Unnatural Amino Acid-Substituted (Hydroxyethyl)urea Peptidomimetics Inhibit -Secretase and Promote the Neuronal Differentiation of Neuroblastoma Cells

-Secretase, exhibiting characteristics of aspartyl protease, mediates the intramembranous proteolysis of -amyloid precursor protein (APP) and Notch, and it is considered to be a prime pharmacological target in the development of therapeutics for Alzheimer’s disease (AD). To identify compounds that block -secretase-mediated proteolysis, we used a highly sensitive cell-based reporter gene assay for -secretase in which Gal4/VP16-tagged C99-APP was expressed as the immediate substrate of -secretase, and Gal4/VP16-tagged APP intracellular domain released by the -secretase cleavage then activated the expression of the Gal4-driven luciferase reporter gene. Using this reporter assay, we demonstrated that the newly synthesized (hydroxyethyl)urea peptidomimetics, which contain unnatural amino acid moieties at positions P1 and/or P3 , can effectively inhibit -secretase activity and significantly reduce A production. The -secretase-dependent S3 cleavage of Notch was also consistently blocked by these (hydroxyethyl)ureas as evidenced by the decreased generation of the Notch intracellular domain, a prerequisite for the activation of Notch signaling. The inhibition of Notch signaling by active Jia compounds efficiently promotes the neuronal differentiation of neuroblastoma cells, intervening in tumorigenesis and the malignancy of neuroblastomas. Our results suggest that (hydroxyethyl)urea peptidomimetics containing unnatural amino acid substitutions could represent a novel class of -secretase inhibitors with enhanced stability, providing the basis for the further development of effective therapeutics for AD and neuroblastomas. -Secretase catalyzes the final proteolytic step in the generation of A (the principal constituent of senile plaques in the AD brain), and it has thus been regarded as a prime therapeutic target for AD. Mounting evidence from pharmacological studies, mutagenesis, affinity labeling, and biochemical isolation strongly suggests that -secretase is an aspartyl protease and that the active site of -secretase is located at the interface of the presenilin (PS) heterodimer (Wolfe and Haass, 2001). The heterodimeric PS consisting of an 30-kDa N-terminal fragment (NTF) and a 20-kDa C-terminal fragment (CTF) is thought to be the active form of PS (Capell et al., 1998). Peptidomimetics that mimic the This study was supported by National Science Council, Taiwan, grants NSC 93-2320-B-001-037, NSC 94-2320-B-001-004, NSC 94-3112-B-001-001, and NSC 95-3112-B-001-006 (to Y.-F.L.) and NSC 92-2320-B-016-046 (to M.-K.H.) and Academia Sinica (to Y.-F.L.). Proteomic mass spectrometry analyses performed by the Core Facilities for Proteomics Research located at the Institute of Biological Chemistry, Academia Sinica, were supported by National Science Council Grant NSC 91-3112-P-001-009-Y and the Academia Sinica. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.024299. ABBREVIATIONS: AD, Alzheimer’s disease; PS, presenilin; NTF, N-terminal fragment; CTF, C-terminal fragment; NICD, Notch intracellular domain; HIV, human immunodeficiency virus; ELISA, enzyme-linked immunosorbent assay; HEK, human embryonic kidney; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; APP, amyloid precursor protein; C99, 99-residue C-terminal fragment of amyloid precursor protein; PBS, phosphate-buffered saline; PLB, passive lysis buffer; DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]S-phenylglycine t-butyl ester; GV, Gal4/VP16 DNA binding domain; GAP-43, growth-associated protein 43; PAGE, polyacrylamide gel electrophoresis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBST, phosphate-buffer saline/Tween 20; A , -amyloid; HRP, horseradish peroxidase; CHAPSO, 3-[(3-cholamidopropyl)dimethyl-ammonio]-2-hydroxy-1-propanesulfonate; siRNA, small interfering RNA; RNAi, RNA interference; ESI, electrospray ionization; MS, mass spectrometry, MS/MS, tandem mass spectrometry; LC, liquid chromatography; OD, optical density; Chy, cyclohexylmethyl; RA, retinoic acid; L-685–458, (2R-(2R*,4R*,5S*))N-(5-(((1,1-dimethylethoxy)carbonyl)amino)-4-hydroxy-1-oxo-6phenyl-2-(phenylmethyl)hexyl)-L-leucyl-L-phenylalaninamide. 