HDAC4; HDAC4-VP16-driven reporter
Myocyte Enhancer Factor-2 (MEF2) transcription factor (specifically binds to the hydrophobic pocket on the MADS-box/MEF2 domain, blocking its interaction with class IIa histone deacetylases (HDACs) such as HDAC4). The estimated Kd for the fluorinated analog NKL54 binding to the MEF2A (1-95) fragment is ~0.5 µM. The apparent IC50 of BML-210 for inhibiting HDAC4:MEF2 interaction in a mammalian two-hybrid assay is ~5 µM. [1]

NB4 cells are inhibited in their growth and proliferation by BML-210 (10, 20 μM; 24, 48 hours) [2]. The amount of S phase NB4 cells was reduced by BML-210 (10, 20 μM; 24, 48 hours), and at 20 μM, G0 BML-210 (10, 20 μM; 24, 48 hours) had a cytotoxic effect on NB4 cells. A dose of 10 μM is sufficient for BML-210 to cause cell death [2]. In NBT4 cells, BML-210 (10, 20 μM; 24, 48 hours) suppresses HDAC expression and activity [2]. HDAC4-VP16 expression is not downregulated by BML-210[1].Cell lines. NB4 cells Concentration: 10, 20 μM Incubation period: 24, 48 hours Outcomes: Reduced NB4 cell growth and proliferation in a time- and dose-dependent manner.

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In a mammalian two-hybrid assay in HeLa cells, BML-210 dose-dependently inhibited the luciferase reporter signal driven by the interaction between the GAL4 DNA-binding domain fused to MEF2D (GAL4-MEF2D) and the VP16 activation domain fused to the MEF2-binding motif of HDAC4 (HDAC4-VP16), with an apparent IC50 of ~5 µM. [1]
BML-210 and its analogs (e.g., NKL54, NKL30, NKL11) similarly inhibited the HDAC4:MEF2-driven reporter signal in transfected monkey kidney cells (COS-7) and mouse embryonic fibroblast cells (NIH 3T3). [1]
In contrast, the pan-HDAC inhibitor Trichostatin A (TSA) at its IC50 (100 nM) did not show significant dose-dependent inhibition of the HDAC4:MEF2-driven reporter signal, suggesting BML-210 acts via a mechanism distinct from catalytic HDAC inhibition. [1]
BML-210 did not significantly reduce the expression level of the HDAC4-VP16 fusion protein in the two-hybrid assay. [1]
Surface Plasmon Resonance (SPR) analysis showed that pre-incubation of purified MEF2A (1-95) with increasing amounts of BML-210 decreased its binding signal to immobilized HDAC4 (AA 155-220). [1]
Fluorescence anisotropy assay using a fluorescein-labeled HDAC4 fragment (AA 155-218) showed that incremental addition of BML-210 caused a gradual decay in anisotropy, indicating displacement of HDAC4 from MEF2. [1]
Chromatin immunoprecipitation (ChIP) assay in HeLa cells co-transfected with FLAG-tagged MEF2C and HDAC4 showed that treatment with 10 µM BML-210 for 6 hours significantly diminished the MEF2C-mediated enrichment of HDAC4 on the endogenous frataxin (FXN) promoter, without significantly affecting MEF2C occupancy itself. [1]
Immunocytochemistry in COS-7 cells showed that overnight treatment with 10 µM of the analog NKL30 induced a more diffuse cytoplasmic localization of HDAC4, reducing the nuclear HDAC4 intensity by 21% compared to vehicle-treated cells, without significantly altering MEF2 nuclear localization. [1]
Treatment of Friedreich's ataxia (FRDA) lymphoid cells (GM15850) with the analog NKL54 also decreased the co-localization of MEF2 and HDAC4. [1]
BML-210 and its analogs belong to the pimeloylanilide o-aminoanilide (PAOA) class of compounds. [1]

BML-210 (20 mg/kg; intraperitoneal injection; three times weekly for two weeks) markedly reduced the amount of body weight and tumor growth. In immunodeficient nude mice, BML-210 has no influence on tumor growth or body weight (Nu/J).

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In this study, researchers show that the epigenetic inhibitors GSK-LSD1, CUDC-101 and BML-210, identified by the screen, display antitumour activities in orthotopic mammary tumours in mice, that they upregulate antigen presentation mediated by the major histocompatibility complex class I on breast tumour cells and that treatment with BML-210 substantially sensitized breast tumours to the inhibitor of the checkpoint programmed death-1. Standardized measurements of tumour-cell killing activity facilitated by tumour-organoid-T-cell screens may help with the identification of candidate immunotherapeutics for a range of cancers[3].

