Cytochrome P450-Mediated Epoxidation of 2-Aminothiazole-Based AKT Inhibitors: Identification of Novel GSH Adducts and Reduction of Metabolic Activation through Structural Changes Guided by in Silico and in Vitro Screening

Abstract

A 2-aminothiazole derivative 1 was developed as a potential inhibitor of the oncology target AKT, a serine/threonine kinase. When incubated in rat and human liver microsomes in the presence of NADPH, 1 underwent significant metabolic activation on its 2-aminothiazole ring, leading to substantial covalent protein binding. Upon addition of glutathione, covalent binding was reduced significantly, and multiple glutathione adducts were detected. Novel metabolites from the in vitro incubates were characterized by LC-MS and NMR to discern the mechanism of bioactivation. An in silico model was developed based on the proposed mechanism and was employed to predict bioactivation in 23 structural analogues. The predictions were confirmed empirically for the bioactivation liability, in vitro, by LC-MS methods screening for glutathione incorporation. New compounds were identified with a low propensity for bioactivation.

Selective Time- and NADPH-Dependent Inhibition of Human CYP2E1 by Clomethiazole

“…Such mechanisms would be consistent with those proposed for other thiazolecontaining compounds (Obach et al, 2008;Sevrioukova and Poulos, 2010;Subramanian et al, 2010;Rock et al, 2014). The chloro-ethyl side chain of CMZ may also be involved in the enzyme inactivation described here.…”

“…A noncovalent, metabolic intermediate complex could also form after NADPH-dependent conversion to a tight-binding metabolite(s). It is interesting to note that CMZ contains a thiazole functional group in common with potent CYP3A4 inhibitors cobicistat and ritonavir and compounds known to undergo metabolic activation and covalent binding to microsomal protein, including ritonavir, sudoxicam, meloxicam, and a series of AKT inhibitors (Obach et al, 2008;Subramanian et al, 2010;Rock et al, 2014). It has been postulated that initial events leading to metabolic activation of the thiazole and subsequent covalent binding are epoxidation of the C4-5 bond (Subramanian et al, 2010) or sulfoxidation (Rock et al, 2014).…”

“…It is interesting to note that CMZ contains a thiazole functional group in common with potent CYP3A4 inhibitors cobicistat and ritonavir and compounds known to undergo metabolic activation and covalent binding to microsomal protein, including ritonavir, sudoxicam, meloxicam, and a series of AKT inhibitors (Obach et al, 2008;Subramanian et al, 2010;Rock et al, 2014). It has been postulated that initial events leading to metabolic activation of the thiazole and subsequent covalent binding are epoxidation of the C4-5 bond (Subramanian et al, 2010) or sulfoxidation (Rock et al, 2014). Although ritonavir exhibits mechanism-based inhibition (Rock et al, 2014), metabolism-independent tight-binding has been suggested as a predominant means of CYP3A4 inactivation whereby the thiazole nitrogen coordinates to both ferric and ferrous forms of the enzyme, decreases heme redox potential, and hinders electron transfer from cytochrome P450 reductase (Sevrioukova and Poulos, 2010).…”

Characterization of Ritonavir-Mediated Inactivation of Cytochrome P450 3A4

“…More recently, two studies have reported that cytochrome P450s can catalyze the bioactivation of thiazole rings to intermediates that form covalent bonds to protein (Subramanian et al, 2010;Obach et al, 2008) via the formation of an epoxide intermediate. The structures of the GSH adducts to bioactivated thiazoles (e.g., sudoxicam, meloxicam, and multiple 2-aminothiazole derivatives) have been determined.…”

Developments in Selective Small Molecule ATP-Targeting the Serine/Threonine Kinase Akt/PKB

“…11) Compound 69 exhibited 64% oral bioavailability with respectable plasma drug exposure (auc = 2600 ng·h/mL, 5 mg/kg) and longer half-life (t 1/2 = 4.3 h). All of these compounds were cleared via oxidation of the thiazole ring [87], therefore their half-lives were similar. In vivo pharmacodynamic (PD) studies in mice six hours after a 30 mg/kg dose, plasma concentration of 69 was about 10-fold over its cellular IC 50 , and significant inhibition of PRAS40 phosphorylation was observed (43%).…”