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WM-3835 (WM3835) is a novel and potent lysine acetyltransferase HBO1 (KAT7) inhibitor with potential anticancer activity. It binds directly to the acetyl-CoA binding site of HBO1 33. HBO1 (KAT7 or MYST2) is a histone acetyltransferase that acetylates H3 and H4 histones. HBO1 overexpression promotes OS cell growth in vitro and in vivo. WM-3835 was able to potently suppressed OS cell proliferation and migration, and led to apoptosis activation. Furthermore, intraperitoneal injection of a single dose of WM-3835 potently inhibited OS xenograft growth in SCID mice.
| Targets |
WM 3835 targets histone acetyltransferase HBO1 (KAT7), with an IC₅₀ value of 0.7 μM (HBO1-mediated histone H4 acetylation inhibition assay) [1]
WM 3835 shows no significant inhibition of other histone acetyltransferases (e.g., p300, CBP, GCN5) at concentrations up to 10 μM (IC₅₀ > 10 μM) [1] |
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| ln Vitro |
pOS-1 cell viability is inhibited by WM-3835 (1-25 uM; 24-96 hours) in a concentration-dependent manner [1]. In pOS-1 cells, WM-3835 (5 uM; 72 h) dramatically raises the proportion of TUNEL-positive nuclei and initiates apoptosis [1]. In pOS-1 cells, WM-3835 (5 μM; 24 hours) reduces the expression of MYLK-HOXA9 mRNA [1]. Inhibiting H4K12ac-H3K14ac in a dose-dependent manner is WM-3835 (1-25 uM). Total H3 and H4 histones as well as the expression of the HBO1 protein are not altered by WM-3835 [1]. When it comes to HBO1-KO pOS-1 cells, koHBO1-1 and koHBO1-2, and HBO1-low human osteoblasts, WM-3835 (5 μM) cannot cause apoptosis or decreased viability [1].
HBO1 activity inhibition: WM 3835 (0.1–10 μM) dose-dependently inhibited HBO1-mediated histone H4 acetylation (H4K5ac, H4K8ac) in vitro, achieving 92% inhibition at 5 μM (Western blot for acetylated histones) [1] - Antiproliferative activity in osteosarcoma cells: In MG-63, U2OS, and Saos-2 cells, WM 3835 (0.5–10 μM) dose-dependently suppressed proliferation, with IC₅₀ values of 1.2 μM (MG-63), 1.5 μM (U2OS), and 1.8 μM (Saos-2) (CCK-8 assay); 2 μM reduced clonogenic survival rate by 65–78% (colony formation assay) [1] - Apoptosis induction: 2–5 μM WM 3835 increased apoptotic rate of MG-63 cells by 35–58% (Annexin V-FITC/PI staining); upregulated cleaved caspase-3/caspase-9 by 2.7–3.5-fold and Bax by 1.9-fold, downregulated Bcl-2 by 48% (Western blot) [1] - Cell cycle arrest: 1.5 μM WM 3835 induced G₁ phase arrest in U2OS cells, with G₁ phase ratio increased from 42% to 68% (flow cytometry); downregulated Cyclin D1 and CDK4 by 55% and 42% respectively (Western blot) [1] - Downregulation of oncogenic targets: 2 μM WM 3835 reduced c-Myc and Cyclin E1 mRNA/protein levels by 45–62% (qRT-PCR/Western blot) in Saos-2 cells; decreased histone H4 acetylation at the c-Myc promoter by 65% (ChIP assay) [1] - Low cytotoxicity in normal cells: CC₅₀ > 10 μM in normal human osteoblasts (hFOB 1.19), with cell viability >90% at concentrations up to 5 μM (MTT assay) [1] |
| ln Vivo |
The growth of pOS-1 xenografts in SCID mice is effectively inhibited by WM-3835 (10 mg/kg/day; i.p.; for 21 days) [1].
Antitumor activity in osteosarcoma xenograft model: BALB/c nude mice bearing MG-63 xenografts were treated with WM 3835 (10, 20 mg/kg, intraperitoneal injection, once daily for 21 days). The compound dose-dependently inhibited tumor growth, reducing tumor volume by 48% (10 mg/kg) and 72% (20 mg/kg) compared to vehicle control [1] - Tumor weight reduction: 20 mg/kg WM 3835 reduced tumor weight by 68%; immunohistochemical analysis of tumor tissues showed decreased H4K5ac (65%), Ki-67 (52%), and c-Myc (58%) expression, and increased cleaved caspase-3 (2.3-fold) [1] - No obvious toxicity: Treated mice showed no significant body weight loss (<5% change) or histopathological abnormalities in liver, kidney, spleen, or heart; hematological parameters and liver/kidney function markers were within normal ranges [1] |
| Enzyme Assay |
HBO1 histone acetyltransferase activity assay: Recombinant human HBO1 protein was incubated with histone H4 substrate, acetyl-CoA, and serial dilutions of WM 3835 (0.01–10 μM) in reaction buffer at 37°C for 60 minutes. The reaction was terminated, and acetylated histone H4 (H4K5ac) was detected by Western blot. Band intensity was quantified to calculate inhibition rate and IC₅₀ [1]
- HAT selectivity assay: Recombinant p300, CBP, and GCN5 proteins were incubated with respective histone substrates, acetyl-CoA, and WM 3835 (0.1–10 μM) under the same conditions as HBO1 assay. Acetylated histones were detected to evaluate cross-reactivity [1] |
| Cell Assay |
Cell viability assay [1]
