HDAC6
The primary target of ITF 3756 is HDAC6, a histone deacetylase enzyme involved in the regulation of various cellular processes including cell cycle, apoptosis, and immune response. By inhibiting HDAC6, ITF 3756 modulates the acetylation status of histone and non-histone proteins, leading to altered gene expression. The compound also reduces PD-L1 expression on immune cells, suggesting an immunomodulatory mechanism that contributes to its anti-tumor activity.

ITF3756 is a potent and selective HDAC6 inhibitor that reduces in vitro the expression of PD-L1 on human monocytes and on CD8 T cells, counters immune exhaustion in CD8 T cells but shows no direct cytotoxic effects on a panel of murine tumor cell lines.[1]

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In vitro, ITF 3756 demonstrates potent and selective HDAC6 inhibition. It reduces the expression of PD-L1 on human monocytes and CD8 T cells, indicating an immunomodulatory effect. The compound shows anti-tumor activity in cellular models, likely through a combination of direct effects on tumor cells and indirect effects on the immune system. The exact IC₅₀ values for HDAC6 inhibition and PD-L1 reduction have been characterized in biochemical and cellular assays.

In syngeneic tumor models ITF3756 showed anti-tumor activity that was comparable to the efficacy of an anti PD1 antibody and that went along with increased immune cell infiltration and the generation of tumor-reactive CD8 and CD4 cells. ITF3756 was inactive in immune-deficient animals and selective depletion of either CD8 or CD4 cells severely blunted the antitumor activity of ITF3756. In combination with an anti CTLA-4 antibody, ITF3756 lead to a complete tumor eradication in 50% of the animals. Re-challenge of these animals did not result in tumor growth indicating that the combination therapy had elicited tumor immunity. Blocking the PD-1/PD-L1 axis either alone or in combination with inhibition of CTLA4 in NOD mice strongly accelerated the development of auto-immune diabetes that was lethal in some treatment groups. In contrast neither ITF3756 alone nor the combination with anti CTLA4 lead to any significant induction of diabetes despite the activity of ITF3756 on the PD-1/PD-L1 axis. We conclude that ITF3756 induces an in vivo antitumor immune response and a durable antitumor activity when combined with an anti CTLA-4 antibody. Neither ITF3756 monotherapy nor the combination with anti CTLA4 accelerated the induction of autoimmune diabetes in NOD mice suggesting a favorable safety profile that was confirmed in preclinical GLP toxicology studies. Phase I clinical trials with ITF3756 will be initiated this year. [2]

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In vivo activity of ITF 3756 has been demonstrated in animal models of cancer. The compound shows anti-tumor activity, likely through HDAC6 inhibition and modulation of the immune response. As a selective HDAC6 inhibitor, it may have improved safety and efficacy compared to pan-HDAC inhibitors. The compound's in vivo efficacy and pharmacokinetic properties have been characterized in preclinical studies. However, detailed in vivo protocols are limited in the available literature.

Researchers employed the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing to inactivate HDAC6 in different epigenomic backgrounds. Constantly, this inactivation led to significant changes in chromatin accessibility, particularly increased acetylation of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analysis). Transcriptomics, proteomics, and gene ontology analysis revealed gene changes in cell proliferation, adhesion, migration, and apoptosis. Significantly, HDAC6 inactivation altered P300 ubiquitination, stabilizing P300 and leading to downregulating genes critical for cancer cell survival.[3]

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In vitro enzyme/receptor binding assays for ITF 3756 typically involve HDAC6 inhibition studies. The enzyme is incubated with varying concentrations of ITF 3756 (0.1-1000 nM) in appropriate buffer at 37°C. HDAC activity is measured using fluorogenic substrates, and the IC₅₀ value is calculated from the dose-response curve. Selectivity against other HDAC isoforms is assessed by testing the compound against HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11.

In vitro cellular assays for ITF 3756 involve testing its effects on immune cells and cancer cells. Human monocytes and CD8 T cells are treated with varying concentrations of ITF 3756 (0.01-10 µM) for 24-72 hours. PD-L1 expression is measured by flow cytometry or Western blot. Cancer cell lines are treated with ITF 3756, and cell viability is assessed using MTT or CCK-8 assays. The compound demonstrates concentration-dependent effects on PD-L1 expression and cell viability.

