Development of a Competition-Binding Assay to Determine Binding Affinity of Molecules to Neuromelanin via Fluorescence Spectroscopy.

Neuromelanin, the polymeric form of dopamine which accumulates in aging neuronal tissue, is increasingly recognized as a functional and critical component of a healthy and active adult human brain. Notorious in plant and insect literature for their ability to bind and retain amines for long periods of time, catecholamine polymers known colloquially as 'melanins' are nevertheless curiously absent from most textbooks regarding biochemistry, neuroscience, and evolution. Recent research has brought attention to the brain pigment due to its possible role in neurodegeneration.

Neuromelanin, one of the most overlooked molecules in modern medicine, is not a spectator.

The loss of pigmented neurons from the human brain has long been the hallmark of Parkinson's disease (PD). Neuromelanin (NM) in the pre-synaptic terminal of dopamine neurons is emerging as a primary player in the etiology of neurodegenerative disorders including PD. This mini-review discusses the interactions between neuromelanin and different molecules in the synaptic terminal and describes how these interactions might affect neurodegenerative disorders including PD.

Saturation Binding of Nicotine to Synthetic Neuromelanin Demonstrated by Fluorescence Spectroscopy.

Neuromelanin (NM) has long been considered as an aging pigment, perhaps an unavoidable and undesirable byproduct of dopaminergic neural transmission. However, NM is carefully packaged into double membrane-bound structures within cells of the substantia nigra and other neural tissues, suggesting a beneficial function to maintaining these stores. It is well established that NM is able to concentrate toxic xenobiotics within pigmented cells due to its unique chemical environment.

Expression and functional analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 isozymes.

The objectives of this study were to compare the drug-metabolizing activity of human CYP2D6.24 (I297L), CYP2D6.26 (I369T), and CYP2D6.

Cytochrome P450-catalyzed pathways in human brain: metabolism meets pharmacology or old drugs with new mechanism of action?

The true importance of cytochrome P450 enzymes, not just in drug metabolism but also in pharmacology, is only beginning to be appreciated. Though originally discovered through their role in the biotransformation of xenobiotics, the P450 enzyme super family is ubiquitous in nature and necessarily evolved around endogenous pathways. The extent of tissue- and cell-specific expression of individual P450 isoforms has led many investigators to hypothesize localized roles in endogenous biochemical pathways for isoforms traditionally thought of as drug-metabolizing.

Expression, purification, and characterization of mouse CYP2d22.

Metabolism of the prototype human CYP2D6 substrates debrisoquine and bufuralol proceeds at a much slower rate in mice; therefore, the mouse has been proposed as an animal model for the human CYP2D6 genetic deficiency. To interpret the molecular mechanism of this deficiency, a cDNA belonging to the CYP2D gene subfamily (Cyp2d22) has been cloned and sequenced from a mouse mammary tumor-derived cell line. In the current study, Cyp2d22 enzyme was overexpressed and purified from insect cells using a baculovirus-mediated system.

The human intestinal cytochrome P450 "pie".

Cytochromes P450 (P450s) 3A, 2C, and 1A2 constitute the major "pieces" of the human liver P450 "pie" and account, on average, for 40, 25, and 18%, respectively, of total immunoquantified P450s (J Pharmacol Exp Ther 270:414-423, 1994). The P450 profile in the human small intestine has not been fully characterized. Therefore, microsomes prepared from mucosal scrapings from the duodenal/jejunal portion of 31 human donor small intestines were analyzed by Western blot using selective P450 antibodies.

CYP2C-catalyzed delta9-tetrahydrocannabinol metabolism: kinetics, pharmacogenetics and interaction with phenytoin.

Delta9-tetrahydrocannabinol (delta9-THC), the primary psychoactive constituent of marijuana, is subject to first pass hepatic metabolism primarily by hydroxylation to yield active and inactive oxygenated products. The primary metabolite is formed via oxidation of the allylic methyl group to yield 11-hydroxy-delta9-THC, which is oxidized further to 11-nor-9-carboxy-delta9-THC. The hydroxylation is thought to be mediated by CYP2C9.