HDAC Activity Analysis[2]
Proteins were isolated using ProteoJET™ Mammalian Cell Lysis Reagent (Fermentas, Vilnius, Lithuania) according to the manufacturer's instructions.
HDAC activity analysis was determined using EpiQuik™ HDAC Activity/Inhibition Assay Kit according to the manufacturer’s instructions. Absorbance was measured using Tecan-Control, Infinite 200 microplate reader at 450 nm. Activity (ng/h/mL) was calculated using formula.[2]

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Drug treatment and luciferase assay[1]
For the two-hybrid assay, cells were treated with 10 µM drug overnight unless indicated otherwise. The amount of DMSO was kept below 0.2% V/V. A luciferase assay was performed according to the manufacturer’s protocol. The luciferase response was normalized against the Renilla Luciferase as an internal control. The data are presented as a mean ± SD (n = 2) of normalized HDAC4:MEF2 luciferase response against the normalized response values for GAL4-VP16 for each condition to correct for non-specific inhibition of the luciferase signal.

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For the ¹⁹F NMR binding assay, a fluorinated analog of BML-210 (NKL54) at concentrations ranging from 0.5 to 5 µM was incubated with the MEF2A (1-95) protein fragment (0.5 to 10 µM) in an aqueous buffer (10 mM HEPES, 250 mM NaCl, 1 mM EDTA, pH 7.66) for 30 minutes. ¹⁹F NMR spectra were acquired at 25°C. The chemical shift of the CF₃ resonance was monitored, with a shift from -62.86 ppm (free compound) to -63.23 ppm (compound bound to MEF2), allowing estimation of the binding constant (K_d ~0.5 µM). [1]
For the Surface Plasmon Resonance (SPR) assay, a fragment of HDAC4 (amino acids 155-220) was immobilized on a CMS sensor chip. Purified MEF2A (1-95) protein was used as the analyte and flowed over the chip at various concentrations to generate binding sensograms. To test inhibition, MEF2A was pre-incubated with increasing amounts of BML-210 before injection, and the decrease in binding signal was monitored. [1]
For the fluorescence anisotropy assay, an HDAC4 fragment (AA 155-218) was site-specifically labeled with 5-Iodoacetamidofluorescein (5-IAF) via a cysteine residue. The binding of increasing concentrations of MEF2A (1-95) to the labeled HDAC4 was monitored by measuring fluorescence anisotropy. To test inhibition, BML-210 was titrated into a pre-formed MEF2-HDAC4 complex, and the decrease in anisotropy (indicating complex dissociation) was measured. [1]

Cell proliferation assay[2]
Cell Types: NB4 cells
Tested Concentrations: 10, 20 μM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: Inhibited cell proliferation and inhibited the growth of NB4 cells in a dose- and time-dependent manner.

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Cell cycle analysis [2]
Cell Types: NB4 cells
Tested Concentrations: 10, 20 μM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: The proportion of S phase of NB4 cells diminished and the proportion of G0/G1 phase increased. 10 μM increased the G0/G1 phase by up to 70% at 24 and 48 hrs (hours).

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Cytotoxicity assay [2]
Cell Types: NB4 cells
Tested Concentrations: 10, 20 μM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: Cytotoxic effects on NB4 cells were dose- and time-dependent.

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Apoptosis analysis [2]
Cell Types: NB4 Cell
Tested Concentrations: 10, 20 μM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: 10 μM dose induced apoptosis.

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Western Blot Analysis[2]
Cell Types: NB4 Cell
Tested Concentrations: 10, 20 μM
Incubation Duration: 24, 48 hrs (hours)
Experimental Results: After 48 hrs (hours) of treatment with 10 μM dose, HDAC1 gene expression was inhibited by up to 36% Aft

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For the mammalian two-hybrid assay, HeLa, COS-7, or NIH 3T3 cells were co-transfected with three plasmids: a reporter plasmid containing a GAL4-responsive luciferase gene, a plasmid expressing the GAL4 DNA-binding domain fused to MEF2D (GAL4-MEF2D), and a plasmid expressing the VP16 transactivation domain fused to the MEF2-binding motif of HDAC4 (HDAC4-VP16). After transfection, cells were treated with compounds (e.g., BML-210, its analogs, or DMSO vehicle) typically overnight at 10 µM. Luciferase activity was measured and normalized to an internal control (e.g., Renilla luciferase). Data were often presented as a percentage of the signal from DMSO-treated control cells. [1]
For chromatin immunoprecipitation (ChIP), HeLa cells were transfected with plasmids expressing FLAG-tagged MEF2C and/or FLAG-tagged HDAC4. After treatment with BML-210 (10 µM, 6 hours) or DMSO, cells were fixed with formaldehyde to crosslink proteins to DNA. Chromatin was sheared by sonication and immunoprecipitated using an anti-FLAG antibody or control IgG. Precipitated DNA was purified and analyzed by quantitative real-time PCR using primers specific for the frataxin (FXN) promoter region to measure enrichment. [1]
For immunocytochemistry and cellular localization analysis, COS-7 cells or FRDA lymphoid cells (GM15850) were treated with compounds (e.g., 10 µM NKL30 or NKL54) or DMSO overnight. Cells were fixed, permeabilized, and stained with primary antibodies against MEF2 and HDAC4, followed by appropriate fluorescently labeled secondary antibodies. Nuclei were often counterstained. Images were acquired by confocal microscopy. For quantification in COS-7 cells, the mean fluorescent intensity of HDAC4 within the nucleus (defined by MEF2 staining) was measured and normalized to the nuclear MEF2 intensity. [1]

