In vitro covalent protein binding

Protocol for In vitro Covalent Protein Binding in Human or Rat Liver Microsomes—Test-Tube Method... 

Cryopreserved human hepatocytes from three male and two female donors or freshly isolated male rat hepatocytes are analyzed for viabilities (75-85%) using the trypan blue exclusion methods. Incubations are performed by suspending the hepatocytes in Krebs-biocarbonate buffer followed by addition of a H-labeled compound in methanol. The specific radioactivity of the compounds is 100 Ci/ mol. The final concentration of test compound in the suspension is 10 xM in a final volume of 1 mL (1x10 cells/mL), and the final concentration of methanol does not exceed 0.2% (v/v). Incubations are allowed to proceed at 37 °C for 1 h, and are quenched with acetonitrile (5 mL). The remaining procedures are the same as described in Section 14.2.2 (the protocol for in vitro covalent protein binding in human or rat liver microsomes a test-tube method). Covalent protein binding values in pmol-equiv./mg protein are estimated based on the residual radioactivity in the protein pellets. 

The protein pellets are then dissolved in 4 mL of 0.1 M sodium hydroxide. The remaining procedures for measurement of radioactivity and protein concentrations of the resulting samples are the same as described in Section 14.2.2 (the protocol for in vitro covalent protein binding in human or rat liver microsomes—a test-tube method). Covalent protein binding in pmol-equiv./mg... 

PROTOCOLS FOR IN VITRO AND IN VIVO COVALENT PROTEIN BINDING STUDIES... 

Duverger-van Bogaert M, Lambotte-Vandepaer M, Mercier M, et al. 1982a. In vitro covalent binding of acrylonitrile to rat liver proteins. Toxicol Lett 13 203-209. 

The reactivity of compounds such as 28 was clearly demonstrated by the peroxidase-catalyzed covalent binding of A -methyW-hydroxyellipticine (27) to proteins (756). Using horseradish peroxidase and hydrogen peroxide, tritiated-27 was converted to the 9-oxoellipticine derivative in the presence of bovine serum albumin (BSA) and human antibovine IgG in vitro. Covalent binding to these proteins was confirmed by gel electrophoresis, combustion, and liquid scintillation analysis. Dissolution of the BSA-ellipticinium derivative with pronase and... 

Soderlund, E.J., Gordon, W.P, Nelson, S.D. Omichinski, J.G, Dybing, E. (1984) Metabolism in vitro of tris(2,3-dibromopropyl) phosphate Oxidative debromination and bis(2.3-dibromopropyl) phosphate formation as correlates of mutagenicity and covalent protein binding. Biochem. Pharmacol., 33, 4017-4023... 

Usui T, Mise M, Hashizume T, Yabuki M, Komuro S. Evaluation of the potential for drug-induced liver injury based on in vitro covalent binding to human liver proteins. Drug Metab Dispos 2009 37 2383-2392. 

Lactam antibiotics, such as cephalosporins, and penicillins, such as ampicillin (11) and aztreonam, covalently modify their protein targets. Alkyne-functionalized versions of these antibiotics, for example, AmpN (12), were used to probe various penicillin-binding proteins in vitro and in vivo using CC-ABPP [36,37],... 

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]. 

Like other aromatic amines 4-chloro-ort/20-toluidine has been shown to undergo metabolic activation resulting in covalent binding to tissue proteins, DNA and RNA both in vivo and in vitro (Hill et al., 1979 Bentley et al., 1986a,b Bimer Neumann, 1988). 

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