KDM1/LSD1

In all four studied RMS cell lines (RD, RH30, RMS13, and TE381.T cells), GSK690 (1-10 μM) and JNJ-26481585 work synergistically to cause cell death[2]. Pro- and antiapoptotic protein balance is altered by GSK690/JNJ-26481585 cotreatment, with 1 μM GSK690 for RD cells and 10 μM GSK690 for RH30 cells[2]. GSK690/JNJ-26481585 cotreatment causes caspase-dependent cell death in RH30 cells at a concentration of 10 μM and RD cells at a concentration of 1 μM [2]. The G2/M arrest induced by JNJ-26481585 is further enhanced by the addition of GSK690 [2].

LSD1 Enzymatic Assay[1]
Assays were performed in Corning 384-well low-flange white flat-bottom polystyrene (no. 3574) microplates in a 10 μL reaction volume consisting of 50 mM TrisHCl, 50 mM NaCl, 1 mM DTT, 0.01% Tween-20, and 1% DMSO with or without compound in a 10-point, 3-fold dilution series, 0.2 μM histone H3(1–21)K4(Me1) biotin peptide substrate and 1 nM LSD1. The reaction was allowed to proceed for 30 min at 25 °C before stopping the reaction with the addition of 0.3 mM tranylcypromine in LANCE detection buffer and quantifying the level of demethylated peptide by the addition of 1 nM europium-α-unmodified H3K4 antibody and 25 nM ULight streptavidin, also in LANCE detection buffer. Following a further 60 min incubation period, the TR-FRET signal was read on a PHERAstar FS plate reader with excitation at 340 nm and emission at 665 nm.

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SPR Binding Assay[1]
Direct binding between compounds and LSD1 was assessed by SPR using Biacore T200, S200, and S51 instruments. LSD1 was immobilized to CM5 or CM7 chips using amine coupling. Interaction experiments were performed at 15 °C in 10 mM HEPES, pH 7.4, 150 mM NaCl, 1% DMSO, 0.05% Tween, at a flow rate of 30 mL/min. Compounds were diluted in the buffer and injected for 15–25 s at increasing concentrations over the prepared surfaces. Sensorgrams were double-referenced by subtracting signals from untreated reference channel and responses from blank injections. Affinities were derived by either dose–response analysis of steady-state responses or regressions analysis of whole sensorgrams (1:1 interaction model including a term for mass transport limitation) using the T200 evaluation software 3.0

Transduction[2]
For BCL-2 overexpression, Phoenix packaging cells were transfected with 20 μg of murine stem cell virus (pMSCV) vector containing murine BCL-2 (mBCL-2) or EV using calcium phosphate transfection as described previously.39 Stable cell lines were generated by lentiviral transduction and selected with 10 μg/ml Blasticidin. For MCL-1 overexpression, cells were transfected with 20 μg of pCMV-Tag3B plasmids, kindly provided by Genentech Inc., containing EV, wild-type MCL-1 (WT), or phospho-mutant MCL-1 (4A) using Lipofectamine 2000 and selected with 0.5 mg/ml G418.

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RNA interference[2]
For transient knockdown by siRNA, cells were reversely transfected with 10 nM (BMF, BIM, NOXA) and 20 nM (BAK) SilencerSelect siRNA using Lipofectamine RNAiMAX reagent and OptiMEM. Following siRNA constructs were used: control siRNA (4390842) and targeting siRNAs (s40385 and s40387 for BMF, s195012 and s223065 for BIM, s10708 and s10709 for NOXA, s1880 and s1881 for BAK).

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Western blot analysis[2]
Western blot analysis was performed as previously described using the following antibodies: mouse anti-NOXA, rat anti-BMF, rabbit anti-MCL-1, mouse anti-BCL-2, rabbit anti-BAK, rabbit anti-caspase-3, rabbit anti-caspase-9, rabbit anti-BIM, mouse anti-PARP, rabbit-anti PUMA, rabbit-anti BCL-xL, mouse anti-GAPDH, rabbit anti-H3K4me2, rabbit anti-acetylated histone H3 and mouse anti-histone H3 or mouse anti-β-Actin. Goat anti-mouse, goat anti-rabbit and goat anti-rat IgG conjugated to horseradish peroxidase and enhanced chemiluminescence or infrared dye-labeled secondary antibodies and infrared imaging were used for detection. For detection of histone modifications cells were lysed using RIPA buffer supplemented with Pierce Nuclease. Representative blots of at least two independent experiments are shown.