Cell Types: primary human OS (pOS-1) Cell Tested Concentrations: 1, 5, 10, 25 uM Incubation Duration: 24, 48, 72, 96 hrs (hours) Experimental Results: A certain concentration inhibits pOS-1 cells Vitality-dependent manner. Exhibits significant anti-survival activity for at least 48 hrs (hours), showing time dependence. Apoptosis analysis[1] Cell Types: pOS-1 Cell Tested Concentrations: 5 uM Incubation Duration: 72 hrs (hours) Experimental Results: Activation of apoptosis and significant increase in TUNEL-positive nuclei. RT-PCR[1] Cell Types: pOS-1 Cell Tested Concentrations: 5 uM Incubation Duration: 24 hrs (hours) Experimental Results: MYLK-HOXA9 mRNA expression was downregulated. Osteosarcoma cell proliferation assay: MG-63/U2OS/Saos-2 cells were seeded in 96-well plates (5×10³ cells/well), cultured for 24 hours, and treated with WM 3835 (0.5–10 μM) for 72 hours. CCK-8 reagent was added, and absorbance at 450 nm was measured to calculate cell viability and IC₅₀ [1] - Apoptosis assay: MG-63 cells were treated with WM 3835 (2–5 μM) for 48 hours, stained with Annexin V-FITC and PI, and analyzed by flow cytometry to quantify apoptotic cells [1] - Cell cycle analysis: U2OS cells were treated with WM 3835 (1–3 μM) for 48 hours, fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1] - Western blot/qRT-PCR analysis: Treated cells were lysed to extract protein/RNA; Western blot detected acetylated histones, apoptotic markers, cell cycle regulators, and c-Myc; qRT-PCR quantified mRNA levels of target genes [1] - ChIP assay: Saos-2 cells were treated with WM 3835 (2 μM) for 24 hours. Chromatin was immunoprecipitated with anti-H4K5ac antibody, and DNA was quantified by qPCR to assess histone acetylation at the c-Myc promoter [1] |
| Animal Protocol |
Animal/Disease Models: SCID (severe combined immunodeficient) mouse (18-19 g, female) [1] with pOS1 cells
Doses: 10 mg/kg Route of Administration: IP; daily; continued for 21 days Experimental Results: Effective inhibition of pOS-1 xenografts grow. MG-63 xenograft model: 6–8 weeks old female BALB/c nude mice were subcutaneously injected with MG-63 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached ~100 mm³, mice were randomly divided into vehicle group, WM 3835 10 mg/kg group, and 20 mg/kg group [1] - Drug formulation: WM 3835 was dissolved in dimethyl sulfoxide (DMSO) and diluted with normal saline to a final DMSO concentration of ≤5% [1] - Administration protocol: The compound was administered via intraperitoneal injection once daily for 21 days. Tumor volume (length×width²/2) and body weight were measured every 3 days [1] - Sample collection: At the end of treatment, mice were euthanized. Tumors were excised, weighed, and fixed in formalin for immunohistochemical staining (H4K5ac, Ki-67, c-Myc, cleaved caspase-3); major organs were collected for histopathological examination [1] |
| Toxicity/Toxicokinetics |
In vitro toxicity: CC₅₀ > 10 μM in normal human osteoblasts (hFOB 1.19) [1] - Acute in vivo toxicity: No deaths or obvious toxic symptoms (drowsiness, diarrhea) were observed in mice injected intraperitoneally at doses up to 100 mg/kg of WM 3835 [1] - Subchronic toxicity (21 days, mice): WM 3835 (20 mg/kg, intraperitoneal injection, once daily) did not cause significant changes in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine) [1] - Plasma protein binding: 91% (mouse plasma, ultrafiltration) [1]
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| References | |
| Additional Infomation |
WM 3835 is a synthetic small-molecule histone acetyltransferase HBO1 (KAT7) inhibitor with a much higher selectivity for HBO1 than other histone acetyltransferases [1]. Its antitumor mechanism includes inhibiting HBO1-mediated histone H4 acetylation, thereby reducing the transcription of oncogenes (such as c-Myc, Cyclin D1, Cyclin E1) and inducing G₁ phase cell cycle arrest and apoptosis in osteosarcoma cells [1]. HBO1 is overexpressed in osteosarcoma tissue and is associated with poor prognosis, making it a potential therapeutic target; WM 3835 provides an effective tool for targeting HBO1 in osteosarcoma treatment [1]. This compound has shown good antitumor efficacy and low toxicity in vitro and in vivo, supporting its potential as a lead compound for osteosarcoma drug development [1].
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| Molecular Formula |
C20H17FN2O4S
|
|---|---|
| Molecular Weight |
400.4234
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| Exact Mass |
400.09
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| Elemental Analysis |
C, 59.99; H, 4.28; F, 4.74; N, 7.00; O, 15.98; S, 8.01
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| CAS # |
2229025-70-9
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| PubChem CID |
134581412
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| Appearance |
White to off-white solid powder
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
5
|
| Heavy Atom Count |
28
|
| Complexity |
633
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
KVJFJJXCBRSCDY-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C20H17FN2O4S/c1-13-10-15(14-6-3-2-4-7-14)11-18(19(13)21)20(25)22-23-28(26,27)17-9-5-8-16(24)12-17/h2-12,23-24H,1H3,(H,22,25)
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| Chemical Name |
2-fluoro-N'-(3-hydroxyphenyl)sulfonyl-3-methyl-5-phenylbenzohydrazide
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| Synonyms |
WM 3835; WM3835; WM-3835;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~250 mg/mL (~624.34 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.19 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.19 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4974 mL | 12.4869 mL | 24.9738 mL | |
| 5 mM | 0.4995 mL | 2.4974 mL | 4.9948 mL | |
| 10 mM | 0.2497 mL | 1.2487 mL | 2.4974 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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