Animal/Disease Models: Female C57BL/6 mice, mouse breast cancer EO771 cells [3]
Doses: 20 mg/kg
Route of Administration: IP ;[3]. Three times a week for two weeks.
Experimental Results: Significant inhibition of tumor growth and weight.

[1]. Inhibition of the function of class IIa HDACs by blocking their interaction with MEF2. Nucleic Acids Res. 2012 Jul; 40(12): 5378–5388.

[2]. The Histone Deacetylase Inhibitor BML-210 Influences Gene and Protein Expression in Human Promyelocytic Leukemia NB4 Cells via Epigenetic Reprogramming. Int J Mol Sci. 2015 Aug; 16(8): 18252–18269.

[3]. An organoid-based screen for epigenetic inhibitors that stimulate antigen presentation and potentiate T-cell-mediated cytotoxicity. Nat Biomed Eng. 2021 Nov;5(11):1320-1335.

BML-210 is a dicarboxylic acid diamide composed of octanoic acid (octanoic acid) coupled with aniline and 1,2-diaminobenzene. It is an EC 3.5.1.98 (histone deacetylase) inhibitor and an antitumor drug. Its function is related to octanoic acid.

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BML-210 (N-(2-aminophenyl)-N'-phenyloctanoic acid) belongs to the pyrrolidine aniline-o-aminoaniline (PAOA) class of compounds. It was initially identified as an HDAC inhibitor that selectively induces histone acetylation but not tubulin acetylation. [1]
This study reveals a novel mechanism of action for BML-210 and related PAOA compounds: they directly bind to the transcription factor MEF2 at the same hydrophobic groove as the interaction site of class IIa HDACs (e.g., HDAC4, HDAC9). By blocking this protein-protein interaction, they prevent the recruitment of class IIa HDACs to MEF2-targeting gene promoters, thus acting as indirect, subtype-selective inhibitors of class IIa HDAC function. [1]
Studies have shown that BML-210 can enhance the expression of fratacin (FXN) in Friedreich ataxia (FRDA) cells, a disease involving epigenetic silencing. This effect is thought to be achieved at least in part by disrupting the recruitment of MEF2-mediated transcriptional co-repressors (such as HDAC4) to the FXN promoter. [1]
BML-210 analogues were synthesized by structure-directed synthesis (e.g., by altering the length of benzene ring substituents and methylene linkers), resulting in compounds with varying degrees of efficacy in inhibiting MEF2:HDAC4 interactions, providing lead compounds for further optimization. [1]
BML-210 has been reported to induce growth inhibition, apoptosis, and differentiation in certain leukemia cell lines under other conditions (not within the scope of this excerpt [1]). [1]

C20H25N3O2

339.4314

339.194

C, 70.77; H, 7.42; N, 12.38; O, 9.43

537034-17-6

9543540

White to off-white solid powder

1.2±0.1 g/cm3

632.5±40.0 °C at 760 mmHg

336.3±27.3 °C

0.0±1.9 mmHg at 25°C

1.633

2.58

3

3

9

25

408

0

O=C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])C1C([H])=C([H])C([H])=C([H])C=1[H])=O)N([H])C1=C([H])C([H])=C([H])C([H])=C1N([H])[H]

RFLHBLWLFUFFDZ-UHFFFAOYSA-N

InChI=1S/C20H25N3O2/c21-17-12-8-9-13-18(17)23-20(25)15-7-2-1-6-14-19(24)22-16-10-4-3-5-11-16/h3-5,8-13H,1-2,6-7,14-15,21H2,(H,22,24)(H,23,25)

N-(2-aminophenyl)-N'-phenyl-octanediamide

BML-210; CAY10433; BML 210; CAY-10433; BML210; BML-210; 537034-17-6; N1-(2-aminophenyl)-N8-phenyloctanediamide; BML-210(CAY10433); N-(2-aminophenyl)-N'-phenyl-octanediamide; CHEBI:61077; Octanediamide, N-(2-aminophenyl)-N'-phenyl-; CAY 10433.

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)

DMSO : ≥ 30 mg/mL (~88.38 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.37 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.)