Thiorphan

All drug doses were calculated according to the active component of the salt. All drugs [morphine sulphate (3.75–120 mg kg⁻¹; Hameln Inc., Hameln, Germany), fentanyl citrate (0.2–4 mg kg⁻¹; Rotexmedica, Trittau, Germany), and naloxone hydrochloride (2 mg kg⁻¹; Ratiopharm, Ulm, Germany), used to precipitate withdrawal] were freshly prepared prior to use and were injected subcutaneously in lightly restrained, unanaesthetized mice at a volume of 10 μl g⁻¹ body weight. For chronic infusion with Alzet osmotic minipumps (1007D), fentanyl citrate salt (2 mg kg⁻¹ day⁻¹) and morphine sulphate salt pentahydrate (17 mg kg⁻¹ day⁻¹) were obtained from Sigma-Aldrich (St. Louis, MO). Drugs were diluted in phosphate-buffered saline for acute injections or dissolved in sterile water and then diluted in phosphate-buffered saline for osmotic pump delivery. For electrophysiology studies, MK-801 was obtained from HelloBio (Princeton, NJ). [Met⁵]enkephalin (ME), bestatin and thiorphan were from Sigma-Aldrich.

NEP activity measurements were performed on primary neurons after 15–28 days of in vitro (DIV15–28) culture as previously described [29 (link)]. Somatostatin (Peptide institute 4023), TT232 (Tocris 3493), recombinant ENSA (abcam ab92932), recombinant NSG-1 (Creative BioMart NSG1–332H), recombinant NUCKS-1 (Creative BioMart NUCKS1–10956M) and diazoxide (Wako 364-98-7) were added as appropriate concentrations, and cells were incubated for a further 24 h. Neurons were then incubated with substrate mixture 50 µM suc-Ala-Ala-Phe-MCA (Sigma S8758), 10 nM benzyloxycarbonyl Z-Leu-Leu-Leucinal (Peptide institute 3175-V) and cOmplete EDTA-Free-Protease inhibitor (Roche Diagnostics 4693132) in 0.2 M MES buffer (pH6.5) with or without Thiorphan (Sigma T6031) for 1 h at 37 ˚C. Following this, 0.1 mM phosphoramidon (Peptide Institute 4082) and 0.1 mg/ml leucine aminopeptidase (Sigma L-5006) were added, and the reaction mixture was incubated at 37 ˚C for a further 30 min. 7-Amino-4-methylcoumarin fluorescence was measured at excitation and emission wavelengths of 380 nm and 460 nm, respectively. Centrifugal 10 and 30 kDa filters (Merck UFC503096, 501096) were used to separate conditioned media obtained from cortical/hippocampal neurons.

Cultured neurons at 19–21 DIV were treated with different compounds before live cell imaging, immunofluorescence or western blot experiments: thiorphan (500 nM, 1 h; Sigma-Aldrich, Sweden), TTX [1 μM, acute (0–1 h) or 48 h], bicuculline [20 μM, acute (0–1 h) or 48 h], picrotoxin (10 μM, 1 h), CNQX (10 μM, 1 h) (Sigma-Aldrich, Sweden), and synthetic Aβ1-42 (Tocris, United Kingdom) and synthetic reverse Aβ42-1 (Tocris, United Kingdom) reconstituted in dimethyl sulfoxide (DMSO) to 250 mM, sonicated 10 min and then centrifuged at 10,000 g for 15 min before adding the supernatant to cell culture media.

All 36 investigated drugs and the two standard solutions (thiorphan and S-acetylthiorphan) were commercially purchased from Sigma-Aldrich (St. Louis, MO, United States), and the detailed information about these compounds is listed in Supplementary Table S1. Acetonitrile, methanol, dimethyl sulfoxide, formic acid, and ammonium acetate were HPLC grade, and along with KCl, KH₂PO₄, NaHCO₃, NaCl, MgCl₂(H2O)₆, (NH₄)₂CO₃, CaCl₂(H₂O)₂, L-cysteine, ethyl acetate, HCl, and NaOH were bought from Merck (Darmstadt, Germany). Bryant and Burkey Medium (BB), de Man Rogosa Sharpe (MRS) broth, glycerol, human salivary α-amylase, porcine pepsin, rabbit gastric extract for gastric lipase, bovine bile, and porcine pancreatin were purchased from Sigma-Aldrich (St. Louis, MO, United States). Modified Gifu anaerobic medium (mGAM) broth was purchased from HyServe GmbH and Co., KG (Germany). Ultrapure water used throughout the study was prepared by a Milli-Q water purification system (Millipore, Molsheim, France).

Whole-cell recordings were done with an Axopatch-1D amplifier in voltage-clamp mode (V_hold = −60 mV). Recording pipettes (1.7–2.1 MΩ, TW150F-3, World Precision Instruments, Saratosa, FL) were filled with internal solution containing (in mM): 115 potassium methanesulfonate or potassium methyl sulfate, 20 KCl, 1.5 MgCl2, 5 HEPES potassium salt, 2 BAPTA, 2 Mg-ATP 0.2 Na-GTP, pH 7.4, 275–280 mOsM. Series resistance was monitored without compensation and accepted for further analysis when < 15 MΩ. Data were collected at 400 Hz with PowerLab (Chart version 5.4.2, AD Instruments, Colorado Springs, CO). [Met⁵]enkephalin was applied by bath superfusion. Bestatin (10 μM, Sigma) and thiorphan (1 μM, Sigma) were included in enkephalin solution to prevent enzymatic degradation. The alpha-2-adrenoceptor agonist, UK14304 (3 µM) and the antagonist Idazoxan (1 µM) were used as controls.

The phycoerythrin (PE)-conjugated anti-CD10 monoclonal antibody (mAb) used for flow cytometric analysis (clone HI10a) was purchased from BD (Franklin Lakes, NJ, US). The CD10 enzymatic inhibitors (phosphoramidon and thiorphan), anticancer drugs (etoposide and gemcitabine), and mitomycin C were obtained from Sigma-Aldrich (St. Louis, Missouri, US). Cell Counting Reagent SF for cell proliferation assay and G418 for the selection of transfected cells were purchased from Nacalai Tesque (Kyoto, Japan).

SH-SY5Y cells seeded in 12-well plates (1 mL; cell density 1 × 10⁶ cells/mL) were incubated with vehicle or compound 8 for 12 h, then the medium was replaced with 400 μL of assay buffer containing 50 μM thiorphan (Sigma) and the cells incubated for 30 min, then 100 μL of the buffer was removed and mixed with 0.8 μL of 2 mM qf-Aβ(12-16)AAC, and the mixture returned to the well. The cells were then incubated for 1 h and 150 μL of the supernatant taken for fluorescence measurement as above.