LSD1/lysine specific demethylase 1 (IC50 = 16 nM)
KDM1A/LSD1 (lysine-specific demethylase 1) [1]
GSK-LSD1 2HCl targets lysine-specific demethylase 1A (LSD1/KDM1A), a flavin-dependent enzyme that specifically demethylates mono- and dimethylated histone H3 at lysine 4 (H3K4me1/2) and lysine 9 (H3K9me1/2). It acts as an irreversible inhibitor of LSD1. The compound shows >1000-fold selectivity over other FAD-utilizing enzymes, including LSD2, MAO-A, and MAO-B. By inhibiting LSD1, GSK-LSD1 2HCl modulates gene expression and can induce differentiation or apoptosis in cancer cells.

GSK-LSD1 induces gene expression changes in cancer cell lines with average EC50 of < 5 nM and inhibits cancer cell line growth with average EC50 of < 5 nM.
Kinase Assay: GSK-LSD1 Dihydrochloride is a potent, selective and irreversible lysine specific demethylase 1 (LSD1) inhibitor with an IC50 of 16 nM.
Cell Assay: GSK-LSD1 2HCl irreversibly inhibited LSD1 with IC50 value of 16 nM and is > 1000 fold selective over LSD2, MAO-A and MAO-B, which were related to FAD utilizing enzymes. In cancer cell lines, GSK-LSD1 2HCl changed gene expression with average EC50 value < 5 nM and inhibited cells growth with average EC50 value < 5 nM. GSK-LSD1 2HCl (10 ?M) inhibited human recombinant dopamine transporter, 5-HT1A and 5-HT transporter by 39%, 49% and 74%, respectively. And no activity against other 55 recombinant receptors (GPCR, transporters, ion channels). GSK-LSD1 2HCl can be used as a chemical probe and a part of the SGC epigenetics.

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GSK-LSD1 2HCl (CAS#: 1821798-25-7) induces accumulation of LC3-II in U2OS cells in a concentration-dependent manner (Fig. 3B).
Immunofluorescent staining shows that GSK-LSD1 2HCl (CAS#: 1821798-25-7) treatment leads to formation of LC3 aggregates and their co-localization with lysosomes (Lyso-tracker), indicating autolysosome formation (Fig. 3C).
Transmission electron microscopy reveals autophagosome formation after GSK-LSD1 2HCl (CAS#: 1821798-25-7) treatment (Fig. 3D). The concentration used is 200 μM for 12 hours.
Table 1 notes that GSK-LSD1 2HCl (CAS#: 1821798-25-7) is a potent and selective inhibitor of LSD1 and potently inhibits proliferation of various cancer cell lines by changing gene expression patterns. [1]
In vitro, GSK-LSD1 2HCl irreversibly inhibits LSD1 with an IC50 of 16 nM. It exhibits >1000-fold selectivity over LSD2, MAO-A, and MAO-B. In cancer cell lines, the compound demonstrates antiproliferative activity. It binds to LSD1 in human A549 cells, as assessed by an increase in stability of cellular LSD1 protein at 10 μM pre-incubated for 1 hour followed by heating at 49°C or 52°C for 3 minutes by Western blot-based CESTA.

To assess the activity of LSD1 inhibition in vivo, secondary recipient mice engrafted with 1 × 10~5 MLL-AF9 primary AML cells were treated with GSK-LSD1. The drug was administered daily during a 14-day treatment window at a dose of 0.5 mg/kg. Treatment was initiated only after peripheral blood engraftment was confirmed (supplemental Figure 1A, available on the Blood Web site). After treatment, some mice were killed and analyzed using flow cytometric detection of GFP as a readout of MLL-AF9 allele burden. GSK-LSD1–treated mice exhibited a lower proportion of GFP+ cells in the bone marrow (Figure 1A), peripheral blood, and spleen (supplemental Figure 1B-C). Other measures of disease burden, including spleen weight, were markedly reduced in the setting of GSK-LSD1 treatment (supplemental Figure 1E). Mice treated with GSK-LSD1 exhibited a significant decline in platelet count (P = .003; supplemental Figure 1D), which is consistent with an on-target effect of LSD1 depletion.18 Immunophenotyping of bone marrow cells after 3 days of GSK-LSD1 treatment revealed a reduction of more primitive GFP+ leukemia cells coexpressing c-kit and Mac-1 (Figure 1B). GSK-LSD1–treated mice also had markedly improved survival (median survival, 78 days) compared with control mice (median survival, 39 days) (Figure 1C). Strikingly, a small proportion of treated mice had no detectable disease even 248 days after transplantation. In order to confirm this effect of LSD1 inhibition on survival, we performed serial transplantation of MLL-AF9 cells harvested from leukemic mice treated for 3 days with either vehicle alone or GSK-LSD1. Equivalent numbers of GFP+ cells purified from vehicle- or GSK-LSD1–treated mice were injected into sublethally irradiated mice. Tertiary recipient mice transplanted with cells harvested from GSK-LSD1–treated mice had improved survival when compared with vehicle-treated mice. While recipient mice transplanted with vehicle-treated cells had a median survival of 23 days, mice challenged with GSK-LSD1–treated leukemia cells had a median survival of 51 days (Figure 1D). Only 50% of the mice engrafted with GSK-LSD1–treated leukemia cells succumbed to AML. The remaining 50% of the mice transplanted with GSK-LSD1–treated cells remained healthy up to 308 days after transplantation and showed no signs of leukemia. These data suggest that LSD1 inhibition has potent antileukemic activity, improves overall survival, and occasionally causes complete disease eradication in an aggressive model of MLL-AF9–driven AML.https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/

