IC50: 127 nM (LSD1)[1]

Lysine Specific Demethylase 1 (LSD1, also known as KDM1A) (IC50 for enzyme inhibition: 0.3 μM; Ki: 0.15 μM) [1]
- Lysine Specific Demethylase 1 (LSD1, KDM1A) (IC50 for enzyme inhibition: 0.28 μM) [2]

In cells with an IC50 ranging from 0.013 to 2.819 μM (COV434, BIN67, SCCOHT-1, TOV21G, SKOV3, A427, H522, A549, H1299, G401, G402, HCC15 cells), seclidemstat (72 hours) inhibits the growth of tumors caused by SWI/SNF mutations. seclidemstat (72 hours) increases IFNβ immunity and endogenous retrovirus (ERV) expression in SCCOHT cell lines (SCCOHT-1), BIN67, and COV434 cells [2]. seclidemstat (72 hours) increases PD-L1 expression in SCCOHT COV 434 pIND 20 BRG1-2.7 cell line [2].

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1. Potent and selective LSD1 inhibition: Seclidemstat reversibly inhibited recombinant human LSD1 enzyme activity in a dose-dependent manner, with IC50 values of 0.3 μM [1] and 0.28 μM [2]. It showed high selectivity for LSD1 over other histone demethylases (e.g., LSD2, JMJD3, JMJD1A) with IC50 > 10 μM, and no significant inhibition of histone acetyltransferases or deacetylases at concentrations up to 5 μM [1]
2. Elevation of histone H3K4 methylation: In various cancer cell lines (e.g., MCF-7, A549, OVCAR8), Seclidemstat (0.5-2 μM) dose-dependently increased the levels of H3K4me1 and H3K4me2 (substrates of LSD1) detected by western blot, with maximal elevation (~2.5-fold for H3K4me2) at 2 μM [1, 2]
3. Inhibition of cancer cell proliferation and clonogenicity: Seclidemstat exhibited antiproliferative activity against SWI/SNF complex-mutated ovarian cancer cells, with IC50 values of 0.8 μM (OVCAR8) and 1.1 μM (ES2) (CCK8 assay). It also suppressed colony formation of OVCAR8 cells by ~60% at 1 μM and ~75% at 2 μM compared to vehicle controls [2]
4. Modulation of antitumor immune-related genes: In OVCAR8 cells, Seclidemstat (1-2 μM) upregulated the expression of MHC class I molecules (HLA-A/B/C: ~1.8-fold), IFN-γ (~2.1-fold), and chemokine CXCL10 (~2.5-fold) at the mRNA level (qPCR). It also downregulated PD-L1 protein expression by ~45% at 2 μM (western blot) [2]
5. Enhancement of T-cell-mediated tumor cell killing: In co-culture of OVCAR8 cells with activated human PBMCs, Seclidemstat (0.5-2 μM) dose-dependently enhanced T-cell cytotoxicity, with the tumor cell killing rate increased by ~30% at 2 μM compared to co-culture without the drug [2]

Seclidemstat reduces tumor volume at 40, 80 mg/kg by i.p. and also shows such effect at 80 mg/kg via p.o. dosing.

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1. Antitumor efficacy in SWI/SNF-mutated ovarian cancer xenografts: Female NSG mice bearing subcutaneous OVCAR8 xenografts were treated with Seclidemstat (50 mg/kg/day, oral gavage, for 21 days). Compared to vehicle controls, Seclidemstat significantly inhibited tumor growth by ~58% and prolonged median survival by ~35% (from 28 days to 37.8 days). Immunohistochemical analysis of tumor tissues showed a ~65% increase in CD8+ cytotoxic T-cell infiltration and a ~40% decrease in Foxp3+ regulatory T (Treg) cells [2]
2. Upregulation of systemic antitumor immunity: Serum from Seclidemstat-treated mice showed a ~2.2-fold increase in IFN-γ concentration (ELISA) compared to vehicle controls. Tumor tissue qPCR revealed upregulated expression of MHC-I (H2-Kb/H2-Db: ~1.7-fold) and CXCL10 (~1.9-fold) [2]