0026-895X/07/7102-588–601$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 2 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 24299/3173365 Mol Pharmacol 71:588–601, 2007 Printed in U.S.A. 588 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from transition state of aspartyl protease catalysis have been derived from sequences around the -cleavage site of APP and are shown to block -secretase activity, indicating that -secretase is an aspartyl protease (Wolfe et al., 1999a). Two highly conserved aspartate residues residing within the sixth and seventh transmembrane domains of presenilins are required for -secretase activity (Wolfe et al., 1999b), suggesting that the active site of this novel aspartyl protease might be located at the heterodimeric interface. A number of compounds designed to interact with the protease active site have been shown to bind directly to both PS subunits (Esler et al., 2000; Li et al., 2000b). Together, these findings strongly support the notion that PS heterodimers constitute the active site of -secretase. Biochemical and genetic analyses further reveal that PSs are part of a multimeric -secretase complex whose constituents include a heterodimeric PS, a mature glycosylated nicastrin (NCT), Aph-1, and Pen-2 (Iwatsubo, 2004). Compelling evidence has shown that the full spectrum of -secretase activity can be reconstituted by the coexpression of human PS, nicastrin, Aph-1, and Pen-2 in yeast (Edbauer et al., 2003), providing definitive proof for the minimal required constituents of a functional -secretase. The -secretase not only mediates the proteolysis of APP but also is critical for the processing of Notch receptor. The Notch signaling pathway is essential for cell fate decisions during development (Mumm and Kopan, 2000). This pathway is initiated by the binding of the Delta-Serrate-Lag2 ligand family, followed by the shedding of extracellular domain (S2 cleavage) mediated by the ADAM family members. The resultant C-terminal membrane-tethered fragment of Notch, termed Notch extracellular membrane truncation (NEXT), is then cleaved within the transmembrane domain (S3 cleavage) by -secretase to release the Notch intracellular domain (NICD). Once released from membrane, NICD bounds to mammalian CBF1/RBPj, Drosophila melanogaster Suppressor of hairless, and Caenorhabditis elegans Lag-1 DNA-binding proteins and converts them from transcriptional repressors to activators, resulting in the expression of Notch downstream target genes. The inhibition of -secretase would likewise block NICD production and reduce Notch signaling (Wolfe et al., 1999b, 2002). (Hydroxyethyl)urea peptidomimetics have recently been identified as a new class of transition state analog inhibitors that mimic the transition state of aspartyl protease catalysis and block -secretase much more efficiently than any of the difluoro ketones or difluoro alcohols (Wolfe et al., 2002). Highly potent and selective inhibitors containing hydroxyethyl isostere have also been developed and used to block such aspartyl proteases as renin and HIV protease (Greenlee, 1990; Huff, 1991). Five HIV protease inhibitors currently available as approved treatments for AIDS all contain the hydroxyethyl isostere (Flexner, 1998), and the core structure of (hydroxyethyl)ureas is known to have apparent low toxicity, a prerequisite for clinical applications. (Hydroxyethyl)urea peptidomimetics have been developed for the affinity isolation and characterization of -secretase, and for probing the active site of this protease (Esler et al., 2004). These compounds can be generated through systemic replacements in five positions (P2, P1 , P2 , P3 , and P4 ) with small, medium, and large hydrophobic L-amino acids (Wolfe et al., 2002). Furthermore, (hydroxyethyl)urea peptidomimetics systematically altered at positions P2-P3 either with hydrophobic Lor D-amino acids are shown to effectively block -secretase activity (Bakshi and Wolfe, 2004; Esler et al., 2004). These studies not only substantiate the loose sequence specificity of -secretase but also raise the possibility that -secretase could be targeted by transition state analog inhibitors encompassing unnatural amino acids that can confer peptidomimetics extraordinary biological stability along with enhanced biological activity and proteolytic resistance. Herein, we report the development of (hydroxyethyl)urea peptidomimetics containing unnatural amino acid substitutions that can effectively block -secretase activity for lowering A production and attenuating Notch signaling so as to promote the neuronal differentiation of neuroblastoma cells. Our studies provide the proof-of-concept for the use of unnatural amino acids as building blocks in the generation of biologically stable -secretase inhibitors for AD treatments and the promotion of neuronal differentiation. Materials and Methods Reagents. The BCA protein assay reagent kit and SuperSignal West Pico and SuperSignal West Dura reagents were purchased from Pierce Chemical (Rockford, IL). The rabbit anti-Notch(Val1744) antibody was from Cell Signaling Technology Inc. (Danvers, MA). Horseradish peroxidase-conjugated anti-rabbit IgG was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidaseconjugated anti-mouse IgG and ECL Western Blotting detection reagents were from GE Healthcare (Little Chalfont, Buckinghamshire, UK). The FuGENE6 transfection reagent, Expand long template polymerase chain reaction system, and polymerase chain reaction nucleotide mix were from Roche Applied Science (Indianapolis, IN). Dual luciferase assay reagents, Steady-Glo luciferase assay reagents, and the pRL-TK vector were from Promega (Madison, WI). Human A 40 and A 42 colorimetric ELISA kits