Differential activation of CYP2C9 variants by dapsone.

Studies have shown that CYP2C9.1 mediated metabolism of flurbiprofen or naproxen is activated by co-incubation with dapsone. However, dapsone activation has not been examined in the known variant forms of CYP2C9.

The relative contribution of monoamine oxidase and cytochrome p450 isozymes to the metabolic deamination of the trace amine tryptamine.

Tryptamine is a trace amine in mammalian central nervous system that interacts with the trace amine TA(2) receptor and is now thought to function as a neurotransmitter or neuromodulator. It had been reported that deamination of tryptamine to tryptophol was mediated by CYP2D6, a cytochrome P450 that is expressed in human brain, suggesting that tryptamine may be an endogenous substrate for this polymorphic enzyme. We were unable to confirm this report and have reinvestigated tryptamine metabolism in human liver microsomes (HLM) and in microsomes expressing recombinant human cytochrome P450 and monoamine oxidase (MAO) isozymes.

Expression, purification, biochemical characterization, and comparative function of human cytochrome P450 2D6.1, 2D6.2, 2D6.10, and 2D6.17 allelic isoforms.

Polymorphism at the cytochrome P450 2D6 (CYP2D6) locus is one of the most widely known causes of pharmacogenetic variability in humans. Our goal is to investigate the intrinsic enzymatic differences that exist among active CYP2D6 isoforms to test the hypothesis that these enzymatic differences are substrate-dependent. Active CYP2D6.

Reduced (+/-)-3,4-methylenedioxymethamphetamine ("Ecstasy") metabolism with cytochrome P450 2D6 inhibitors and pharmacogenetic variants in vitro.

"Ecstasy" [(+/-)-3,4-methylenedioxymethamphetamine or MDMA] is a CNS stimulant, whose use is increasing despite evidence of long-term neurotoxicity. In vitro, the majority of MDMA is demethylenated to (+/-)-3,4-dihydroxymethamphetamine (DHMA) by the polymorphic cytochrome P450 2D6 (CYP2D6). We investigated the demethylenation of MDMA and dextromethorphan (DEX), as a comparison drug, in reconstituted microsomes expressing the variant CYP2D6 alleles (*)2, (*)10, and (*)17, all of which have been linked to decreased enzyme activity.

Polymorphic variants (CYP2C9*3 and CYP2C9*5) and the F114L active site mutation of CYP2C9: effect on atypical kinetic metabolism profiles.

CYP2C9 wild-type protein has been shown to exhibit atypical kinetic profiles of metabolism that may affect in vitro-in vivo predictions made during the drug development process. Previous work suggests a substrate-dependent effect of polymorphic variants of CYP2C9 on the rate of metabolism; however, it is hypothesized that these active site amino acid changes will affect the kinetic profile of a drug's metabolism as well. To this end, the kinetic profiles of three model CYP2C9 substrates (flurbiprofen, naproxen, and piroxicam) were studied using purified CYP2C9*1 (wild-type) and variants involving active site amino acid changes, including the naturally occurring variants CYP2C9*3 (Leu359) and CYP2C9*5 (Glu360) and the man-made mutant CYP2C9 F114L.

(+)-N-3-Benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital: new potent and selective in vitro inhibitors of CYP2C19.

Highly potent and selective CYP2C19 inhibitors are not currently available. In the present study, N-3-benzyl derivatives of nirvanol and phenobarbital were synthesized, their respective (+)- and (-)-enantiomers resolved chromatographically, and inhibitor potencies determined for these compounds toward CYP2C19 and other human liver cytochromes P450 (P450s). (-)-N-3-Benzyl-phenobarbital and (+)-N-3-benzyl-nirvanol were found to be highly potent, competitive inhibitors of recombinant CYP2C19, exhibiting K(i) values of 79 and 250 nM, respectively, whereas their antipodes were 20- to 60-fold less potent.