Summary Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta42 levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta40 levels while concomitantly elevating levels of Abeta38 and Abeta37. Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta42; chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g. intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).

Alzheimer disease ␤-amyloid (A␤) peptides are generated via sequential proteolysis of amyloid precursor protein (APP) by BACE1 and ␥-secretase. A subset of BACE1 localizes to cholesterol-rich membrane microdomains, termed lipid rafts. BACE1 processing in raft microdomains of cultured cells and neurons was characterized in previous studies by disrupting the integrity of lipid rafts by cholesterol depletion. These studies found either inhibition or elevation of A␤ production depending on the extent of cholesterol depletion, generating controversy. The intricate interplay between cholesterol levels, APP trafficking, and BACE1 processing is not clearly understood because cholesterol depletion has pleiotropic effects on Golgi morphology, vesicular trafficking, and membrane bulk fluidity. In this study, we used an alternate strategy to explore the function of BACE1 in membrane microdomains without altering the cellular cholesterol level. We demonstrate that BACE1 undergoes S-palmitoylation at four Cys residues at the junction of transmembrane and cytosolic domains, and Ala substitution at these four residues is sufficient to displace BACE1 from lipid rafts. Analysis of wild type and mutant BACE1 expressed in BACE1 null fibroblasts and neuroblastoma cells revealed that S-palmitoylation neither contributes to protein stability nor subcellular localization of BACE1. Surprisingly, non-raft localization of palmitoylation-deficient BACE1 did not have discernible influence on BACE1 processing of APP or secretion of A␤. These results indicate that post-translational S-palmitoylation of BACE1 is not required for APP processing, and that BACE1 can efficiently cleave APP in both raft and non-raft microdomains.

Voriconazole is an effective antifungal drug, but adverse drug-drug interactions associated with its use are of major clinical concern. To identify the mechanisms of these interactions, we tested the inhibitory potency of voriconazole with eight human cytochrome P450 (CYP) enzymes. Isoform-specific probes were incubated with human liver microsomes (HLMs) (or expressed CYPs) and cofactors in the absence and the presence of voriconazole. Preincubation experiments were performed to test mechanism-based inactivation. In pilot experiments, voriconazole showed inhibition of CYP2B6, CYP2C9, CYP2C19, and CYP3A (half-maximal [50%] inhibitory concentrations, <6 M); its effect on CYP1A2, CYP2A6, CYP2C8, and CYP2D6 was marginal (<25% inhibition at 100 M voriconazole). Further detailed experiments with HLMs showed that voriconazole is a potent competitive inhibitor of CYP2B6 (K i < 0.5), CYP2C9 (K i ‫؍‬ 2.79 M), and CYP2C19 (K i ‫؍‬ 5.1 M). The inhibition of CYP3A by voriconazole was explained by noncompetitive (K i ‫؍‬ 2.97 M) and competitive (K i ‫؍‬ 0.66 M) modes of inhibition. Prediction of the in vivo interaction of voriconazole from these in vitro data suggests that voriconazole would substantially increase the exposure of drugs metabolized by CYP2B6, CYP2C9, CYP2C19, and CYP3A. Clinicians should be aware of these interactions and monitor patients for adverse effects or failure of therapy.Voriconazole, a derivative of fluconazole, belongs to the second generation of triazole antifungal drugs and has improved potency and a spectrum of antifungal activity that is expanded compared with the potency and activity of fluconazole (56, 59). Currently, orally or intravenously administered voriconazole is considered the first-line therapy for invasive aspergillosis (39,56,59). In addition, voriconazole is widely used for the management of patients infected with a broad range of other fungal pathogens, particularly patients who are intolerant of or who developed resistance to other conventional antifungal therapies (59).However, despite its proven efficacy, the goal of optimal therapy with voriconazole is made difficult by the occurrence of clinically important drug-drug interactions. Several clinical studies and case reports have documented that voriconazole substantially reduces the clearance of several drugs, including warfarin (49), phenytoin (48), midazolam (53), diazepam (52), immunosuppressant drugs (cyclosporine, sirolimus, and tacrolimus) (46, 47), efavirenz (37), methadone (36), ibuprofen (27), diclofenac (26), fentanyl and alfentanil (54), oxycodone (22), and omeprazole (47). Considering the mechanisms of clearance of the drugs affected (3), many of these drug-drug interactions appear to be attributable to pharmacokinetic changes that can be understood in terms of inhibition of the cytochrome P450 (CYP) system. Indeed, in vitro studies by Niwa et al (41,43) have documented that voriconazole inhibits CYPs 2C9, 2C19, and 3A, while its effect on the activity of other CYPs (CYPs 1A2, 2D6, and 2E1) was marginal. However, th...

Despite the promising neuro-regenerative capacities of stem cells, there is currently no licensed stem cell-based product in the repair and regeneration of peripheral nerve injuries. Here, we explored the potential use of human gingiva-derived mesenchymal stem cells (GMSCs) as the only cellular component in 3D bio-printed scaffold-free neural constructs that were transplantable to bridge facial nerve defects in rats. We showed that GMSCs have the propensity to aggregate into compact 3D-spheroids that could produce their own matrix. When cultured under either 2D- or 3D-collagen scaffolds, GMSC spheroids were found to be more capable of differentiating into both neuronal and Schwann-like cells than their adherent counterparts. Using a scaffold-free 3D bio-printer system, nerve constructs were printed from GMSC spheroids in the absence of exogenous scaffolds and allowed to mature in a bioreactor. In vivo transplantation of the GMSC-laden nerve constructs promoted regeneration and functional recovery when used to bridge segmental defects in rat facial nerves. Our findings suggest that GMSCs represent an easily accessible source of MSCs for 3D bio-printing of scaffold-free nervous tissue constructs with promising potential application for repair and regeneration of peripheral nerve defects.