Mechanism-based inhibition,

In this chapter, mechanism-based inhibition is discussed in its broadest sense, where an inhibitor is converted by the enzyme catalytic mechanism to form an enzyme-inhibitor complex. Other terms used in the literature for mechanism-based inhibitors include suicide inhibitors, suicide substrate inhibitors, alternate substrates, substrate inhibitors, and enzyme inactivators, as well as irreversible, catalytic, or cat inhibitors. The terms alternate substrate inhibition and suicide inhibition are used here to describe the two major subclasses of mechanism-based inhibition. 

Alternate substrates are processed by an enzyme s normal catalytic pathway to form a stable covalent enzyme-inhibitor intermediate, such as an acyl-enzyme in the case of serine proteases, where the complex is essentially trapped in a potential energy well. As such, the inhibition is both time dependent and active-site directed. Theoretically, alternate substrates are reversible inhibitors, since the enzyme is essentially unchanged rather, it is suspended at a point within the catalytic process. However, in practical terms, the enzyme-inhibitor complex can be of such stability as to render the inhibition virtually irreversible. 

Suicide inhibitors are also processed by an enzyme s catalytic mechanism, but in this case, enzyme catalysis of the relatively unreactive inhibitor uncovers a latent reactive moiety. This intermediate then reacts 

The on rate, kon, is equivalent to k, and the off rate, off is equivalent to the sum of all pathways of E-I breakdown, in this case, A i - - k2. It is possible that multiple products are formed, and the rates of formation of these should be included in the koff term. A progress curve or continuous assay is the best way to determine the kon and Ki of an alternate substrate. Addition of an alternate substrate inhibitor to an enzyme assay results in an exponential decrease in rate to some final steady-state turnover of substrate (Fig. 13.1). In an individual assay, both the rate of inhibition (kobs) and the final steady-state rate (C) will depend on the concentration of inhibitor. Care must be taken to have a sufficient excess of inhibitor over enzyme concentration present, since the inhibitor is consumed during the process. Where possible, working at assay conditions well below the of the assay substrate simplifies the kinetics, as the substrate will not interfere in the inhibition. If the 

The second-order rate constant kon is the slope of a plot of kobs versus [1] for inhibitor at nonsaturating concentrations, where [S] Km.  

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  

reacting with nucleophilic amino acids in the active site  

co-ordination of the heme iron to form a metabolite-intermediate complex (MIC). 

A comprehensive overview of MBI chemistry is not within the scope of this section, but the reader is referred to recent reviews that discuss this topic in more detail [47-50]. 

This further incubation should be sufficiently long enough to observe measurable metabolite formation. There is some debate as to the most ideal dilution scheme that should be followed, but the general consensus is that a higher dilution (e.g., 10-fold) reduces the influence of competitive inhibition. Also the concentration of probe substrate should ideally be at least 5 Km, the purpose being that the high probe concentration together with the dilution step minimizes competitive inhibition of 

After a chemical scaffold tests positive in the IC50 shift assay, it becomes necessary to provide a quantitative measure of the potency and rate of the suspected MBI. ATDI 

Typically, microsomal fractions are used for TDI analysis, but in many cases TDI is performed in rCYPs. The ability to use rCYPs for TDI relies on the accurate ability to extrapolate the rCYP data [186]. The second requirement for successful extrapolation of rCYP data is that the clearance pathways of the inhibitor in question are understood. For example, raloxifene has been shown to be a strong MBI of CYP3A4 in microsomes and rCYPs, but in hepatocytes the most predominant pathway is glucuronadation, which would effectively mask any CYP3A4 inactivation observed in 

Additional Mechanistic Tools for Investigating Mechanism-Based Inhibition 

In addition to the TDI experiment, the partition ratio measures the TDI efficiency. Specifically, the partition ratio is the number of inactivation kinetic events (k nact) versus the number of substrate turnover events per unit enzyme (kcat) [161], Thus, the most potent partition ratio is zero. The most common experimental setup for determining the partition ratio is the titration method that increases the inhibitor concentration relative to a known amount of enzyme. After the incubations, a secondary incubation containing a probe substrate similar to the TDI experiment is used to define the remaining activity. For accurate determination of the partition ratio from the titration method, it is assumed that the inhibitor is 100% metabolized  

Even with the knowledge of the reactive moieties that are suspected to trigger M BI, there are numerous potential pathways for the chemistry to lead to protein inactivation [174,196]. Differentiating these mechanisms can facilitate the generation of alternate and safer chemical scaffolds. The UV-Vis spectrophotometer has been a key instrument in activity and functional characterization for CYPs for the past 40 years, as indicated by the derivation of its name, pigment 45 0 being the signature UV band present when reduced in the presence of CO [5,197]. This technique has been 

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Following concurrent administration of two drugs, especially when they are metabolized by the same enzyme in the liver or small intestine, the metabolism of one or both drugs can be inhibited, which may lead to elevated plasma concentrations of the dtug(s), and increased pharmacological effects. The types of enzyme inhibition include reversible inhibition, such as competitive or non-competitive inhibition, and irreversible inhibition, such as mechanism-based inhibition. The clinically important examples of drug interactions involving the inhibition of metabolic enzymes are listed in Table 1 [1,4]. 