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Quantitative real-time PCR[2]
Total RNA was isolated by using peqGOLD Total RNA kit according to the manufacturer's instructions. For cDNA-synthesis, 1 μg of total RNA was used to synthesize the corresponding cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit according to the manufacturer's protocol with the use of the random primers. For quantification of gene expression levels, SYBR-green based quantitative real-time PCR was performed using the 7900GR fast real-time PCR system. Data were normalized on 28S-rRNA expression as a reference. Analysis of the melting curves served as control for the specificity of the amplified products. Relative expression levels of the target transcript were calculated compared to the reference transcript by using the ΔΔct-method. At least three independent experiments in duplicate were performed for each gene.

[1]. Development of (4-Cyanophenyl)glycine Derivatives as Reversible Inhibitors of Lysine Specific Demethylase 1. J Med Chem. 2017 Oct 12;60(19):7984-7999.

[2]. Concomitant epigenetic targeting of LSD1 and HDAC synergistically induces mitochondrial apoptosis in rhabdomyosarcoma cells. Cell Death Dis. 2017 Jun 15;8(6):e2879.

Inhibition of lysine specific demethylase 1 (LSD1) has been shown to induce the differentiation of leukemia stem cells in acute myeloid leukemia (AML). Irreversible inhibitors developed from the nonspecific inhibitor tranylcypromine have entered clinical trials; however, the development of effective reversible inhibitors has proved more challenging. Herein, we describe our efforts to identify reversible inhibitors of LSD1 from a high throughput screen and subsequent in silico modeling approaches. From a single hit (12) validated by biochemical and biophysical assays, we describe our efforts to develop acyclic scaffold-hops from GSK-690 (1). A further scaffold modification to a (4-cyanophenyl)glycinamide (e.g., 29a) led to the development of compound 32, with a Kd value of 32 nM and an EC50 value of 0.67 μM in a surrogate cellular biomarker assay. Moreover, this derivative does not display the same level of hERG liability as observed with 1 and represents a promising lead for further development.[1]

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The lysine-specific demethylase 1 (LSD1) is overexpressed in several cancers including rhabdomyosarcoma (RMS). However, little is yet known about whether or not LSD1 may serve as therapeutic target in RMS. We therefore investigated the potential of LSD1 inhibitors alone or in combination with other epigenetic modifiers such as histone deacetylase (HDAC) inhibitors. Here, we identify a synergistic interaction of LSD1 inhibitors (i.e., GSK690, Ex917) and HDAC inhibitors (i.e., JNJ-26481585, SAHA) to induce cell death in RMS cells. By comparison, LSD1 inhibitors as single agents exhibit little cytotoxicity against RMS cells. Mechanistically, GSK690 acts in concert with JNJ-26481585 to upregulate mRNA levels of the proapoptotic BH3-only proteins BMF, PUMA, BIM and NOXA. This increase in mRNA levels is accompanied by a corresponding upregulation of BMF, PUMA, BIM and NOXA protein levels. Importantly, individual knockdown of either BMF, BIM or NOXA significantly reduces GSK690/JNJ-26481585-mediated cell death. Similarly, genetic silencing of BAK significantly rescues cell death upon GSK690/JNJ-26481585 cotreatment. Also, overexpression of antiapoptotic BCL-2 or MCL-1 significantly protects RMS cells from GSK690/JNJ-26481585-induced cell death. Furthermore, GSK690 acts in concert with JNJ-26481585 to increase activation of caspase-9 and -3. Consistently, addition of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) significantly reduces GSK690/JNJ-26481585-mediated cell death. In conclusion, concomitant LSD1 and HDAC inhibition synergistically induces cell death in RMS cells by shifting the ratio of pro- and antiapoptotic BCL-2 proteins in favor of apoptosis, thereby engaging the intrinsic apoptotic pathway. This indicates that combined treatment with LSD1 and HDAC inhibitors is a promising new therapeutic approach in RMS.[2]

C24H24CLN3O

405.919864654541

405.16

2436760-79-9

GSK 690;2101305-84-2

135397147

Typically exists as Light yellow to yellow solids at room temperature

2

4

5

29

527

1

Cl.O(C1C=NC(C2C=CC(C)=CC=2)=C(C2C=CC(C#N)=CC=2)C=1)C[C@H]1CNCC1

DBTSJYXXWZFXNJ-FSRHSHDFSA-N

InChI=1S/C24H23N3O.ClH/c1-17-2-6-21(7-3-17)24-23(20-8-4-18(13-25)5-9-20)12-22(15-27-24)28-16-19-10-11-26-14-19;/h2-9,12,15,19,26H,10-11,14,16H2,1H3;1H/t19-;/m1./s1

4-[2-(4-methylphenyl)-5-[[(3R)-pyrrolidin-3-yl]methoxy]pyridin-3-yl]benzonitrile;hydrochloride

GSK 690 (Hydrochloride); 2436760-79-9; EX-A8792;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 125 mg/mL (307.94 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.12 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)