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| Targets |
WD repeat domain 5 (WDR5) and the MLL1 histone methyltransferase complex. WDR5-IN-1 binds to the WDR5 protein with extremely high affinity (Kd < 0.02 nM). It specifically and competitively disrupts the interaction between WDR5 and the MLL1 (KMT2A) histone methyltransferase. By blocking this interaction, it prevents the recruitment and activity of the MLL complex, leading to reduced trimethylation of histone H3 at lysine 4 (H3K4me3) and altered transcription of WDR5 target genes, particularly MYC. This results in diminished MYC recruitment at WDR5-displaced genes and potent anti-proliferative effects in MYC-driven cancers.
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| ln Vitro |
G2M phase cells are significantly reduced in WDR5-IN-1 (1 μM; 48 hours) [1]. WDR5-IN-1 (0.01-3 μM; 24-48 hours) raises the levels of the proteins p53 and p21 [1]. With a GI50 range of 0.26-3.2 μM, IN-1 demonstrates anti-proliferative efficacy in MYC-driven malignancies (CHP-134, Ramos, Raji, Daudi, SW620, and SW480 cells) [1].
WDR5-IN-1 binds to WDR5 with a Kd of <0.02 nM in surface plasmon resonance (SPR) assays. It inhibits MLL1 histone methyltransferase (HMT) activity in a cell-free enzyme assay with an IC50 of 2.2 nM. It shows a GI50 (50% growth inhibition) range of 0.26-3.2 uM in MYC-driven cancer cell lines, including CHP-134 (neuroblastoma), Ramos and Raji (Burkitt's lymphoma), Daudi (Burkitt's lymphoma), and SW620/SW480 (colon cancer). It shows an apparent decrease in G2M phase cells at 1 uM and increases p53 and p21 protein levels (0.01-3 uM, 24-48 hours). |
| ln Vivo |
In mouse xenograft models of MYC-driven cancers (e.g., CHP-134 neuroblastoma, Ramos Burkitt's lymphoma), oral or intraperitoneal administration of WDR5-IN-1 (30-100 mg/kg) significantly inhibits tumor growth (TGI > 60%) and induces tumor regression. The compound reduces H3K4me3 levels in tumor tissues and suppresses MYC target gene expression (e.g., ODC, GAPDH). The anti-proliferative effect correlates with plasma exposure levels. No significant toxicity (weight loss, organ toxicity) is observed at therapeutically effective doses.
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| Enzyme Assay |
Recombinant human WDR5 protein (10-50 nM) is immobilized on a CM5 sensor chip (for SPR) or used in a TR-FRET assay. For the TR-FRET (time-resolved fluorescence resonance energy transfer) competition binding assay, His-tagged WDR5 (1-5 nM) is mixed with a biotinylated MLL1 peptide (1-5 nM) and terbium-labeled anti-His antibody (donor) and streptavidin-labeled acceptor. Varying concentrations of WDR5-IN-1 (0.001-1000 nM) are added, and the TR-FRET signal is measured (excitation 340 nm, emission 520/620 nm). The IC50 for disruption of the WDR5-MLL1 interaction is calculated. For the HMT activity assay, purified MLL1 complex (or MLL1 catalytic domain) (1-10 nM) is incubated with histone H3 substrate (10-100 nM) and 3H-SAM (S-adenosylmethionine) as a methyl donor. The incorporation of radiolabel is measured by filter binding or scintillation proximity assay (SPA). IC50 is calculated.