MDR-ABC Transporters Mechanism-based Inhibition Meglitinide-related Compounds Melanin-concentrating Hormone Melanocortins... 

Zhong W, Uss AS, Ferrari E, Lau JY, Hong Z (2000) De novo initiation of RNA synthesis by hepatitis C virus nonstructural protein 5B polymerase. J Virol 74 2017-2022 Zhou S, Yung Chan S, Cher Goh B, Chan E, Duan W, Huang M, McLeod HL (2005) Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs. Clin Pharma-cokinet 44 279-304... 

The third mechanism that results in slow binding behavior is covalent inactivation of the enzyme by affinity labeling or mechanism-based inhibition (Scheme... 

Figure9.8 Drug-drug interaction predictions involving reversible (closed symbols) or mechanism-based inhibition (open symbols).

Kunze, K.L. and Trager, W.F. (1993) Isoform-selective mechanism-based inhibition of human cytochrome P450 1A2 by furafylline. Chemical Research in Toxicology, 6 (5), 649-656. 

Irreversible CYP inhibition can arise from different chemical mechanisms. However, a common initial step is the metabolic activation of a substrate into a reactive metabolite that is trapped within the active site of the CYP to form a tightly bound complex causing a long-lasting inactivation of enzyme activity. Enzymatic activity can be restored only through the new synthesis of the enzyme. For this reason, irreversible CYP inhibition is often referred to as mechanism-based inhibition , metabolite-based inhibition or suicide inhibition . 

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. 

In the above-mentioned examples, the prediction of CYP-mediated compound interactions is a starting point in any metabolic pathway prediction or enzyme inactivation. This chapter presents an evolution of a standard method [1], widely used in pharmaceutical research in the early-ADMET (absorption, distribution, metabolism, excretion and toxicity) field, which provides information on the biotransformations produced by CYP-mediated substrate interactions. The methodology can be applied automatically to all the cytochromes whose 3 D structure can be modeled or is known, including plants as well as phase II enzymes. It can be used by chemists to detect molecular positions that should be protected to avoid metabolic degradation, or to check the suitability of a new scaffold or prodrug. The fully automated procedure is also a valuable new tool in early-ADMET where metabolite- or mechanism based inhibition (MBI) must be evaluated as early as possible. 

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]. 

CYP enzymes are induced, resulting in reduced plasma drug levels. Alternatively, CYP enzymes could also undergo mechanism-based inhibition, whereby a CYP enzyme can be completely inactivated by covalent bonding to a component of the herb. Furthermore, botanicals can elicit a biphasic cellular response, whereby CYP activity may be inhibited initially, followed by induction after prolonged incubation or repeated administration. Such factors would need to be considered in future studies in order to establish the true risk of ginseng in herb-drug interactions. 

A number of reports also describe the prediction of mechanism-based inhibition (MBI) [17,18]. In this type of model, MBI is determined in part by spectral shift and inactivation kinetics. Jones et al. applied computational pharmacophores, recursive partitioning and logistic regression in attempts to predict metabolic intermediate complex (MIC) formation from structural inputs [17]. The development of models that accurately predict MIC formation will provide another tool to help reduce the overall risk of DDI [19]. 

Liquid chromatography/ultraviolet detection Monoamine oxidase Mechanism-based inhibition Metabolic intermediate complex Molybdenum-containing oxidase Multi parameter optimization... 

Bertelsen, K.M., Venkatakrishnan, K., Von Moltke, L.L., Obach, R.S. and Greenblatt, D.J. (2003) Apparent mechanism-based inhibition of human CYP2D6 in vitro by paroxetine comparison with fluoxetine and quinidine. Drug Metabolism and Disposition The Biolo cal Fate of Chemicals, 31, 289-293. 

MECHANISM-BASED INHIBITION Drug D can t dock because Drug has comploxed and... 

Jones DR, Hall SD. Mechanism based inhibition of cytochrome P450 in vitro kinetics and in vitro-in vivo correlations. In Rodrigues AD, ed. Drug-Drug Interactions From Basic Pharmacokinetics Concepts to Marketing Issues. New York, NY Marcel Dekker, 2001. 

Wang, Y.-H., Jones, D. R., Hall, S. D. Differential Mechanism-Based Inhibition of CYP3A4 and CYP3A5 by Verapamil. Drug Metab. Dispos. 2005, 33, 664—671. 

Richter T, Murdter TE, Heinkele G, et al. Potent mechanism-based inhibition of human CYP2B6 by clopidogrel and ticlopidine. J Pharmacol Exp Ther 2004 308 189-197. 

Mechanism-based inhibition should be irreversible. Dialysis, ultrafiltration, or washing the protein (e.g., by isolating microsomes by centrifugation and resuspending them in drug-free buffer) will not restore enzyme activity, and the inhibition is highly resistant to sample dilution. Mechanism-based inhibition should be saturable. The rate of inactivation is proportional to the concentration of the inactivator until all enzyme molecules are saturated, in accordance with Michaelis-Menten kinetics. Additionally, the decrease in enzymatic activity over time should follow pseudo-first-order kinetics. 

Substrates should protect against mechanism-based inhibition. The addition of an alternative substrate or competitive inhibitor with good affinity for the enzyme will prevent or at least decrease the rate of inactivation. There should be stoichiometric (ideally one-to-one) binding of inactivator to enzyme. 

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