IC50: 16 nM (LSD1)[1]
GSK-LSD1 dihydrochloride specifically targets lysine-specific demethylase 1 (LSD1), also known as KDM1A. LSD1 is a histone demethylase that removes methyl groups from histone H3 at lysine 4 (H3K4me1/2) and lysine 9 (H3K9me1/2), functioning within transcriptional co-repressor complexes. By irreversibly inhibiting LSD1, the compound alters gene expression patterns, leading to antiproliferative effects in various cancer cell lines.

GSK-LSD1 demonstrates selectivity over other similarly related FAD-utilizing enzymes, such as LSD2, and the monoamine oxidases MAO-A and MAO-B, by more than 1000 times[1]. Enzyme activity of KDM1A/LSD1 can be inhibited by GSK-LSD1. In U2OS cells, GSK-LSD1 stimulates the production of LC3-II. When GSK-LSD1 is administered, autophagosome production is demonstrated by electronic microscopy. Through altering gene expression patterns, GSK-LSD1 potently suppresses the proliferation of many cancer cell lines[2].

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In vitro, GSK-LSD1 dihydrochloride potently inhibits LSD1 enzyme activity with an IC50 of 16 nM. In U2OS cells, it stimulates the production of LC3-II and induces autophagosome formation, as demonstrated by electron microscopy. The compound effectively inhibits proliferation across various cancer cell lines by modulating gene expression patterns. It shows remarkable selectivity, being more than 1000-fold selective for LSD1 over other closely related FAD-dependent enzymes.

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 dihydrochloride demonstrates significant antitumor activity in mouse models. In secondary recipient mice engrafted with MLL-AF9 primary AML cells, daily treatment at 0.5 mg/kg for 14 days reduced the proportion of GFP+ leukemia cells in bone marrow, peripheral blood, and spleen. Treated mice showed markedly reduced spleen weight and a significant decline in platelet count, consistent with on-target LSD1 inhibition. Survival was substantially improved (median 78 days vs. 39 days in controls), with some mice remaining disease-free beyond 248 days.

The in vitro enzyme inhibition assay for GSK-LSD1 dihydrochloride typically involves measuring LSD1 demethylase activity using a fluorescence-based or ELISA-based format. Recombinant LSD1 enzyme is incubated with a biotinylated histone H3K4me2 peptide substrate in the presence of varying concentrations of the compound. The reaction is initiated by the addition of the substrate and terminated after incubation at 37°C. Demethylation is detected using a specific antibody against the demethylated product, and IC50 values are calculated from dose-response curves.

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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Cellular activity is assessed in U2OS cells, where GSK-LSD1 dihydrochloride is administered at various concentrations. After treatment, cells are analyzed for LC3-II production via Western blotting and for autophagosome formation using transmission electron microscopy. Antiproliferative effects are evaluated across multiple cancer cell lines using standard cell viability assays such as MTT or CellTiter-Glo, with IC50 values determined after 72-96 hours of treatment. Gene expression changes are analyzed by RNA sequencing or qPCR to assess the impact of LSD1 inhibition.

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 the in vivo efficacy model, secondary recipient mice are engrafted with 1 × 10⁵ MLL-AF9 primary AML cells. After confirming peripheral blood engraftment, animals are treated with GSK-LSD1 dihydrochloride at 0.5 mg/kg daily via intraperitoneal or oral administration for 14 days. Disease burden is assessed by flow cytometric detection of GFP-positive cells in bone marrow, peripheral blood, and spleen. Spleen weight is recorded as a secondary measure of disease burden, and survival is monitored over time.

Pharmacokinetic properties of GSK-LSD1 dihydrochloride have not been extensively detailed in publicly available literature. As a small molecule with a molecular weight of 289.24 g/mol (free base), it is expected to have moderate oral bioavailability. The compound is soluble in DMSO at ≥62.5 mg/mL (216.08 mM). For in vivo studies, it is typically formulated in suitable vehicles for administration. Detailed PK parameters such as half-life, clearance, and volume of distribution would require specific studies that are not widely reported in the public domain.

Toxicity data for GSK-LSD1 dihydrochloride is primarily derived from in vivo efficacy studies. In mouse models, the compound was well-tolerated at the studied dose of 0.5 mg/kg daily for 14 days. A significant decline in platelet count was observed in treated mice, which is consistent with an on-target effect of LSD1 depletion rather than off-target toxicity. No other major toxicities were reported in these studies. As with all research compounds, GSK-LSD1 dihydrochloride is for research use only and not for human therapeutic applications.

[1]. Purich D. The Inhibitor Index A Desk Reference on Enzyme Inhibitors, Receptor Antagonists, Drugs, Toxins, Poisons, Biologics, and Therapeutic Leads. ISBN 9781138739215.

[2]. Inhibition of H3K4 demethylation induces autophagy in cancer cell lines. Biochim Biophys Acta. 2017 Aug 8;1864(12):2428-2437.

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

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GSK-LSD1 dihydrochloride is an irreversible, mechanism-based inhibitor of LSD1 that covalently modifies the FAD cofactor in the enzyme's active site. The compound induces autophagy in cancer cells through the production of LC3-II and autophagosome formation. It has been studied in the context of acute myeloid leukemia (AML) and other malignancies where LSD1 is dysregulated. The compound is available as a dihydrochloride salt for improved solubility and stability, and is intended for research purposes only, not for clinical use.

C14H22CL2N2

289.243881702423

288.116

2102933-95-7

GSK-LSD1;1431368-48-7

91663353

White to light yellow solid powder

4

2

3

18

217

2

C1(N[C@H]2[C@H](C3=CC=CC=C3)C2)CCNCC1.Cl.Cl

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

N-[(1R,2S)-2-phenylcyclopropyl]piperidin-4-amine;dihydrochloride

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

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 : ≥ 62.5 mg/mL (216.08 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.19 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 (7.19 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 (7.19 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.)