No abstract

Presenilins are integral membrane protein involved in the production of amyloid ␤-protein. Mutations of the presenilin-1 and -2 gene are associated with familial Alzheimer's disease and are thought to alter ␥-secretase cleavage of the ␤-amyloid precursor protein, leading to increased production of longer and more amyloidogenic forms of A␤, the 4-kDa ␤-peptide. Here, we show that radiolabeled ␥-secretase inhibitors bind to mammalian cell membranes, and a benzophenone analog specifically photocross-links three major membrane polypeptides. A positive correlation is observed among these compounds for inhibition of cellular A␤ formation, inhibition of membrane binding and cross-linking. Immunological techniques establish N-and C-terminal fragments of presenilin-1 as specifically cross-linked polypeptides. Furthermore, binding of ␥-secretase inhibitors to embryonic membranes derived from presenilin-1 knockout embryos is reduced in a gene dose-dependent manner. In addition, C-terminal fragments of presenilin-2 are specifically cross-linked. Taken together, these results indicate that potent and selective ␥-secretase inhibitors block A␤ formation by binding to presenilin-1 and -2.␤-Amyloid precursor protein (␤APP) 1 is a transmembrane protein that undergoes processing to A␤ by proteolytic activities known as ␤-and ␥-secretases (for review, see Refs. 1-3). The ␤-secretase cleavage occurs in the extracellular domain by a recently identified aspartyl protease variously termed BACE, memapsin, and Asp2 (4 -9), whereas the heterogeneous ␥-secretase cleavage occurs in the transmembrane domain (2, 10). Dominant mutations in either of the two human presenilin (PS-1 and PS-2) genes lead to familial Alzheimer's disease (AD). PS-1 and -2 are polytopic membrane proteins (for review, see Refs. 11-13). Presenilins are proteolytic processed. In vivo, only small amounts of the holoprotein can be detected, primarily in the nuclear envelope, whereas 30-kDa N-terminal and 20-kDa C-terminal fragments of presenilin are observed in all mammalian tissues and cell lines analyzed so far. Coimmunoprecipitation experiments revealed that presenilin fragments are assembled into a high molecular weight complex together with other proteins (for review see 11-13). The proposed mechanism through which the presenilin mutations cause AD is an alteration in the predominant ␥-secretase cleavage site which increases the amount of the longer, more amyloidogenic A␤ 1-42(43) fragments produced (11-13). A null mutation of the mouse PS-1 selectively reduces ␥-secretase activity (14), and site-directed mutagenesis of PS-1 and PS-2 at two conserved aspartyl residues, which resemble the catalytic center of aspartyl proteases, also reduces ␥-secretase activity (15, 16). These observations indicate that PS-1 and PS-2 either stimulate the activity of ␥-secretase by trafficking to appropriate cellular compartments, serve as cofactors of the ␥-secretase, or are ␥-secretase themselves.Here, we report that a series of potent and selective ␥-secretase inhibitors bind to mam...

ABSTRACT:Ketoconazole has generally been used as a standard inhibitor for studying clinical pharmacokinetic drug-drug interactions (DDIs) of drugs that are primarily metabolized by CYP3A4/5. However, ketoconazole at therapeutic, high concentrations also inhibits cytochromes P450 (P450) other than CYP3A4/5, which has made the predictions of DDIs less accurate. Determining the in vivo inhibitor concentration at the enzymatic site is critical for predicting the clinical DDI, but it remains a technical challenge. Various approaches have been used in the literature to estimate the human hepatic free concentrations of this inhibitor, and application of those to predict DDIs has shown some success. In the present study, a novel approach using cryopreserved human hepatocytes suspended in human plasma was applied to mimic the in vivo concentration of ketoconazole at the enzymatic site. Ketoconazole is an antifungal medicine and is also a potent CYP3A4/5 (CYP3A4) inhibitor in humans. Ketoconazole is widely used in vitro as a selective CYP3A4 inhibitor at low concentrations (Pelkonen et al., 1998;Li et al., 1999). However, at high concentrations it also inhibits cytochrome P450 (P450) isozymes other than CYP3A4 (Venkatakrishnan et al., 2001;Stresser et al., 2004) and UDP glucuronosyltransferase (Yong et al., 2005), as well as transporters such as sodium taurocholate cotransporting polypeptide, Pglycoprotein (Pgp), breast cancer resistance protein, and multidrug resistance-associated protein 2 (Azer et al., 1995;Salphati and Benet 1998, Achira et al., 1999;Xia et al., 2005b). For the purpose of overcoming possible drug resistance, clinical usage of ketoconazole involves much higher concentrations (ϳ25-fold) than its in vitro efficacious concentration (Schaefer-Korting et al., 1984). For studying the worst-case scenario of CYP3A4-mediated drug-drug interaction (DDI) potential on a compound, dosing of ketoconazole at 200 mg b.i.d. is recommended by the Pharmaceutical Research and Manufacturers of America (Bjornsson et al., 2003). At such a high dose, the maximum plasma concentration (C max ) could reach as high as 20 M (Rochlitz et al., 1988;Hsu and Chen, 1997). Other dose regimens, such as 200 mg/day, have also been reported. These doses showed plasma C max ranging from 3.2 to 10 M with most of the values about 10 M (Schaefer- Korting et al., 1984;Ito et al., 1998;Pelkonen et al., 1998;Hardman et al., 2001;Blanchard et al., 2004). Ketoconazole treatment has been found to completely inhibit CYP3A4 activity in vivo (Obach et al., 2006), but the cross-inhibition to P450s other than CYP3A4 has not yet been thoroughly shown in vivo as it has been in vitro. The cross-P450 isozyme inhibition by ketoconazole in vivo is usually overlooked when predicting DDIs between ketoconazole and CYP3A4 substrates, and ketoconazole is still treated as a selective CYP3A4 inhibitor in vivo.P450-reactive phenotyping is a quantitative measurement of the relative contributions of each P450 to the overall metabolism of a drug, when this drug is primar...