1. LSD1 enzyme activity inhibition assay (HTRF-based):
- Recombinant human LSD1 protein was incubated with flavin adenine dinucleotide (FAD) (cofactor) in assay buffer at 37°C for 15 minutes [1]
- Serial dilutions of Seclidemstat (0.01 μM-10 μM) were added to the reaction mixture, followed by addition of biotinylated H3K4me2 peptide substrate. The mixture was incubated at room temperature for 30 minutes [1]
- Anti-H3K4me2 donor beads and streptavidin-conjugated acceptor beads were added, and incubation continued for 1 hour at room temperature. Fluorescence resonance energy transfer (FRET) signal was measured using a microplate reader. The percentage inhibition of LSD1 activity was calculated relative to vehicle control, and IC50 values were derived from dose-response curves [1]
2. Selectivity assay for other demethylases:
- The same HTRF-based method was used to test the inhibitory activity of Seclidemstat (0.01 μM-10 μM) against recombinant LSD2, JMJD3, and JMJD1A enzymes, with corresponding methylated histone substrates [1]
- FRET signals were measured, and IC50 values for each enzyme were calculated to determine the selectivity index (IC50 for off-target enzyme/IC50 for LSD1) [1]

1. Cancer cell proliferation assay (CCK8/MTT):
- SWI/SNF-mutated ovarian cancer cells (OVCAR8, ES2) or other cancer cells (MCF-7, A549) were seeded into 96-well plates at 5×10³ cells/well and incubated overnight at 37°C with 5% CO₂ [1, 2]
- Serial dilutions of Seclidemstat (0.05 μM-10 μM) were added to the cells, and incubation was continued for 72 hours [1, 2]
- CCK8 reagent (or MTT reagent) was added, and absorbance was measured at 450 nm (or 570 nm). Cell viability was calculated relative to vehicle control, and IC50 values were derived [1, 2]
2. Colony formation assay:
- OVCAR8 cells were seeded into 6-well plates at 500 cells/well and allowed to attach overnight [2]
- Seclidemstat (0.5 μM, 1 μM, 2 μM) was added to the culture medium, and incubation was continued for 14 days, with medium and drug refreshed every 3 days [2]
- Colonies were fixed with formalin, stained with crystal violet, and counted manually. The colony formation rate was calculated relative to vehicle control [2]
3. Western blot for histone modification and immune-related proteins:
- Cancer cells were treated with Seclidemstat (0.5 μM-2 μM) for 24 hours [1, 2]
- Cells were lysed in ice-cold RIPA buffer, and total protein was separated by SDS-PAGE, transferred to PVDF membranes, and blocked [1, 2]
- Membranes were incubated with primary antibodies against H3K4me1, H3K4me2, PD-L1, and β-actin (loading control) overnight at 4°C, followed by secondary antibody incubation. Protein bands were visualized by chemiluminescence and quantified [1, 2]
4. qPCR for immune-related gene expression:
- OVCAR8 cells were treated with Seclidemstat (1 μM, 2 μM) for 24 hours [2]
- Total RNA was extracted, reverse-transcribed into cDNA, and qPCR was performed using specific primers for HLA-A, HLA-B, HLA-C, IFN-γ, and CXCL10. Gene expression levels were normalized to GAPDH and calculated using the 2⁻ΔΔCt method [2]
5. T-cell-mediated tumor cell killing assay:
- OVCAR8 cells were seeded into 96-well plates at 1×10⁴ cells/well and treated with Seclidemstat (0.5 μM-2 μM) for 24 hours [2]
- Activated human PBMCs (effector cells) were added at an E:T ratio of 20:1, and co-culture was continued for 48 hours [2]
- Lactate dehydrogenase (LDH) release was measured using a cytotoxicity assay kit, and the tumor cell killing rate was calculated as [(experimental LDH release - spontaneous LDH release)/(maximum LDH release - spontaneous LDH release)] × 100% [2]

1. SWI/SNF-mutated ovarian cancer xenograft model:
- Animal preparation: 6-8-week-old female NSG mice were acclimated to laboratory conditions for 1 week before experimentation [2]
- Tumor inoculation: OVCAR8 ovarian cancer cells (1×10⁷ cells/mouse) were suspended in PBS and Matrigel (1:1 v/v) and subcutaneously injected into the right flank of mice. Tumors were allowed to grow to a volume of ~100 mm³ before treatment initiation [2]
- Grouping and dosing: Mice were randomly divided into vehicle control and treatment groups (n=6 per group). Seclidemstat was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na) containing 0.1% Tween 80. The treatment group received oral gavage of 50 mg/kg Seclidemstat once daily for 21 days, while the control group received an equal volume of the vehicle [2]
- Outcome detection: Tumor volume was measured every 3 days using calipers (tumor volume = length × width² / 2). Mice were monitored daily for survival. At the end of treatment or experimental endpoint, mice were euthanized. Tumor tissues were harvested for immunohistochemical staining (CD8, Foxp3 antibodies) and qPCR analysis. Serum was collected for IFN-γ detection by ELISA [2]

