HDAC5 ( IC50 = 4.22 nM ); HDAC4 ( IC50 = 11.9 nM ); HDAC6 ( IC50 = 55.7 nM ); HDAC1 ( IC50 = 320 nM ); HDAC11 ( IC50 = 852 nM ); HDAC2 ( IC50 = 881 nM ); HDAC8 ( IC50 = 1278 nM )

LMK235 (5 and 20 mg/kg) restores postmitotic granule neurons' maturation and survival in Cdkl5 -/Y mice. In Cdkl5 -/Y mice, LMK235 also promotes the development of synapses in the dentate gyrus and hippocampal regions. Additionally, in Cdkl5 -/Y mice, LMK235 restores hippocampal-dependent learning and memory[3].

In accordance with the company's standard operating procedure, the in vitro inhibitory activity of compounds against seven human HDAC isoforms (1, 2, 4 C2A, 5 C2A, 6, 8, and 11) is assessed using a fluorescent-based assay. Using three-fold serial dilution and ten distinct concentrations, starting at 10 μM, the IC50 values are calculated. Vorinostat and TSA are the reference compounds.

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Whole-Cell HDAC Inhibition Assay[1]
The cellular HDAC assay was based on an assay published by Ciossek et al. and Bonfils et al with minor modifications. Briefly, human cancer cell lines Cal27sens/Cal27 CisR, Kyse510sens/Kyse510 CisR, A2780/A2780 CisR, and MDA-MB231sens/CisR were seeded in 96-well tissue culture plates at a density of 1.5 × 104 cells/well in a total volume of 90 μL of culture medium. After 24 h, cells were incubated for 18 h with increasing concentrations of test compounds such as Compound 19i (LMK235). The reaction was started by adding 10 μL of 3 mM Boc-Lys(ε-Ac)-AMC to reach a final concentration of 0.3 mM. The cells were incubated with the Boc-Lys(ε-Ac)-AMC for 3 h under cell culture conditions. After this incubation, 100 μL/well stop solution (25 mM Tris-HCl (pH 8), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1% NP40, 2.0 mg/mL trypsin, 10 μM vorinostat) was added and the mixture was developed for 3 h under cell culture conditions. Fluorescence intensity was measured at an excitation of 320 nm and emission of 520 nm in a NOVOstar microplate reader.

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HDAC IC50 Profiling[1]
The in vitro inhibitory activity of compounds 19e, 19h, and Compound 19i (LMK235) against seven human HDAC isoforms (1, 2, 4 C2A, 5 C2A, 6, 8, and 11) were performed with a fluorescent based assay according to the company’s standard operating procedure. The IC50 values were determined using 10 different concentrations with 3-fold serial dilution starting at 10 μM. TSA and vorinostat were used as reference compounds.

Improved MTT assay is used to determine the rate of cell survival under test substance action. Based on the ability of living cells to convert yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into violet formazan, which can be measured spectrophotometrically, the assay is implemented. Briefly put, 96-well plates are seeded with 5000, 7000, 8000, and 10,000 cells/well of A2780, Cal27, Kyse510, and MDA-MB-231 cell lines. Cells are exposed to higher concentrations of the test compounds after a 24-hour period. Following a 72-hour incubation period, the addition of MTT solution (5 mg/mL in phosphate buffered saline) is used to assess cell survival. In DMSO, the formazan precipitate dissolves. The absorbance in a FLUOstar microplate reader is measured at 544 and 690 nm[1].

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Primary hippocampal neurons and treatments[3]
Hippocampal neurons were prepared from P1 Cdkl5 -/Y (n = 4) and Cdkl5 +/Y male (n = 4) mice. Briefly, hippocampi were dissected from mouse brains under a dissection microscope and treated with trypsin for 15 min at 37 °C and DNase for 2 min at room temperature before triturating mechanically with a fire-polished glass pipette to obtain a single-cell suspension. Approximately 1.2 x 105 cells were plated on coverslips coated with poly-L-lysine in 6-well plates and cultured in Neurobasal medium supplemented with B27 and glutamine. Cells were maintained in vitro at 37 °C in a 5% CO2-humified incubator and fixed for immunostaining or western blot analysis at day 10 after plating (DIV10). 2 nM Compound 19i (LMK235) was administrated on alternate days starting from DIV2.

C57BL/6-BALB/c mice
20 mg/kg
i.p.

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Colony and treatments[3]
Mice for experiments were produced by crossing Cdkl5 +/− females with Cdkl5 -/Y males and Cdkl5 +/− females with Cdkl5 +/Y males; animals were genotyped by PCR of genomic DNA as previously described and littermate controls were used for all experiments. The day of birth was designated postnatal day (P) zero and animals that were 24 h of age were considered as 1-day-old animals (P1). After weaning, mice were housed 3 to 5 per cage on a 12-h light/dark cycle in a temperature-and humidity-controlled environment with food and water provided ad libitum.[3]
Starting from postnatal day 40 (P40), Cdkl5 +/Y and Cdkl5 -/Y male mice were treated with vehicle (PBS) or Compound 19i (LMK235) (N-((6 (hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide) 5 mg/kg or 20 mg/kg administered i.p. daily for 8 or 16 days. Animals were sacrificed on P48 or P56.