were from BioSource International (Camarillo, CA). All other reagents were of at least reagent grade and were obtained from standard suppliers. Synthesis of (Hydroxyethyl)ureas. The (hydroxyethyl)urea peptidomimetics containing unnatural amino acid moieties were synthesized using methods described previously with some modifications (Getman et al., 1993) (Scheme 1). In brief, a commercially available Boc-protected amino acid derivative 1, which fits the desired structural feature of the P1 residue, was reduced to -aminoaldehyde 2 via the Weinreb amide and then transferred to the corresponding epoxide 4 through alkene 3. The epoxide was ringopened by treatment with benzylamine or cyclohexylmethylamine under heated reflux to give the amino alcohols 7 and 8. These intermediates were condensed with isocyanates 9a and 9b (in turn obtained from -amino methyl esters and phosgene) at room temperature to provide the (hydroxyethyl)urea P1-P2 isosteres 10a, 10b, and 11. C-Terminal incorporation of P3 residues was accomplished by hydrolysis of the methyl ester with LiOH and subsequent coupling of P3 residues 14a g with the HATU coupling reagent in DMF to yield the desired (hydroxyethyl)urea isosteres (see Table 1 as Jia compounds). In this synthesis scheme, the epoxide was treated with a variety of alkylamines that came up with P1-P1 isosteres in which the P1 site was modified with nitrogen at the backbone and an unnatural cyclohexyl side chain that is not seen in essential amino acids. Moreover, the selected amines and unnatural amino acid derivatives 14a g were incorporated to the P1-P2 fragments to yield entire P1-P3 (hydroxyethyl)ureas. Through this synthesis scheme, a collection of (hydroxyethyl)urea peptidomimetics that contained unnatural amino acid moieties mainly distributed either at the P1 and/or P3 positions was generated. The characterization and the purity of each compound were confirmed by H NMR spectros-Secretase Inhibitors Induce Neuronal Differentiation 589 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from copy, high-resolution mass spectroscopy, and high-pressure liquid chromatography. Cell Culture and Cell Lines. Human embryonic kidney (HEK)293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.1 mg/ml penicillin and streptomycin. T-REx293 cells were purchased from Invitrogen and cultured in DMEM supplemented with 10% FBS and 5 g/ml blasticidin. The generation of stably transfected cell lines, T16, T20, N7, and -30 has been described previously (Kimberly et al., 2003; Liao et al., 2004; Bakshi et al., 2005). Cells of the SH-SY5Y human neuroblastoma cell line were grown in DMEM/Ham’s F-12 supplemented with 10% FBS. Cells were incubated in a humidified incubator at 37°C in 5% CO2. Cell-Based -Secretase Assays. The generation of these stable cell lines has been reported previously (Liao et al., 2004). To examine the inhibitory effects of the compounds specifically on -secretase, T16 or T20 cells were trypsinized and washed with serum-free DMEM before plating onto 12-well microplates in 1 ml/well DMEM supplemented with 10% FBS at 5 10 cells/well. After incubation at 37°C overnight, cells were treated with 10 M concentrations of individual (hydroxyethyl)urea peptidomimetics in culture medium containing 1% DMSO and 1 g/ml tetracycline, the inducer of APP695-Gal4/VP16 and the 99-residue C-terminal fragment of APPGal4/VP16 (C99-GV) expressions, and they were incubated at 37°C for 24 h or various intervals as specified. For the dose-response studies, various concentrations of (hydroxyethyl)urea peptidomimetics were included in the medium as specified. Cells incubated with the culture medium containing 1 g/ml tetracycline and 1% DMSO were used to define the basal level of -secretase activity, whereas cells treated with DMSO-containing medium without tetracycline were used to estimate the nonspecific background emission of the luciferase signal. To terminate the reactions, cells were harvested with PBS containing 20 mM EDTA and lysed in 100 l of 1 passive lysis buffer (PLB; Promega). Cell debris was removed by centrifugation at 13,200g for 5 min, and luciferase activity in clarified lysates was determined by mixing 20 l of lysates and 20 l of Steady-Glo luciferase assay reagent in a 96-well LumiNunc microplate (Nalge Nunc, International, Rochester, NY). After incubation at room temperature for 5 min, emitted luminescence in individual microwells was determined by an MLX Microplate luminometer (Dynex Technologies, Chantilly, VA) and subsequently normalized by the protein content of the lysates. The protein content of clarified lysates was determined using the BCA protein assay kit (Pierce Chemical) following the manufacturer’s instructions. The normalized luciferase signal emitted by T20 cells in regular culture medium without tetracycline was referred as 1-fold of activation. Known -secretase inhibitors, such as compound E and DAPT (Seiffert et al., 2000; Dovey et al., 2001), were included as positive controls of -secretase