HDAC9 ( IC50 = 23 nM ); HDAC7 ( IC50 = 43 nM ); HDAC5 ( IC50 = 97 nM ); HDAC4 ( IC50 = 157 nM ); HDAC8 ( IC50 = 42000 nM ); HDAC6 ( IC50 = 82000 nM )
Target: The target of TMP269 is class IIa histone deacetylases (HDACs), including HDAC4, HDAC5, HDAC7, and HDAC9. It shows minimal inhibition of class I HDACs (e.g., HDAC1, IC50 > 100 μM) [1]

TMP269 can be used as a tool to identify the endogenous substrates of class IIa HDAC enzymes, and at 10 μM, it has no effect on the mitochondrial activity and/or viability of human CD4+ T cells.[1]
TMP269 blocks the G protein-coupled receptor/PKD1-induced cell cycle progression, DNA synthesis, and proliferation in intestinal epithelial cells IEC-18.[2]

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TMP269 selectively inhibits class IIa HDACs, leading to increased histone acetylation in cells. In various cell lines, treatment with TMP269 (1-10 μM) upregulated the expression of genes associated with differentiation and anti-proliferation, as demonstrated by PCR and western blot analyses [1]
In intestinal epithelial cells, TMP269 (5 μM) blocked the phosphorylation and nuclear extrusion of class IIa HDACs induced by protein kinase D1 (PKD1), thereby retaining HDACs in the nucleus and inhibiting mitogenic signaling. This was associated with reduced cell proliferation, as measured by cell counting and BrdU incorporation assays [2]
In multiple myeloma cells, TMP269 (2.5-10 μM) enhanced endoplasmic reticulum (ER) stress-mediated cell death. It increased the expression of ER stress markers (e.g., CHOP, GRP78) and activated caspase-3/7, leading to apoptosis. Combination with ER stress inducers (e.g., bortezomib) synergistically increased cell death, as shown by flow cytometry and viability assays [3]
In triple-negative breast cancer cells, TMP269 (5 μM) downregulated HDAC9 expression, which in turn upregulated microRNA-206. This resulted in reduced invasive and angiogenic potential, as evidenced by transwell invasion assays and tube formation assays with endothelial cells [4]
In neuronal cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic ischemia/reperfusion injury, TMP269 (1-5 μM) reduced cell death and oxidative stress. It increased the expression of neuroprotective factors (e.g., Bcl-2) and decreased pro-apoptotic proteins (e.g., Bax), as shown by western blot and ROS detection assays [5]

In a mouse model of cerebral ischemia/reperfusion injury induced by middle cerebral artery occlusion (MCAO), intraperitoneal injection of TMP269 (10 mg/kg) reduced infarct volume and neurological deficit scores. It also decreased oxidative stress markers (e.g., MDA) and increased antioxidant enzyme activity (e.g., SOD) in brain tissues. Additionally, TMP269 upregulated Bcl-2 and downregulated Bax in the ischemic cortex, as demonstrated by immunohistochemistry and western blot [5]

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Level of histone H2A acetylation in the motor cortex increases after TMP269 injection[5]
Histone H2A acetylation was measured by western blot assays. TMP269 up-regulated H2A acetylation compared with the I/R group (P < 0.001), and the optimal concentration was 4 mg/kg (Figure 1A). In addition, immunohistochemistry showed that the number of positive cells in the infarct area was increased and the nucleus was more deeply stained in the 4 mg/kg TMP269 group compared with the I/R group (Figure 1B). Quantitative analysis confirmed that acetyl-H2A was significantly upregulated in the 4 mg/kg TMP269 group compared with the I/R group (P < 0.001; Figure 1B).

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Different concentrations of TMP269 ameliorate cerebral injury after ischemic stroke to varying degrees[5]
Ischemia in the left hemisphere was induced by middle cerebral artery occlusion and infarct size was determined at 24 hours after reperfusion. TTC assay results demonstrated that infarct volume was reduced by TMP269 compared with the I/R group (P < 0.001) and that 4 mg/kg TMP269 produced the biggest reduction in infarct size (Figure 2).