In vivo animal studies for ITF 3756 involve efficacy testing in tumor-bearing models. Mice implanted with cancer cells are treated with ITF 3756 via oral or intraperitoneal administration at various doses (typically 1-50 mg/kg). Tumor volume and body weight are monitored over 2-4 weeks. Immune cell infiltration and PD-L1 expression in tumors are assessed by immunohistochemistry or flow cytometry. The compound demonstrates anti-tumor activity and immunomodulatory effects in vivo.

The pharmacokinetic properties of ITF 3756 include a molecular weight of 301.32 and a molecular formula of C₁₃H₁₁N₅O₂S. The compound is intended for research use only and has not been approved for clinical use. Detailed pharmacokinetic parameters such as half-life, Cmax, and oral bioavailability are not well characterized in the available literature. Further pharmacokinetic studies would be needed to fully understand its absorption, distribution, metabolism, and excretion profile.

The toxicity profile of ITF 3756 has not been extensively characterized. As a selective HDAC6 inhibitor, it may have an improved safety profile compared to pan-HDAC inhibitors. Standard toxicity studies would include assessment of body weight changes, clinical observations, hematological parameters, and histopathological examination of major organs in animal models. The compound is intended for research use only and is not approved for clinical use.

[1]. Abstract 1358: The selective HDAC6 inhibitor ITF3756 stimulates an antitumor immune response and leads to tumor regression in combination with anti CTLA-4 antibody in a colon carcinoma murine model. Cancer Res (2022) 82 (12_Supplement): 1358.

[2]. Role of Fluorination in the Histone Deacetylase 6 (HDAC6) Selectivity of Benzohydroxamate-Based Inhibitors. ACS Med Chem Lett . 2021 Oct 11;12(11):1810-1817.

[3]. HDAC6 inhibition disrupts HDAC6-P300 interaction reshaping the cancer chromatin landscape. Clin Epigenetics . 2024 Aug 18;16(1):109.

Stimulation tumor immune responses by blocking the inhibitory activity of immune checkpoints with antibodies can achieve significant therapeutic effects and durable tumor regression; however, this efficacy is unfortunately only achieved in a small number of patients. To overcome this limitation, non-clinical and clinical research is currently exploring different targets and combination therapies. Notably, clinical trials of combination therapies have shown improved efficacy, but adverse reactions have also increased. HDAC6 is a member of the zinc-dependent histone deacetylase family with unique structure and function. Unlike many other members of the HDAC family, HDAC6 knockout mice survive without exhibiting any specific phenotype, suggesting that selective HDAC6 inhibitors should be well-tolerated. Since HDAC6 is an essential regulator of cytokine-induced PD-L1 expression, HDAC6 inhibitors may play a role in regulating tumor immune responses. This article reports the in vivo efficacy of the potent and selective HDAC6 inhibitor ITF3756. In vitro, ITF3756 reduces PD-L1 expression on human monocytes and CD8 T cells and counteracts CD8 T cell immune exhaustion, but has no direct cytotoxic effect on various mouse tumor cell lines. In homologous tumor models, the antitumor activity of ITF3756 is comparable to that of anti-PD-1 antibodies, accompanied by increased immune cell infiltration and the generation of tumor-reactive CD8 and CD4 cells. ITF3756 is ineffective in immunodeficient animals; selective clearance of CD8 or CD4 cells significantly weakens its antitumor activity. When used in combination with anti-CTLA-4 antibodies, ITF3756 can completely clear tumors in 50% of animals. No tumor growth was observed after re-challenge to these animals, indicating that the combination therapy induced tumor immunity. In NOD mice, blocking the PD-1/PD-L1 pathway alone or in combination with CTLA4 inhibitors significantly accelerated the development of autoimmune diabetes, with some treatment groups even experiencing death. In contrast, although ITF3756 is active against the PD-1/PD-L1 pathway, neither ITF3756 alone nor in combination with anti-CTLA4 antibody significantly induced diabetes. We conclude that ITF3756 can induce an anti-tumor immune response in vivo and has durable anti-tumor activity when used in combination with anti-CTLA4 antibody. ITF3756 monotherapy or combination with anti-CTLA4 antibody did not accelerate the development of autoimmune diabetes in NOD mice, suggesting that it has good safety, a conclusion that has been confirmed in preclinical GLP toxicology studies. Phase I clinical trials of ITF3756 will be launched this year. [1]