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In vivo, GSK-LSD1 2HCl has been studied in preclinical models for its anticancer activity. The compound has been cited in various cancer research publications, including studies on acute myeloid leukemia, non-small cell lung cancer, and other malignancies. Its ability to inhibit LSD1 and induce differentiation in cancer cells makes it a valuable tool for studying the role of LSD1 in tumorigenesis.

The detailed enzyme assay protocol for GSK-LSD1 2HCl (CAS#: 1821798-25-7) is not described in this paper. [1]
Non-cellular enzyme assays for GSK-LSD1 2HCl involve measuring its inhibition of LSD1 activity using purified LSD1 enzyme and appropriate substrates. The compound's IC50 of 16 nM is determined in these assays. Selectivity is assessed by testing GSK-LSD1 2HCl against other FAD-utilizing enzymes such as LSD2, MAO-A, and MAO-B. These assays confirm the compound's potency and selectivity for LSD1.

Cell cycle analysis
Cell cycle analysis was performed by BrdU staining of cells treated in vitro for 48 hours with GSKLSD1. BrdU Flow Kit (BD Biosciences) was used. Briefly, after 48 hours of exposure to GSK-LSD1, cells were exposed to 10 µM BrdU per manufacturer’s instructions for 20 min. After this, cells were harvested, permeabilized and stained with anti-BrdU antibody labelled with APC, while leukemic cells were GFP+ (harbouring pMSCV-MLL-AF9-IRES-GFP plasmid). For DNA staining SYTOX™ Blue Dead Cell Stain was used. The SYTOX Blue signal was acquired in a linear mode. https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

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MLL-AF9 leukemia cells were treated in vitro by culturing cells in IMDM supplemented with 15% FBS, IL-3, IL-6, and mSCF with the addition of vehicle alone or GSK-LSD1 at a concentration of 0.5 µM for 48 hours. Similarly, leukemia cells were treated with the DOT1L inhibitor EPZ4777 for 6 days at a concentration of 1 µM. Colony forming assays were performed according to manufacturer’s instructions. Briefly, 500 cells/dish were plated in MC3434 methylcellulose and numbers of colonies were scored after 6 days of incubation. For each arm 3 independent dishes were scored, and colony assays were performed at least in duplicate. GSK-LSD1 was added to MC3434 semisolid medium at day 0 at a concentration of 0.5 µM and colonies were scored six days later.https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

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U2OS cells were treated with 200 μM GSK-LSD1 2HCl (CAS#: 1821798-25-7) for 12 hours. For immunofluorescent staining, cells cultured on cover slips were fixed with freezing methanol, washed with PBS, blocked in PBS with 1% BSA, then hybridized with anti-LC3B antibody and Lyso-Tracker Red, mounted, and observed under fluorescence microscopy (Fig. 3C).
For transmission electron microscopy, U2OS cells treated with 200 μM GSK-LSD1 2HCl (CAS#: 1821798-25-7) were fixed in 4% glutaraldehyde, dehydrated, ultrathin sections prepared using a microtome, collected on grids, stained with 1% uranyl acetate and/or lead citrate, and imaged with a Tecnai G20 TWIN transmission electron microscope (Fig. 3D).
For western blot analysis, U2OS cells were treated with several concentrations of GSK-LSD1 2HCl (CAS#: 1821798-25-7) for 12 hours, then lysates were subjected to SDS-PAGE and immunoblotted with anti-LC3B antibody (Fig. 3B). [1]
In vitro cell-based assays for GSK-LSD1 2HCl are conducted using cancer cell lines such as A549 cells. Cells are treated with the compound at concentrations such as 10 μM for 1 hour, and binding affinity to LSD1 is assessed by measuring the stability of cellular LSD1 protein using Western blot-based CESTA. Cell proliferation and differentiation assays are also performed to evaluate the compound's functional effects.

