CYP2A5, a mouse cytochrome P450 monooxygenase that shows high similarities to human CYP2A6 and CYP2A13 in protein sequence and substrate specificity, is expressed in multiple tissues, including the liver, kidney, lung, and nasal mucosa. Heterologously expressed CYP2A5 is active in the metabolism of both endogenous substrates, such as testosterone, and xenobiotic compounds, such as nicotine and cotinine. To determine the biological and pharmacological functions of CYP2A5 in vivo, we have generated a Cyp2a5-null mouse. Homozygous Cyp2a5-null mice are viable and fertile; they show no evidence of embryonic lethality or developmental deficits; and they have normal circulating levels of testosterone and progesterone. The Cyp2a5-null mouse and wild-type mouse were then used for determination of the roles of CYP2A5 in the metabolism of nicotine and its major circulating metabolite, cotinine. The results indicated that the Cyp2a5-null mouse has lower hepatic nicotine 5Ј-hydroxylation activity in vitro, and slower systemic clearance of both nicotine and cotinine in vivo. For both compounds, a substantially longer plasma half-life and a greater area under the concentration-time curve were observed for the Cyp2a5-null mice, compared with wild-type mice. Further pharmacokinetics analysis confirmed that the brain levels of nicotine and cotinine are also influenced by the Cyp2a5 deletion. These findings provide direct evidence that CYP2A5 is the major nicotine and cotinine oxidase in mouse liver. The Cyp2a5-null mouse will be valuable for in vivo studies on the role of CYP2A5 in drug metabolism and chemical toxicity, and for future production of CYP2A6-and CYP2A13-humanized mouse models.

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Prodrug nanoassemblies, which can refrain from large excipients, achieve higher drug loading and control drug release, have been placed as the priority in drug delivery system. Reasoning that glutathione (GSH) and reactive oxygen species (ROS) are highly upgraded in tumor tissues which makes them attractive targets for drug delivery system, we designed and synthetized a novel prodrug which utilized mono thioether bond as a linker to bridge linoleic acid (LA) and docetaxel (DTX). This mono thioether-linked conjugates (DTX-S-LA) could self-assemble into nanoparticles without the aid of much excipients. The mono thioether endowed the nanoparticles redox sensitivity resulting in specific release at the tumor tissue. Our studies demonstrated that the nanoassemblies had uniform particle size, high stability and fast release behavior. DTX-S-LA nanoassemblies outperformed DTX solution in pharmacokinetic profiles for it had longer circulation time and higher area under curve (AUC). Compared with DTX solution, the redox dual-responsive nanoassemblies had comparable cytotoxic activity. Besides, the antitumor efficacy was evaluated in mice bearing 4T1 xenograft. It turned out this nanoassemblies could enhance anticancer efficacy by increasing the dose because of higher tolerance. Overall, these results indicated that the redox sensitivity nanoassemblies may have a great potential to cancer therapy.

G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme.

ABSTRACT:3-Methylindole (3MI), a respiratory tract toxicant, can be metabolized by a number of cytochromes P450 (P450), primarily through either dehydrogenation or epoxidation of the indole. In the present study, we assessed the bioactivation of 3MI by recombinant CYP2A13, a human P450 predominantly expressed in the respiratory tract. Four metabolites were detected, and the two principal ones were identified as indole-3-carbinol (I-3-C) and 3-methyloxindole (MOI). Bioactivation of 3MI by CYP2A13 was verified by the observation of three glutathione (GSH) adducts designated as GS-A1 (glutathione adduct 1), GS-A2 (glutathione adduct 2), and GS-A3 (glutathione adduct 3) in a NADPH-and GSH-fortified reaction system. GS-A1 and GS-A2 gave the same molecular ion at m/z 437, an increase of 305 Da over 3MI. Their structures are assigned to be 3-glutathionyl-S-methylindole and 3-methyl-2-glutathionyl-S-indole, respectively, on the basis of the mass fragmentation data obtained by high-resolution mass spectrometry. Kinetic parameters were determined for the formation of I-3-C (V max ‫؍‬ 1.5 nmol/min/nmol of P450; K m ‫؍‬ 14 M), MOI (V max ‫؍‬ 1.9 nmol/min/nmol of P450; K m ‫؍‬ 15 M) and 3-glutathionyl-S-methylindole (V max ‫؍‬ 0.7 nmol/min/nmol of P450; K m ‫؍‬ 13 M). The structure of GS-A3, a minor adduct with a protonated molecular ion at m/z 453, is proposed to be 3-glutathionyl-S-3-methyloxindole. We also discovered that 3MI is a mechanism-based inactivator of CYP2A13, given that it produced a time-, cofactor-, and 3MI concentration-dependent loss of activity toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with a relatively low K I value of ϳ10 M and a k inact of 0.046 min ؊1 . Thus, CYP2A13 metabolizes 3MI through multiple bioactivation pathways, and the process can lead to a suicide inactivation of CYP2A13.3-Methylindole (3MI) is a potent pneumotoxicant and nasal toxicant in several animal species studied, including ruminants, rabbits, and rodents (Adams et al., 1988;Carlson and Yost, 1989;Yost, 1989;Gaskell, 1990). The pulmonary toxicity of 3MI has been attributed to bioactivation by cytochrome P450 (P450) enzymes, which catalyze the formation of reactive intermediates that can bind to cellular proteins (Yost, 1989) and DNA (Regal et al., 2001). Evidence for P450 involvement comes from studies on in vitro metabolism of 3MI using vaccinia-expressed P450s (Thornton-Manning et al., 1991Lanza and Yost, 2001), as well as studies in which P450 inhibitors were found to decrease covalent binding and toxicity associated with 3MI metabolism (Huijzer et al., 1989). Bioactivation of 3MI results in the formation of at least three potentially toxic species via two distinct pathways: 3-methyleneindolenine (MEI), via dehydrogenation, and both 2,3-epoxy-3MI and 3-hydroxy-3-methylindolenine (HMI), via epoxidation on the pyrrole moiety (Scheme 1). Extensive studies seemed to show that dehydrogenation of 3MI is the major route of metabolism that results in toxicity (Skiles and Yost, 1996; reviewed in Yost, 2001). A study of the me...