Lysine-specific demethylase 1 (LSD1/KDM1A) (IC₅₀ = 2.1 nmol/L)
> Selective for LSD1 over monoamine oxidases MAO-A and MAO-B [1]

Recombinant human LSD1 is irreversibly inhibited by T-3775440, with a kinact/KI value of 1.7×105 (sec−1 M−1). With an IC50 value of 2.1 nM, T-3775440 exhibits remarkable selectivity for LSD1 in comparison to other monoamine oxidases like MAO-A and MAO-B. Multiple cell line growth is inhibited by T-3775440 as early as day 3 of therapy. Interestingly, T-3775440 therapy frequently increased the granulocyte/macrophage markers CD86 and CD11b on TF-1a and HEL92.1.7 cells, whereas it decreased the erythrocyte markers CD235a and CD71. While just a slight increase in cell surface CD86 expression was seen, T-3775440 also dramatically elevated CD86 mRNA expression in CMK11-5 cells in a concentration-dependent manner. Treatment with T-3775440 breaks the connection between LSD1 and GFI1B in a concentration-dependent way. T-3775440 raises the amounts of dimethylated H3K4 at PI16 and decreases LSD1 binding, but not GFI1B binding [1].

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T-3775440 potently inhibited proliferation of acute erythroid leukemia (AEL) and acute megakaryoblastic leukemia (AMKL) cell lines, with responses observed as early as 3 days of treatment [1]
T-3775440 treatment induced cell cycle arrest (increased G₁ and sub-G₁ populations) and apoptosis in sensitive cell lines (TF-1a, HEL92.1.7, CMK11-5, M07e), as shown by Western blot analysis of p27 and cleaved PARP [1]
T-3775440 induced transdifferentiation of erythroid/megakaryocytic leukemia cells towards granulomonocytic-like lineage, evidenced by morphological changes, upregulation of monocytic gene signatures (e.g., CD86, ITGAM), and downregulation of erythroid markers (e.g., CD235a, GATA1) [1]
Gene set enrichment analysis showed that T-3775440 upregulated SPI1/PU.1 target genes and downregulated erythroid signature genes [1]
T-3775440 disrupted the physical interaction between LSD1 and the transcription factor GFI1B in a concentration-dependent manner, as shown by immunoprecipitation assays [1]
T-3775440 increased dimethylated H3K4 levels at the PI16 promoter, a GFI1B target gene, indicating chromatin-level effects [1]
Knockdown of SPI1 attenuated T-3775440-induced transdifferentiation, apoptosis, and growth suppression, supporting a SPI1-dependent mechanism [1]
Knockdown of both LSD1 and GFI1B phenocopied T-3775440 effects, enhancing CD86 expression and growth inhibition [1]
In colony-forming assays using normal human bone marrow, T-3775440 selectively inhibited erythroid progenitor proliferation (IC₅₀ ~0.1–0.14 μmol/L) without significantly affecting myeloid progenitors [1]

A single oral dose of 3 to 30 mg/kg of T-3775440 causes dose-dependent upregulation of CD86 mRNA expression in HEL92.1.7 cell tumor xenografts. Direct biomarker testing of PI16 expression levels was done to investigate the targeting effect of this drug in malignancies. Predictably, therapy with T-3775440 markedly reversed the inhibition of PI16. With dosages of 20 and 40 mg/kg, respectively, T-3775440 demonstrated a ~0% 15-day T/C value, indicating a strong anticancer activity in a TF-1a (AEL) tumor xenograft model. The additive AEL model of HEL 92.1.7 and the AMKL model of CMK11-5 had strong anticancer effects in T-3775440 as well, leading to nearly total suppression of tumor development throughout dosage. A mechanism-based side effect of LSD1 inhibition is thought to be responsible for the study's findings, which showed that T-3775440 therapy in mice caused a brief drop in platelets followed by a notable rebound. Body weight differences between vehicle and T-3775440-treated tumor xenograft model mice were statistically significant at the higher dose in a dosing regimen of 5 days on and 2 days off. Nonetheless, all subcutaneous tumor xenograft models showed tolerability of effective T-3775440 dosages [1].

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T-3775440 (oral, single dose 3–30 mg/kg) dose-dependently upregulated CD86 mRNA expression in HEL92.1.7 xenograft tumors, demonstrating target engagement [1]
T-3775440 (oral, once daily, 5 days on/2 days off, for 2 weeks) significantly suppressed tumor growth in AEL (TF-1a, HEL92.1.7) and AMKL (CMK11-5) subcutaneous xenograft models, with T/C values as low as <0% at 40 mg/kg in the TF-1a model [1]
T-3775440 treatment reversed repression of the PI16 gene in vivo, consistent with in vitro findings [1]

Recombinant human LSD1 enzyme activity was assessed to determine inhibitory potency and mechanism [1]
A surface plasmon resonance (SPR) biosensing assay was used to study the interaction between recombinant LSD1 and a GFI1B SNAG domain peptide. T-3775440 pretreatment diminished the SPR signal, indicating direct disruption of the LSD1-GFI1B interaction [1]