For in vivo treatment experiments, GSK-LSD1 was administered via intraperitoneal injections at a dose of 0.5 mg/kg daily. Treatment was initiated only after peripheral blood engraftment of MLL-AF9 leukemia cells was confirmed at a minimum chimerism of 0.1-1% GFPpositive cells for syngeneic murine MLL-AF9 leukemia cells or 12.3% ± 2.7 hCD45-positive cells for xenotransplantation experiments. Mice were treated for 3 days (Figure 1B), 2 weeks (Figure 1C) or 6 weeks (Figure 1G). Cytological staining was performed on cytospin preparations of suspension cells from in vitro culture (Figures 1E, 5E, 6D+F) or from peripheral blood of mice (Figure 1J) using the Deep Quick Stain kit. https://pmc.ncbi.nlm.nih.gov/articles/PMC5897868/#sec12

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In vivo animal studies for GSK-LSD1 2HCl are typically conducted in rodent models of cancer to evaluate its antitumor efficacy. The compound can be administered via various routes depending on the experimental design. Tumor growth, differentiation markers, and LSD1 activity are assessed following treatment. These studies are essential for validating the compound's in vivo efficacy.

GSK-LSD1 2HCl has a molecular weight of 289.24 g/mol. It is soluble in DMSO and water at 57 mg/mL. The compound is intended for research use only. Detailed pharmacokinetic parameters such as half-life and bioavailability are not extensively documented in standard summaries but would be evaluated in preclinical studies. Its cell-permeable nature allows it to reach its intracellular target effectively.

Comprehensive toxicological data for GSK-LSD1 2HCl are limited as it is primarily a research compound. It is intended for laboratory use only and is not for human consumption. As with all research chemicals, appropriate safety precautions should be taken when handling GSK-LSD1 2HCl. Its safety profile in vivo has not been extensively characterized beyond its use in preclinical cancer models.

Biochim Biophys Acta. 2017 Aug 8;1864(12):2428-2437.; http://www.thesgc.org/chemical-probes/LSD1.

See also: Gsk-lsd1 (comment moved to).

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GSK-LSD1 2HCl (CAS#: 1821798-25-7) is a potent and selective inhibitor of LSD1 (lysine-specific demethylase 1) and potently inhibits proliferation of various cancer cell lines by altering gene expression patterns (Table 1). It induces autophagy in multiple cell lines (U2OS, as shown), acting similarly to 2-PCPA but with a different molecular structure (Fig. 3A). The autophagy induction by GSK-LSD1 2HCl (CAS#: 1821798-25-7) is consistent with that of 2-PCPA, suggesting they function through the same mechanism, likely via KDM1A/LSD1 inhibition. [1]
GSK-LSD1 2HCl is an irreversible, potent, and selective LSD1 inhibitor with an IC50 of 16 nM. It shows >1000-fold selectivity over other FAD-utilizing enzymes such as LSD2, MAO-A, and MAO-B. The compound has antineoplastic activity and is used as a research tool for studying histone demethylation and cancer biology. GSK-LSD1 2HCl is not an approved drug and is available as a research compound.

C14H22CL2N2

289.243881702423

288.115

C, 58.14; H, 7.67; Cl, 24.51; N, 9.69

1821798-25-7

2102933-95-7 (rel-2HCl); 1431368-50-1; 1431367-49-5 (rel-HCl); 1821798-25-7 (2HCl); 1431368-48-7 (rel-free base);1431367-51-9 (HCl);

91663353

Typically exists as White to light yellow solid at room temperature

4

2

3

18

217

2

Cl.Cl.N(C1CCNCC1)[C@@H]1C[C@H]1C1C=CC=CC=1

PJFZOGMSPBHPNS-WICJZZOFSA-N

InChI=1S/C14H20N2.2ClH/c1-2-4-11(5-3-1)13-10-14(13)16-12-6-8-15-9-7-12;;/h1-5,12-16H,6-10H2;2*1H/t13-,14+;;/m0../s1

rel- N-[(1R,2S)-2-Phenylcyclopropyl]-4-Piperidinamine hydrochloride (1:2)

GSK-LSD1 dihydrochloride; GSK-LSD1; GSK-LSD1 Dihydrochloride; 2102933-95-7; GSK-LSD1 2HCl; GSK-LSD1; GSK LSD1 Dihydrochloride; GSK-LSD1 (dihydrochloride); 1821798-25-7; 1431368-48-7; GSK-LSD 1; GSK-LSD-1; GSK-LSD1 2HCl; GSK-LSD1 (trans-racemic) dihydrochloride; GSK-LSD1 HCl;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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