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Purity: ≥98%
GSK 690 is a novel, potent and selective reversible inhibitor of lysine specific demethylase 1 (LSD1), with a Kd value of 9 nM and a biochemical IC50 of 37 nM. 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.
| Targets |
LSD1/lysine specific demethylase 1
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|---|---|
| ln Vitro |
Cell death is induced in all four RMS cell lines tested (RD, RH30, RMS13, and TE381.T cells) by GSK690 (1-10μM) in conjunction with JNJ-26481585-206. GSK690/JNJ-26481585 co-treatment, which uses 1 μM GSK690 for RD cells and 10 μM GSK690 for RH30 cells, alters the ratio of pro- to anti-apoptotic proteins [2]. GSK690/JNJ-26481585 co-treatment uses 1 μM GSK690 for RD cells and 10 μM GSK690 for RH30 cells to cause caspase-dependent cell death [2]. The G2/M blockage induced by JNJ-26481585 was further improved by the addition of GSK690 [2].
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| Enzyme Assay |
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. 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 |
| Cell Assay |
Clonogenic Assays[1]
Clonogenic assays of human leukemic cell lines were performed in methylcellulose medium with no supplemental growth factors. Cells were seeded at 2.5 × 103 and colonies enumerated after 7 days in culture. CD86 and Apoptosis Assays[1] For CD86 assays, leukemic cell lines were incubated in culture medium containing compounds or DMSO (vehicle control) for 48 h at a density of 1 × 105/mL. Cells were pelleted, resuspended in 100 mL of SM buffer (Phenol red free RPMI, 5 mM EDTA, and 2% BSA) containing 0.25 μL of CD86-PerCP EF710 (Clone IT2.2) and incubated at 4 °C for 10 min. Cells were washed, resuspended in SM buffer, and analyzed by flow cytometry. To determine the effects of compounds on apoptosis, leukemic cell lines were incubated with 10 μM compound or vehicle for 4 days. Apoptosis was assessed using a BD Pharmingen PerCP-Cy5.5 annexin V/7-AAD Kit according to manufacturer’s instructions. All FACS analyses were performed using either an LSR model II or NovoCyte flow cytometer or FACSArray bioanalyzer |
| References |
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| Additional Infomation |
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]
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| Molecular Formula |
C24H23N3O
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|---|---|
| Molecular Weight |
369.458925485611
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| Exact Mass |
369.184
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| Elemental Analysis |
C, 78.02; H, 6.27; N, 11.37; O, 4.33
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| CAS # |
2101305-84-2
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| Related CAS # |
GSK 690 Hydrochloride;2436760-79-9
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| PubChem CID |
72793898
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
530.5±50.0 °C at 760 mmHg
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| Flash Point |
274.6±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.640
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| LogP |
3.75
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
28
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| Complexity |
527
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O(C1=CN=C(C2C=CC(C)=CC=2)C(C2C=CC(C#N)=CC=2)=C1)C[C@H]1CNCC1
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| InChi Key |
IQVDLEXWAPYWDT-LJQANCHMSA-N
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| InChi Code |
InChI=1S/C24H23N3O/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/t19-/m1/s1
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| Chemical Name |
(R)-4-[5-(Pyrrolidin-3-ylmethoxy)-2-p-tolyl-pyridin-3-yl]-benzonitrile
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| Synonyms |
GSK690; GSK 690; CHEMBL3134377; GSK-690; (R)-4-(5-(Pyrrolidin-3-ylmethoxy)-2-(p-tolyl)pyridin-3-yl)benzonitrile; SCHEMBL18952126; GSK 690; GSK-354; GSK-690.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7067 mL | 13.5333 mL | 27.0665 mL | |
| 5 mM | 0.5413 mL | 2.7067 mL | 5.4133 mL | |
| 10 mM | 0.2707 mL | 1.3533 mL | 2.7067 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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