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5 protocols using ory 1001

1

Evaluation of LSD1 Inhibitors in Research

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GSK-LSD1 (CAS #2102933–95-7), SP-2577 (CAS #1423715–37-0), and IMG-7289 (CAS #1990504–72-7) were provided by GlaxoSmithKline, Salarius Pharmaceuticals, and Imago BioSciences, respectively through materials transfer agreements. TCP and RN-1 were purchased through Enzo Life Sciences (Farmingdale, NY) and Cayman Chemical (Ann Arbor, MI). ORY-1001 and ORY-2001 were purchased through Cayman Chemical (Ann Arbor, MI) and MedChemExpress (Monmouth Junction, NJ). All LSD1 inhibitors were diluted per manufacturer’s instructions, aliquoted into single-use vials, and stored at −80°C. Table 1 illustrates the chemical structures for LSD1 inhibitors utilized in this study.
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2

ORY-1001 Injection Improves Maze Memory

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ORY-1001 (Cayman Chemical, 19136) was dissolved in PBS at 1mg/ml by vortexing. Mice at 17–20 g body weight were injected 9.0 μg/kg/day; mice at 20–28g body weight were injected 10.0 μg/kg/day. Adult animals were injected for 14 consecutive days followed by a non-match to sample T-maze task. During the behavior task, mice were injected with the same dose at 6 pm daily.
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3

Pharmacokinetics of LSD1 Inhibitor Derivatives

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LSD1 inhibitors used in this study include RN-1 (Calbiochem, San Diego, CA), ORY-1001 (Cayman Chemical, Ann Arbor, MI), OG-L002 (Selleck Chemicals, Houston, TX), S2101 (Millipore, Temecula, CA) and two N-alkylated derivatives of S2101, S2116 and S2157 (synthesized by Tokyo Chemical Industry, Tokyo, Japan) (15 (link)). A pharmacokinetic study was conducted with a single intraperitoneal injection of either S2116 or S2157 at 50 mg/kg to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice (Charles River Laboratories, Wilmington, MA), followed by serial sampling of blood at 1, 2, 4, 8 and 24 hours after administration. Brain tissues were harvested at 0.5 hour and homogenized in deionized water. Plasma and brain samples were subjected to LC-MS/MS analysis to determine the concentrations of each drug.
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4

Synaptic and Ion Channel Modulation

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Synaptic and ion channel inhibition experiments were performed with bicuculline (10 μM, Cat# 14340, Sigma), picrotoxin (10 μM, Cat# P1675, Sigma), NBQX (10 μM, Cat# N183, Sigma), D-APV (40 μM, Cat# A8054, Sigma), Nifedipine (10 μM, Cat# N7634, Sigma), TTX (1 μM, Cat# 1078, Tocris) and Mefloquine (25 μM, Cat# M2319, Sigma). The rescue experiments were performed with Tranylcypromine (TCP, 600 nM, Sigma, P8511) and ORY-1001 (0.6–0.9 nM, Cayman Chemical, 19136). All the compounds were added to the culture media as described in the results.
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5

Evaluating M-CSF, RANKL, and TNF Signaling

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Human and murine M-CSF, sRANKL, and TNF were purchased from Peprotech (Rocky Hill, NJ, USA). Inhibitors, SP2509, RN-1, ORY-1001, and Torin1, were purchased from Cayman Chemical Company (Ann Arbor, MI). The antibodies used were as follows: LSD1, phospho-NFkB p65, Phospho-4E-BP1, HIF1a, E2F1, PHD-2 (Cell Signaling Technologies, Danvers, MA), p38, c-Myc (BioLegend, San Diego, California) and Swine Anti-Rabbit Immunoglobulins/HRP (DAKO, Hovedstaden, Denmark).
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