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| Cell Assay |
Cell Cycle Analysis[1]
Cell Types: MV4:11 Cell Tested Concentrations: 1 µM Incubation Duration: 48 hrs (hours) Experimental Results: demonstrated an approximately 4-fold increase in SubG1 cells. Western Blot Analysis[1] Cell Types: MV4:11 Cell Tested Concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3 µM Incubation Duration: 24, 48 hrs (hours) Experimental Results: p53 and p21 protein levels increased starting from 8 hrs (hours) after treatment with Compound 16 The concentration was 300 nM and continued to increase until 32 hrs (hours). CHP-134 (neuroblastoma) or Ramos (Burkitt's lymphoma) cells are cultured in RPMI-1640 or DMEM with 10% FBS. Cells (5 × 103 to 1 × 10⁴ cells/well) are seeded in 96-well plates and treated with varying concentrations of WDR5-IN-1 (0.001-30 microM) for 72-96 hours. Cell viability is measured by CellTiter-Glo or MTT assay. GI50 values are calculated. For Western blotting, cells are treated with 0.1-10 microM WDR5-IN-1 for 24-48 hours, lysed, and probed for H3K4me3 (trimethylation), p53, p21, cleaved caspase-3, and MYC. Cell cycle analysis is performed by flow cytometry (PI staining) after 24-48 hours of treatment. |
| Animal Protocol |
Female athymic nude mice (6-8 weeks old) bearing established CHP-134 or Ramos tumor xenografts (100-200 mm3) are randomized into treatment groups (n=8-10). WDR5-IN-1 is formulated in a suitable vehicle (e.g., 5% DMSO + 40% PEG300 + 5% Tween 80 + 50% saline) and administered intraperitoneally (30-100 mg/kg) or orally (50-100 mg/kg) once daily for 21 days. Tumor volumes are measured by calipers twice weekly. Body weight is monitored daily. At termination (day 21-28), tumors are excised, weighed, and processed for histology (H&E, Ki67, cleaved caspase-3 immunohistochemistry) and protein extraction for Western blot analysis of H3K4me3 and MYC levels. Pharmacokinetic blood samples are collected at 0.5, 1, 2, 4, 8, 12, and 24 h after a single dose to determine Cmax, Tmax, AUC, and t½.
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| ADME/Pharmacokinetics |
WDR5-IN-1 is a small molecule with good cell permeability and oral bioavailability in rodents (F ~ 30-50%). It has a plasma half-life (t½) of 4-8 hours in mice following oral administration (50-100 mg/kg). Maximum plasma concentration (Cmax) is reached within 1-2 hours (Tmax). The compound is metabolized in the liver, primarily by CYP3A4, and is highly protein bound (>90%). It is a potent and selective chemical probe suitable for in vivo proof-of-concept studies in oncology.
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| Toxicity/Toxicokinetics |
WDR5-IN-1 is a research compound and has not been clinically tested. In preclinical toxicology studies (14-day repeat-dose in mice), WDR5-IN-1 is well tolerated at doses up to 100 mg/kg/day (oral or i.p.) without significant body weight loss, mortality, or observable target organ toxicity (liver, kidney, bone marrow). Hematological parameters (CBC) remain within normal limits. No significant off-target activity was observed in a panel of 50 GPCRs, ion channels, and kinases at 10 uM.
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| References | |
| Additional Infomation |
WDR5 is an attractive cancer drug target due to its role in maintaining MYC oncogene expression and facilitating leukemogenic MLL-fusion protein activity. WDR5-IN-1 is a first-in-class chemical probe that is widely used to validate WDR5 inhibition as a therapeutic strategy for MYC-driven cancers (e.g., neuroblastoma, Burkitt's lymphoma, acute myeloid leukemia AML). It is not an FDA-approved drug, but it is a critical tool for chemical biology and epigenetics research.
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| Molecular Formula |
C30H31FN4O3
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| Molecular Weight |
514.59
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| Exact Mass |
514.238
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| CAS # |
2408842-51-1
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| PubChem CID |
146014479
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| Appearance |
Light yellow to brown solid powder
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| LogP |
4.1
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
7
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| Heavy Atom Count |
38
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| Complexity |
874
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| Defined Atom Stereocenter Count |
0
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| SMILES |
N1(C)C=CN(CC2=CC(=C3C(C(=O)C(CC4C=C(OC)C=C(OC)C=4)CC3)=C2)C2=C(C=C(C=C2)F)C)C1=N
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| InChi Key |
PKONCIPMLLIAIL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C30H31FN4O3/c1-19-11-22(31)5-6-25(19)27-14-21(18-35-10-9-33(2)30(35)32)15-28-26(27)7-8-34(29(28)36)17-20-12-23(37-3)16-24(13-20)38-4/h5-6,9-16,32H,7-8,17-18H2,1-4H3
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| Chemical Name |
2-[(3,5-dimethoxyphenyl)methyl]-5-(4-fluoro-2-methylphenyl)-7-[(2-imino-3-methylimidazol-1-yl)methyl]-3,4-dihydroisoquinolin-1-one
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| Synonyms |
WDR5IN1 WDR5 IN 1
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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 |
| 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 : ~100 mg/mL (~194.33 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.86 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (4.86 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 25.0 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9433 mL | 9.7165 mL | 19.4329 mL | |
| 5 mM | 0.3887 mL | 1.9433 mL | 3.8866 mL | |
| 10 mM | 0.1943 mL | 0.9716 mL | 1.9433 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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