[1]. Histone deacetylase (HDAC) inhibitors with a novel connecting unit linker region reveal a selectivity profile for HDAC4 and HDAC5 with improved activity against chemoresistant cancer cells. J Med Chem. 2013 Jan 24;56(2):427-36.

[2]. HDAC5, a potential therapeutic target and prognostic biomarker, promotes proliferation, invasion and migration in human breast cancer. Oncotarget. 2016 Jun 21;7(25):37966-37978.

[3]. HDAC4: a key factor underlying brain developmental alterations in CDKL5 disorder. Hum Mol Genet. 2016 Sep 15;25(18):3887-3907.

The synthesis and biological evaluation of new potent hydroxamate-based HDAC inhibitors with a novel alkoxyamide connecting unit linker region are described. Biological evaluation includes MTT and cellular HDAC assays on sensitive and chemoresistant cancer cell lines as well as HDAC profiling of selected compounds. Compound 19i (LMK235) (N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide) showed similar effects compared to vorinostat on inhibition of cellular HDACs in a pan-HDAC assay but enhanced cytotoxic effects against the human cancer cell lines A2780, Cal27, Kyse510, and MDA-MB231. Subsequent HDAC profiling yielded a novel HDAC isoform selectivity profile of 19i in comparison to vorinostat or trichostatin A (TSA). 19i shows nanomolar inhibition of HDAC4 and HDAC5, whereas vorinostat and TSA inhibit HDAC4 and HDAC5 in the higher micromolar range.[1]

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Purpose: Histone deacetylase 5 (HDAC5) is an important protein in neural and cardiac diseases and a potential drug target. However, little is known regarding the specific role of HDAC5 in breast cancer (BC). We aimed to evaluate HDAC5 expression in human breast tumors and to determine the effects of the inhibition of HDAC5 expression in BC cells. Experimental design: HDAC5 expression was evaluated in BC patients and was correlated with clinical features and with patient prognosis. Functional experiments were performed using shRNA and the selective HDAC inhibitor LMK-235 for HDAC5 knockdown and inhibition in BC cells. The synergistic effects of LMK-235 with the proteasome inhibitor bortezomib were also examined. Results: HDAC5 was extensively expressed in human BC tissues, and high HDAC5 expression was associated with an inferior prognosis. Knockdown of HDAC5 inhibited cell proliferation, migration, invasion, and enhanced apoptosis. The HDAC5 inhibitor LMK-235 inhibited cell growth and induced apoptosis, while the inclusion of bortezomib synergistically enhanced the efficacy of LMK-235. Conclusions: Our findings indicate that HDAC5 is a promising prognostic marker and drug target for BC and that the combination of LMK-235 and bortezomib presents a novel therapeutic strategy for BC.[2]

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Cyclin-dependent kinase-like 5 (CDKL5) is a Ser/Thr protein kinase predominantly expressed in the brain. Mutations of the CDKL5 gene lead to CDKL5 disorder, a neurodevelopmental pathology that shares several features with Rett Syndrome and is characterized by severe intellectual disability. The phosphorylation targets of CDKL5 are largely unknown, which hampers the discovery of therapeutic strategies for improving the neurological phenotype due to CDKL5 mutations. Here, we show that the histone deacetylase 4 (HDAC4) is a direct phosphorylation target of CDKL5 and that CDKL5-dependent phosphorylation promotes HDAC4 cytoplasmic retention. Nuclear HDAC4 binds to chromatin as well as to MEF2A transcription factor, leading to histone deacetylation and altered neuronal gene expression. By using a Cdkl5 knockout (Cdkl5 -/Y) mouse model, we found that hypophosphorylated HDAC4 translocates to the nucleus of neural precursor cells, thereby reducing histone 3 acetylation. This effect was reverted by re-expression of CDKL5 or by inhibition of HDAC4 activity through the HDAC4 inhibitor LMK235. In Cdkl5 -/Y mice treated with LMK235, defective survival and maturation of neuronal precursor cells and hippocampus-dependent memory were fully normalized. These results demonstrate a critical role of HDAC4 in the neurodevelopmental alterations due to CDKL5 mutations and suggest the possibility of HDAC4-targeted pharmacological interventions.[3]

C15H22N2O4

294.35

294.157

C, 61.21; H, 7.53; N, 9.52; O, 21.74

1418033-25-6

71520717

White to off-white solid powder

1.2±0.1 g/cm3

1.538

2.05

3

4

8

21

326

0

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

VRYZCEONIWEUAV-UHFFFAOYSA-N

InChI=1S/C15H22N2O4/c1-11-8-12(2)10-13(9-11)15(19)17-21-7-5-3-4-6-14(18)16-20/h8-10,20H,3-7H2,1-2H3,(H,16,18)(H,17,19)

N-[6-(hydroxyamino)-6-oxohexoxy]-3,5-dimethylbenzamide

LMK 235; LMK235; N-((6-(hydroxyamino)-6-oxohexyl)oxy)-3,5-dimethylbenzamide; LMK235; N-[6-(hydroxyamino)-6-oxohexoxy]-3,5-dimethylbenzamide; CHEMBL2312168; 6-{[(3,5-dimethylphenyl)formamido]oxy}-N-hydroxyhexanamide; LMK-235

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 (7.07 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 (7.07 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 (7.07 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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Solubility in Formulation 4: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 3.0mg/ml (10.19mM)

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