␥-Secretase is a multimeric complex consisted of presenilins (PSs) and three other proteins. PSs appear to be key contributors for the enzymatic center, the potential target of a number of recently developed ␥-secretase inhibitors. Using radiolabeled and unlabeled inhibitors as ligands, this study was aimed to determine the in situ distribution of ␥-secretase in the brain. Characterization using PS-1 knock-out mouse embryos revealed 50 and 80% reductions of ␥-secretase inhibitor binding density in the heterozygous (PS-1 ϩ/Ϫ ) and homozygous (PS-1 Ϫ/Ϫ ) embryos, respectively, relative to the wild type (PS-1). The pharmacological profile from competition binding assays suggests that the ligands may target at the N-and C-terminal fragments of PS essential for ␥-secretase activity. In the adult rat brain, the binding sites existed mostly in the forebrain, the cerebellum, and discrete brainstem areas and were particularly abundant in areas rich in neuronal terminals, e.g., olfactory glomeruli, CA3-hilus area, cerebellar molecular layer, and pars reticulata of the substantia nigra. In the developing rat brain, diffuse and elevated expression of binding sites occurred at the early postnatal stage relative to the adult. The possible association of binding sites with neuronal terminals in the adult brain was further investigated after olfactory deafferentation. A significant decrease with subsequent recovery of binding sites was noted in the olfactory glomeruli after chemical damage of the olfactory epithelium. The findings in this study support a physiological role of PS or ␥-secretase complex in neuronal and synaptic development and plasticity.

The in vitro and in vivo disposition of DPC 423 was investigated in mice, rats, dogs and humans and the metabolites characterized by LC/MS, LC/NMR and high field-NMR. The rodents produced several metabolites that included an aldehyde (M1), a carboxylic acid (M2), a benzyl alcohol (M3), glutamate conjugates (M4 and M5), an acyl glucuronide (M6) and its isomers; a carbamyl glucuronide (M7); a phenol (M8) and its glucuronide conjugate (M9), two glutathione adducts (M10 and M11), a sulfamate conjugate (M12), isomers of an oxime metabolite (M13), and an amide (M14). Humans and dogs produced less complex metabolite profiles than rats. While unchanged DPC 423 was the major component in plasma and urine samples, differences in the metabolic disposition of this compound among species were noted. M1 is believed to be rapidly oxidized to the carboxylic acid (M2), which forms the potentially reactive acyl glucuronide (M6). The formation of novel glutamate conjugates (M4 and M5) and their role in depleting endogenous glutathione have been described previously. The carbamyl glucuronide M7, found as the major metabolite in rats and in other species, was considered nonreactive and was easily hydrolyzed to the parent compound in the presence of beta-glucuronidase. The identification of GSH adducts M10 and M11 led us to postulate the existence of at least two reactive intermediates responsible for their formation, an epoxide and possibly a nitrile oxide, respectively. Although the formation of GSH adducts such as M10 from epoxides has been described before, there are no reports to date describing the existence of a GSH adduct (M11) of an oxime. The formation of a sulfamate conjugate (M12) formed by direct coupling of sulfate to the nitrogen of benzylamine is described. A mechanism is proposed for the formation of the oxime (M13) that involves sequential oxidation of the benzylamine to the corresponding hydroxylamine and nitroso intermediate. The rearrangement of the nitroso intermediate is believed to produce the oxime (M13). In vitro studies suggested that both the oxime (M13) and the aldehyde (M1) were precursors to the carboxylic acid (M2). This is the first demonstration of carboxylic acid formation via an oxime intermediate produced from an amine. The stability of DPC423 in plasma obtained from several species was studied. Significant species differences in the plasma stability of DPC 423 were observed. The formation of the aldehyde metabolite (M1) was found to be catalyzed by a semicarbazide-sensitive monoamine oxidase (SSAO) found in plasma of rabbits, dogs, and rhesus monkeys. Rat, chimpanzee, and human plasma did not form M1.