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TMP269 counteracts abnormal endothelial cell permeability[5]
TMP269 increased expression of the tight-junction proteins, ZO-1, Occludin and Claudin-5 compared with the I/R group (P < 0.01; Figure 3A). Blood-brain barrier integrity was assessed by observing the extravasation of Evans blue which is found in the brain when the blood-brain barrier is not intact (Figure 3B). Under green excitation, Evans blue produces bright red fluorescence. Almost no red fluorescence was observed in the sham group. The I/R group had the highest fluorescence intensity, and TMP269 groups showed decreased fluorescence intensity compared with the I/R group. Immunohistochemistry showed positive neurofilament staining after I/R, which was still present but weaker in the TMP269 group (Figure 3C) compared with the I/R group (P < 0.001).

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TMP269 regulates tissue kallikrein expression[5]
Tissue kallikrein expression was increased in the TMP269 groups compared with the I/R group (P < 0.01; Figure 4A). In addition, immunohistochemistry showed an increased number of positive cells in the infarct area and deeply stained plasma in the 4 mg/kg TMP269 group (Figure 4B). Quantitative analysis demonstrated significant upregulation of tissue kallikrein in the 4 mg/kg TMP269 group compared with the I/R group (P < 0.001; Figure 4B).

To measure HDAC inhibitory activity, recombinant class IIa HDACs (HDAC4, HDAC5, HDAC7, HDAC9) and class I HDACs (HDAC1) were incubated with a fluorogenic substrate in the presence of varying concentrations of TMP269. The release of fluorescent product was monitored over time, and IC50 values were calculated as the concentration required to reduce enzyme activity by 50% [1]
At Reaction Biology Corp., dose-response studies are carried out using ten concentrations in a triplicate dilution series, starting from a maximum final compound concentration of 100 μM in the reaction mixture. The same basic idea underlies all assays, including the HDAC9 assay previously mentioned: HDAC deacetylates acetylated or trifluoroacetylated lysine residues on fluorogenic peptide substrates. The substrate used by HDAC1, HDAC2, HDAC3, HDAC6, HDAC10, and HDAC11 was based on p53 residues 379–382 (Arg–His–Lys–Lys(Ac)). The diacetylated peptide substrate for HDAC8 is Arg-His-Lys(Ac)-Lys(Ac) and is based on residues 379–382 of p53. The class IIa HDAC-specific fluorogenic substrate, Boc-Lys(trifluoroacetyl)-AMC, was utilized in the HDAC4, HDAC5, HDAC7, and HDAC9 assays. Assay buffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1 mg/mL BSA) containing 1% DMSO final concentration is used for all reactions. 50 μM HDAC substrate is used in these reactions, which are then incubated for two hours at 30 °C and developed using trichostatin A and trypsin.

For assessing histone acetylation, cells were treated with TMP269 (1-10 μM) for 24-48 hours, followed by western blot analysis using antibodies against acetylated histones (e.g., H3K9ac) [1]
In intestinal epithelial cells, cells were pre-treated with TMP269 (5 μM) before PKD1 activation. Nuclear and cytoplasmic fractions were isolated, and the localization of class IIa HDACs was analyzed by western blot. Cell proliferation was assessed by counting cells at 24 and 48 hours and by measuring BrdU incorporation [2]
In multiple myeloma cells, cells were treated with TMP269 (2.5-10 μM) alone or in combination with bortezomib. Cell viability was measured by MTT assay, apoptosis by annexin V/PI staining via flow cytometry, and ER stress markers by western blot [3]
In triple-negative breast cancer cells, cells were treated with TMP269 (5 μM) for 48 hours. HDAC9 expression was measured by western blot, microRNA-206 by qPCR. Invasion was assessed using transwell chambers with Matrigel, and angiogenic potential by co-culturing with endothelial cells in tube formation assays [4]
In OGD/R-treated neuronal cells, cells were exposed to TMP269 (1-5 μM) during reoxygenation. Cell viability was measured by CCK-8 assay, ROS levels by DCFH-DA staining, and protein expression by western blot [5]

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The RosetteSep Human CD4+ T cell enrichment kit's manufacturer's instructions are followed to isolate human CD4+ T cells from whole blood via negative selection. The cells are then re-suspended in T-cell culture medium (10% FBS, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, 10 U/10 mg penicillin/streptomycin, 0.5% DMSO in RPMI) and plated at 50,000 cells/well with IL-2 (10 BRMP units/mL) and 100,000 human T-expander Dynabeads for a 72-hour period. According to the manufacturer's instructions (Cell Proliferation Assay Kit I (MTT)), mitochondrial function or cell viability is determined and is expressed as a percentage of control (no inhibitor) wells.

