be metabol-ically converted to the triphosphate (3b) and released into the medium before it will inhibit viral DNA polymerase. Further discussion on prodrugs may be found in volume 2, chapter 14. 

In these cases, as with affinity labels, nonspecific covalent modification of residues other than those located in the active site cannot be excluded. A second test for a metabolically activated affinity label is to add an additional aliquot of fresh enzyme to the incubation buffer. The fresh enzyme should be inactivated at a higher rate than that of the first equivalent of enzyme because there is more reactive species present in solution. By contrast, the mechanism-based inhibitor should show no difference in rate until the concentration of inhibitor is depleted. It should also be noted that the observation of such rate increases necessitates that the reactive species is relatively stable and is not immediately quenched by the incubation buffer. 

Sahali-Sahly Y, Balani SK, Lin JH, et al. In vitro studies on the metabolic activation of the furanopyridine L-754,394, a highly potent and selective mechanism-based inhibitor of cytochrome P450 3A4. Chem Res Toxicol. 1996 9(6) 1007-1012. 

Further biotransformations of A" VPA involve both the liver microsomal CYP enzymes and the fatty acid (3-oxidation pathway (Figure 33.29). The mixed-function-oxidase system metabolizes the unsaturated metabolite to a -butyrolactone derivative through a chemically reactive entity that is a mechanism-based inhibitor of CYP. The alkylation of the prosthetic heme by means of the radical occurs prior to formation of the epoxide. Thus, the epoxide is not involved in the CYP inhibition. 

Many fluorinated, mechanism-based inhibitors are amino acid derivatives [3, 81]. These target enzymes involved in amino acid metabolism, for example decarboxylases, transaminases or monoamine oxidases. 

The CYP inhibition assay utilizes the 96-well plate format with a robotic system, where both incubation and analysis are performed in the same plates. The setup of the sample plates is shown in Figure 4.1. For each compound, both direct inhibition and metabolism/mechanism-based inhibition, which is caused by a metabolite of the NCE that is either a more potent direct reversible inhibitor (metabolism-based) or a time-dependent irreversible inhibitor (mechanism-based), are evaluated. Both direct and mechanism-based inhibitors could result in inhibitory DDIs [51,52],... 

Figure 7.35. Reversible and mechanism-based inhibitors of enzymes involved in the metabolism of fatty acids and related endogenous substrates.

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