Cell proliferation and viability were assessed using CellTiter-Glo luminescent assay after compound treatment for designated periods. IC₅₀ values were calculated using a 4-parameter logistic model [1]
For cell cycle analysis, cells were fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry [1]
Quantitative RT-PCR was performed to measure mRNA expression of target genes (e.g., CD86, GYPA, GATA1, GFI1B, PI16) using TaqMan probes and normalized to GAPDH [1]
Microarray analysis was conducted on RNA from treated and control cells to assess genome-wide expression changes. Differentially expressed genes were identified and subjected to pathway and gene set enrichment analyses [1]
Flow cytometry was used to evaluate surface marker expression (e.g., CD86, CD235a) on cells after treatment [1]
Chromatin immunoprecipitation (ChIP) assay was performed to assess LSD1 and GFI1B binding and histone modification (H3K4me2) at specific gene loci (e.g., PI16 promoter) [1]
Immunoprecipitation of chromatin fractions was used to examine protein-protein interactions (LSD1-GFI1B-CoREST complex) [1]
Western blotting was performed to detect protein levels (e.g., p27, cleaved PARP, LSD1, GFI1B, CoREST, β-actin) in whole-cell extracts or immunoprecipitates [1]
Colony-forming cell (CFC) assays were performed using human bone marrow cells in methylcellulose-based medium to assess effects on hematopoietic progenitors [1]

Female C.B17/Icr-scid/scid Jcl mice were used for subcutaneous xenograft models [1]
AML cells (2×10⁶ cells) mixed with Matrigel were inoculated subcutaneously into the left flank of mice [1]
When mean tumor volume reached ~150–350 mm³, mice were randomized into groups [1]
T-3775440 or vehicle was administered orally once daily on a schedule of 5 days on/2 days off for 2 weeks [1]
Tumor dimensions were measured twice weekly with calipers, and volume was calculated as (length × width²) × 0.5 [1]
For pharmacodynamic studies, mice bearing HEL92.1.7 xenografts received a single oral dose of T-3775440 (3–30 mg/kg). Tumors were harvested at specified times for mRNA analysis [1]

In mice, T-3775440 treatment resulted in a transient decrease in platelet count, followed by a rebound, which is believed to be a mechanism effect of LSD1 inhibition[1]. No significant effect on red blood cell count was observed after a single dose[1]. In some xenograft models, weight loss was observed at higher doses, but effective doses were generally well tolerated[1]. The platelet effect is reversible and is considered clinically manageable[1].

[1]. A Novel LSD1 Inhibitor T-3775440 Disrupts GFI1B-Containing Complex Leading to Transdifferentiation and Impaired Growth of AML Cells. Mol Cancer Ther. 2017 Feb;16(2):273-284.

T-3775440 is a novel, irreversible, selective LSD1 inhibitor whose structure is derived from the cyclopropylamine moiety and is designed to bind to FAD in the enzyme's catalytic core [1]. It exerts a unique mechanism of action by disrupting the LSD1-GFI1B complex, leading to the transdifferentiation of erythroid/megakaryocytic leukemia cells into granulocyte-monocyte-like cells, thereby inhibiting cell growth and inducing apoptosis [1]. It exhibits potent and selective activity against the AEL and AMKL subtypes of acute myeloid leukemia (AML), which are characterized by high invasiveness, poor prognosis, and limited treatment options [1]. This study provides a theoretical basis for the clinical evaluation of LSD1 inhibitors, especially in leukemias expressing GFI1B [1].

C18H23CLN4O

346.854422807693

346.156

1422535-52-1

1422620-34-5;1422535-52-1 (HCl);

86729932

Light yellow to yellow solid powder

3

3

6

24

431

2

Cl.O=C(C1C=NN(C)C=1)NC1C=CC(=CC=1)[C@@H]1C[C@H]1NCC1CC1

XYFPAGOQZFSLFH-MCJVGQIASA-N

InChI=1S/C18H22N4O.ClH/c1-22-11-14(10-20-22)18(23)21-15-6-4-13(5-7-15)16-8-17(16)19-9-12-2-3-12;/h4-7,10-12,16-17,19H,2-3,8-9H2,1H3,(H,21,23);1H/t16-,17+;/m0./s1

N-(4-((1S,2R)-2-((Cyclopropylmethyl)amino)cyclopropyl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide hydrochloride

T3775440 HCl; T-3775440 HCl; T 3775440 HCl; T-3775440 Hydrochloride;

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 (e.g. under nitrogen), 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 : ≥ 30.18 mg/mL (~87.01 mM)
H2O : ~8.33 mg/mL (~24.02 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.21 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 25.0 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: ≥ 2.5 mg/mL (7.21 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 25.0 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: ≥ 2.5 mg/mL (7.21 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 25.0 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.)