In the mouse MCAO model, mice were randomly divided into sham, vehicle, and TMP269 groups. TMP269 was administered intraperitoneally at 10 mg/kg immediately after reperfusion and once daily for 3 days. Neurological deficits were scored daily, and infarct volume was measured by TTC staining at 72 hours after injury. Brain tissues were collected for biochemical and molecular analyses [5]

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Group allocation and TMP269 administration[5]
Rats were randomly divided into sham (n = 24), I/R (cerebral I/R, n = 24), 1 mg/kg TMP269 (cerebral I/R combined with 1 mg/kg TMP269, n = 12), 4 mg/kg TMP269 (cerebral I/R combined with 4 mg/kg TMP269, n = 24), 10 mg/kg TMP269 (cerebral I/R combined with 10 mg/kg TMP269, n = 12), and 16 mg/kg TMP269 (cerebral I/R combined with 16 mg/kg TMP269, n = 12) groups. TMP269 was intraperitoneally administered half an hour before induction of ischemia.

[1]. Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group. Nat Chem Biol. 2013 May;9(5):319-25.

[2]. Protein kinase D1 mediates class IIa histone deacetylase phosphorylation and nuclear extrusion in intestinal epithelial cells: role in mitogenic signaling. Am J Physiol Cell Physiol. 2014 May 15;306(10):C961-71.

[3]. Class IIa HDAC inhibition enhances ER stress-mediated cell death in multiple myeloma. Leukemia. 2015 Sep;29(9):1918-1927.

[4]. Neuroprotective mechanism of TMP269, a selective class IIA histone deacetylase inhibitor, after cerebral ischemia/reperfusion injury. Neural Regen Res. 2019 Sep 24;15(2):277–284.

TMP269 is a selective class IIa HDAC inhibitor with a non-chelating zinc-binding group, distinguishing it from other HDAC inhibitors that chelate zinc. Its selectivity for class IIa HDACs makes it a valuable tool to study the specific roles of these enzymes in cell signaling, differentiation, and disease [1]
It exerts diverse biological effects, including inhibition of cell proliferation, enhancement of ER stress-mediated apoptosis, suppression of cancer invasion/angiogenesis, and neuroprotection against ischemia/reperfusion injury, through modulation of class IIa HDAC-dependent gene expression.
TMP269 is a novel and selective class IIa histone deacetylase (HDAC) inhibitor.

C25H21F3N4O3S

514.52

514.128

C, 58.36; H, 4.11; F, 11.08; N, 10.89; O, 9.33; S, 6.23

1314890-29-3

53344908

White to off-white solid powder

1.3±0.1 g/cm3

1.574

5.81

1

10

6

36

749

0

S1C([H])=C(C2C([H])=C([H])C([H])=C([H])C=2[H])N=C1C1(C([H])([H])N([H])C(C2=C([H])C([H])=C([H])C(C3=NOC(C(F)(F)F)=N3)=C2[H])=O)C([H])([H])C([H])([H])OC([H])([H])C1([H])[H]

HORXBWNTEDOVKN-UHFFFAOYSA-N

InChI=1S/C25H21F3N4O3S/c26-25(27,28)22-31-20(32-35-22)17-7-4-8-18(13-17)21(33)29-15-24(9-11-34-12-10-24)23-30-19(14-36-23)16-5-2-1-3-6-16/h1-8,13-14H,9-12,15H2,(H,29,33)

N-[[4-(4-phenyl-1,3-thiazol-2-yl)oxan-4-yl]methyl]-3-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]benzamide

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: ≥ 2.5 mg/mL (4.86 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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 (Please use freshly prepared in vivo formulations for optimal results.)