Covalent binding of reactive metabolite

Gillette, J.R. 1994. Commentary. Perspective on the role of chemically reactive metabolites of foreign compounds in toxicity. I. Correlation of changes in covalent binding of reactivity metabolites with changes in the incidence and severity of toxicity. Biochem. Pharmacol. 23, 2785. 

From a purely pragmatic perspective, it is clear that reactive metabolites are linked with toxicity and that a circumstantial link can be made to idiosyncratic toxicides. Consequently, even though the mechanism of this toxicity is not fully understood, since assays are available to measure the potential for bioactivation in an ideal world one would not carry this liability forward. Conversely, it is not an ideal world, all drug molecules have challenges and the definition of therapeutic index (i.e., the ratio between the toxic exposure and the therapeutic exposure) is critical. Covalent binding of reactive metabolites to macromolecules is a crude measure and not a full predictor of toxicity and it is well known that toxicity can be ameliorated by a lower dose. Furthermore, the so-called definitive assays require radiolabeled drug material which is expensive and generally slow to produce. 

The most direct mechanism of hepatotoxicity is through specific interaction of a chemical with a key cellular component and consequential modulation of its function. More common mechanisms, however, involve secondary effects of toxicant interaction. These include depletion of cellular molecules, such as ATP and GSH free radical and oxidant damage, in particular to membrane lipids covalent binding of reactive metabolites to critical cellular molecules and collapse of regulatory ion gradients. The following discussion will highlight how these cellular and molecular mechanisms contribute to specific types of chemically induced hepatic lesions. 

Guengerich, F.P., Principles of covalent binding of reactive metabolites and examples of activation of bis-electrophiles by conjugation, Arch. Biochem. Biophys., 433(2), 369, 2005. 

Covalent binding of reactive metabolites Effect on intracellular calcium... 

Because different chemically reactive metabolites react with various tissue nucleophiles at relatively different rates, it seemed likely to us that measuring the total covalent binding of reactive metabolites to proteins would not provide a reliable estimate of the relative toxicity of the chemically reactive metabolites. Indeed it seemed entirely possible that a chemically reactive metabolite could react extensively with protein and still be nontoxic. Moreover, it also seemed possible that a toxicant might be converted to a chemically reactive metabolite which combined with protein even though the toxicity is caused directly by the parent substance. 

Pearson, P.G., Omichinski, J.G., McClanahan, R.H., Soderlund, E.J., Dybing, E. Nelson, S.D. (1993a) Metabolic activation of tris(2,3-dibromopropyl) phosphate to reactive intermediates. 1. Covalent binding and reactive metabolite formation in vitro. Toxicol, appl. Pharmacol., 118, 186-195... 

FIGURE 16.4 General scheme for the role played by reactive drug metabolites in causing a variety of adverse reactions. The reactive metabolites usually account for only a small fraction of total drug metabolism and are too unstable to be chemically isolated and analyzed. In many cases, covalent binding of these metabolites to tissue macromolecules only occurs after their formation exceeds a critical threshold that overcomes host detoxification and repair mechanisms. 

If toxicity is linked to the covalent binding of reactive drug metabolites to tissue macromolecules, then it is logical that the search for the end-products... 

Horseradish peroxidase can be inactivated by protein as well as heme modifications. The reaction of horseradish peroxidase with phenylhydrazine is instructive in this regard (Ator and Ortiz de Montellano, 1987). Enzyme inactivation correlates well with covalent binding of 2 equivalents of radiolabeled phenylhydrazine but is associated with conversion of only a small fraction of the prosthetic group to the meso-phenyl adduct. Covalent binding of a metabolite of phenylhydrazine to the protein is therefore primarily responsible for enzyme inactivation, although the identity of the reactive species and the site at which it reacts are not known. The factors that determine whether the heme group (e.g., methylhydrazine, sodium azide) or the protein (e.g., phenylhydrazine) is the primary site of the inactivation reaction also remain elusive. 

When only small amounts of reactive metabolites are formed, severe toxic hepatitis does not occur. However, mild toxicity may still occur in metabolically susceptible patients, as shown by a clinically silent increase in serum transaminase activity. This mild toxicity causes the release of hepatic proteins, which have been modified by covalent binding with reactive metabolites (Pessayre and Larrey 2007). 

The concept may also be expanded to include situations in which the chemically reactive metabolite that reacts with proteins also is converted to another metabolite that causes toxicity. In this situation any treatment that causes a change in the fraction of the dose that is converted to another metabolite will cause parallel changes in the covalent binding of the metabolite to protein and the severity of the toxicity. But a treatment that preferentially alters the conversion of the chemically reactive metabolite to the toxic metabolite would cause inversely related changes in the magnitude of the covalent binding and the severity of the toxicity. 

The consequences of covalent binding of reactive drug metabolites to proteins as it relates to lADRs remain poorly understood, even after... 

Reactive Metabolites of PAHs. A wide variety of products have been identified as metabolites of PAHs. These include phenols, quinones, trans-dihydrodiols, epoxides and a variety of conjugates of these compounds. Simple epoxides, especially those of the K-region, were initially favored as being the active metabolites responsible for the covalent binding of PAH to DNA. Little direct experimental support exists for this idea (62.63,64) except in microsomal incubations using preparation in which oxidations at the K-region are favored (65,66). Evidence has been presented that a 9-hydroxyB[a]P 4,5-oxide may account for some of the adducts observed in vivo (67.68) although these products have never been fully characterized. 

Cribb AE, Nuss CE, Alberts DW, et al. Covalent binding of sulfamethoxazole reactive metabolites to human and rat liver subcellular fractions assessed by immunochemical detection. Chem Res Toxicol 1996 9(2) 500-507. 

Irreversible inhibition of CYPs is particularly worrisome as its consequences cannot be predicted easily or quantified from in vitro data the in vivo effect of an irreversible inhibitor is usually greater than that predicted based on affinity alone. Moreover, irreversible inhibition is generally the consequence of the production of reactive metabolites (electrophiles), which can also bind covalently to endogenous proteins and, in rare cases, trigger serious autoimmune reactions [4]. 

Utility of Reactive Metabolite Trapping and Covalent Binding Studies in Drug Discovery... 

While the detection of adducts (GSH, amino, and/or cyano) and covalent binding to hepatic tissue is indicative of the formation of reactive metabolites, the data need to be placed in proper context prior to making a decision on discarding a drug candidate... 

While covalent binding studies have an advantage over the reactive metabolite assay in that they provide a quantitative estimate of covalently bound drug to proteins and therefore an indirect measure of reactive metabolite formation, there are no studies to date which show a correlation between the extent of covalent binding and/or reactive metabolite formed and the probability that a drug is... 

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