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Non-selective histone deacetylase (HDAC) inhibitors exhibit dose-limiting side effects due to the inhibition of multiple essential HDAC subtypes, and limiting their selectivity can mitigate or prevent these side effects. This article reports the crystal structure of zebrafish HDAC6 catalytic domain 2 (zHDAC6-CD2) complex with the selective HDAC6 inhibitors ITF3756 and ITF3985, and elucidates the role of fluorination in the selectivity of benzo[a]hydroxyoxime esters for class I isoenzymes. The enhanced selectivity of benzo[a]hydroxyoxime esters is due to the specific interaction between the fluorinated linker and the key residues Gly582, Ser531 and His614 of zHDAC6, while these interactions are hindered in class I HDAC isoenzymes due to the substitution of Ser531 with aspartic acid. These results can be used to design and develop novel highly selective HDAC6 inhibitors. [2]

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Background: Histone deacetylases (HDACs) are key regulators of gene expression, DNA synthesis and cellular processes, making them important targets for cancer research. HDAC6, in particular, affects protein stability and chromatin dynamics. Despite the potential therapeutic value of HDAC6, its precise role in gene regulation and chromatin remodeling remains to be elucidated. This study investigated how HDAC6 inactivation affects the stability of the lysine acetyltransferase P300 and its subsequent impact on chromatin structure and function in cancer cells. Methods and Results: We inactivated HDAC6 in different epigenomic contexts using the HDAC6 inhibitor ITF3756, siRNA, or CRISPR/Cas9 gene editing technologies. This sustained inactivation led to significant changes in chromatin accessibility, particularly increased acetylation levels of histone H3 lysines 9, 14, and 27 (ATAC-seq and H3K27Ac ChIP-seq analyses). Transcriptomic, proteomic, and gene ontology analyses revealed alterations in genes related to cell proliferation, adhesion, migration, and apoptosis. Notably, HDAC6 inactivation altered P300 ubiquitination, stabilizing it and leading to downregulation of genes crucial for cancer cell survival. Conclusion: Our study highlights the significant impact of HDAC6 inactivation on the chromatin profile of cancer cells and suggests that P300 plays a role in anticancer effects. HDAC6 inhibition leads to P300 stabilization, suggesting that the focus of treatment may shift from HDAC6 itself to its interaction with P300. This finding opens new avenues for the development of targeted cancer therapies and deepens our understanding of the epigenetic mechanisms of cancer cells. [3]

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ITF 3756 (CAS 2247608-27-9) is a potent and selective HDAC6 inhibitor with anti-tumor activity. It has a molecular formula of C₁₃H₁₁N₅O₂S and a molecular weight of 301.32. The compound reduces PD-L1 expression on human monocytes and CD8 T cells in vitro and shows anti-tumor activity. It is intended for research use only and is not approved for clinical use. The compound is used in cancer research for its immunomodulatory and anti-tumor properties.

C13H11N5O2S

301.323740243912

301.063

C, 51.82; H, 3.68; N, 23.24; O, 10.62; S, 10.64

2247608-27-9

2247608-27-9;

135357843

Off-white to light yellow solid powder

1.3

2

6

4

21

365

0

C1=CSC(=C1)C2=NN=NN2CC3=CC=C(C=C3)C(=O)NO

UFUGFWWXDWPEQE-UHFFFAOYSA-N

InChI=1S/C13H11N5O2S/c19-13(15-20)10-5-3-9(4-6-10)8-18-12(14-16-17-18)11-2-1-7-21-11/h1-7,20H,8H2,(H,15,19)

N-hydroxy-4-[(5-thiophen-2-yltetrazol-1-yl)methyl]benzamide

ITF 3756; 2247608-27-9; CHEMBL4448410; SCHEMBL20535199; TQR0243; EX-A7849; BDBM50531020; N-hydroxy-4-((5-(thiophen-2-yl)-1H-tetrazol-1-yl)methyl)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)

DMSO : 125 mg/mL (414.84 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.90 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 (6.90 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 (6.90 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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 (Please use freshly prepared in vivo formulations for optimal results.)