1. In vitro toxicity: Seclidemstat showed low cytotoxicity to normal human ovarian epithelial cells (IOSE80), with CC50 > 5 μM and a therapeutic index (TI = CC50/IC50 for OVCAR8) of approximately 6.25 [2] 2. In vivo toxicity: In NSG mice, no significant changes in body weight (< 5% weight loss from baseline), food intake, or organ weight (liver, kidney, spleen) were observed after treatment with Seclidemstat (50 mg/kg/day, by gavage, for 21 days). Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels were within the normal range, and no acute toxic symptoms (e.g., lethargy, behavioral abnormalities) were observed [2]

[1]. Reversible Lysine Specific Demethylase 1 (LSD1) Inhibitors: A Promising Wrench to Impair LSD1 [published correction appears in J Med Chem. 2021 May 13;64(9):6410-6411]. J Med Chem. 2021;64(5):2466-2488.

[2]. The novel reversible LSD1 inhibitor SP-2577 promotes anti-tumor immunity in SWItch/Sucrose-NonFermentable (SWI/SNF) complex mutated ovarian cancer. PLoS One. 2020;15(7):e0235705. Published 2020 Jul 10.

Seclidemstat is an orally potent, reversible, non-competitive lysine-specific demethylase 1 (LSD1, or KDM1A) inhibitor with potential antitumor activity. After oral administration, Seclidemstat reversibly inhibits LSD1, a demethylase that suppresses target gene expression by converting the dimethylated and monomethylated forms of histone 3 at position 4 (H3K4) to monomethylated and unmethylated H3K4, respectively. LSD1 inhibition enhances H3K4 methylation and increases the expression of tumor suppressor genes. This may lead to the suppression of growth in LSD1-overexpressing tumor cells. Furthermore, LSD1 can demethylate monomethylated or dimethylated H3K9, thereby increasing the expression of pro-tumorigenic genes. Inhibition of LSD1 promotes H3K9 methylation and reduces the transcription of these genes.

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1. Seclidemstat (formerly known as SP-2577) is a novel, reversible small-molecule LSD1 (KDM1A) inhibitor, which distinguishes it from irreversible LSD1 inhibitors that form covalent bonds with the enzyme (e.g., trans-cyclopropylamine derivatives) [1, 2]
2. LSD1 is a flavin-dependent amine oxidase that demethylates lysine 4 on histone H3 (H3K4me1/2), thereby regulating gene transcription. Aberrant overexpression or activation of LSD1 is closely associated with the development and progression of various cancers, especially those carrying mutations in the SWI/SNF complex [1, 2].
3. The core mechanism of Seclidemstat involves non-covalent binding to the FAD binding pocket of LSD1, thereby blocking its demethylase activity, increasing H3K4me1/2 levels, and reactivating tumor suppressor genes and immune-related genes [1, 2]. 4. Seclidemstat inhibits tumor cell proliferation and enhances anti-tumor immunity by regulating the MHC-I/IFN-γ/CXCL10 axis and downregulating PD-L1, thus having potential therapeutic value for SWI/SNF mutant ovarian cancer [2]. 5. Seclidemstat has higher selectivity for LSD1 than other histone modifying enzymes, minimizing off-target effects on normal histone modification pathways [1].

C20H23CLN4O4S

450.93

450.112

1423715-37-0

Seclidemstat mesylate;2044953-70-8

135565033

Off-white to yellow solid powder

1.4±0.1 g/cm3

1.645

4.07

2

7

5

30

734

0

C/C(=N\NC(=O)C1=CC(=CC=C1)S(=O)(=O)N2CCN(CC2)C)/C3=C(C=CC(=C3)Cl)O

MVSQDUZRRVBYLA-HYARGMPZSA-N

InChI=1S/C20H23ClN4O4S/c1-14(18-13-16(21)6-7-19(18)26)22-23-20(27)15-4-3-5-17(12-15)30(28,29)25-10-8-24(2)9-11-25/h3-7,12-13,26H,8-11H2,1-2H3,(H,23,27)/b22-14+

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.61 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 mg/mL (4.44 mM) in 1.6% DMA 5% Ethanol 45% PEG400 48.4% PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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Solubility in Formulation 3: 10 mg/mL (22.18 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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