- WDR5 (WD repeat-containing protein 5) – MLL (Mixed Lineage Leukemia) protein-protein interaction (WIN site). [3]
Binding affinity: Kd = 0.45 ± 0.02 μM (determined by Isothermal Titration Calorimetry, ITC). [3]
Binding affinity (Kdisplacement): 3.0 ± 0.1 μM (determined by fluorescence polarization-based peptide displacement assay). [3]
ΔTm (thermal shift): 8.4 ± 0.1 °C (determined by Differential Scanning Fluorimetry, DSF). [3] - Selectivity: Shows no inhibitory effect on other histone methyltransferases including SETD7, G9a, EHMT1, SUV39H2, SETD8, PRMT3, and PRMT5 at concentrations up to 100 μM. [3]

- Inhibition of MLL HMT Activity: WDR5-0103 inhibits the methyltransferase activity of the pre-formed MLL-WDR5-RbBP5 trimeric complex in a concentration-dependent manner. The IC50 values are 39 ± 10 μM, 83 ± 10 μM, and 280 ± 12 μM at MLL complex concentrations of 125 nM, 500 nM, and 1000 nM, respectively. The inhibitory effect is diminished with increasing protein concentration, consistent with a competitive mechanism of action. [3]
- Inhibition of Tetrameric MLL Complex Activity: For the more active MLL-WDR5-RbBP5-ASH2L tetrameric complex, the inhibitory effect of WDR5-0103 is less pronounced but still concentration-dependent, with more potent inhibition observed at lower enzyme concentrations (e.g., 10 nM). [3]
- Reversal of ABCB1-mediated Multidrug Resistance: In ABCB1-overexpressing multidrug-resistant cancer cells (KB-V1, NCI-ADR-RES, MDR19-HEK293), WDR5-0103 (at non-toxic concentrations of 1-10 μM) sensitizes cells to ABCB1 substrate drugs (paclitaxel, vincristine, colchicine) in a concentration-dependent manner. For example, in KB-V1 cells, the IC50 for colchicine decreased from 1467.80 nM to 350.91 nM (4.2-fold reversal) with 10 μM WDR5-0103. [2]
- Reversal of ABCG2-mediated Multidrug Resistance: In ABCG2-overexpressing multidrug-resistant cancer cells (S1-MI-80, NCI-H460/MX20, R482-HEK293), WDR5-0103 (1-10 μM) sensitizes cells to ABCG2 substrate drugs (mitoxantrone, SN-38, topotecan) in a concentration-dependent manner. For example, in S1-MI-80 cells, the IC50 for mitoxantrone decreased from 27.23 μM to 1.08 μM (25.2-fold reversal) with 10 μM WDR5-0103. [2]
- Inhibition of Drug Efflux Function: WDR5-0103 (20 μM) significantly increases the intracellular accumulation of the fluorescent ABCB1 substrate calcein in ABCB1-overexpressing cells (KB-V1, NCI-ADR-RES, MDR19-HEK293) and the fluorescent ABCG2 substrate PhA in ABCG2-overexpressing cells (S1-MI-80, NCI-H460/MX20, R482-HEK293), without affecting accumulation in parental sensitive cells. [2]
- No Effect on Transporter Protein Expression: Treatment with WDR5-0103 (1-10 μM for 72 hours) does not significantly alter the protein expression levels of ABCB1 in KB-V1 and NCI-ADR-RES cells, or ABCG2 in S1-MI-80 and NCI-H460/MX20 cells. [2]
- Restoration of Drug-Induced Apoptosis: WDR5-0103 (10 μM) significantly restores colchicine-induced apoptosis in KB-V1 cells (from ~20% to ~54% total apoptosis) and topotecan-induced apoptosis in S1-MI-80 cells (from ~8% to ~17% total apoptosis), without inducing significant apoptosis by itself. [2]

- Alzheimer's Disease Mouse Models (P301S Tau mice and 5xFAD mice): Systemic administration of WDR5-0103 (2.5 mg/kg, intraperitoneal, once daily for 3 days) significantly reduces the elevated H3K4me3 level in the prefrontal cortex (PFC) of both P301S Tau mice and 5xFAD mice (5-6 months old) back to control levels, without affecting H3K27me3 levels. [1]
- Cognitive Function Improvement in P301S Tau Mice: In the Novel Object Recognition Test (NORT), WDR5-0103-treated P301S Tau mice showed a significantly improved discrimination ratio for the novel object. In the Barnes maze test, WDR5-0103-treated P301S Tau mice spent significantly more time exploring the correct hole and showed a significantly improved spatial memory index. Doses of 1, 2.5, and 5 mg/kg all improved performance, with 2.5 mg/kg giving slightly better results. No significant changes in locomotion, open-field, or rotarod tests were observed. [1]
- Cognitive Function Improvement in 5xFAD Mice: WDR5-0103 treatment (2.5 mg/kg, i.p., three times) significantly improved the discrimination ratio in NORT and spatial memory index in the Barnes maze test in 5xFAD mice (5-6 months old). [1]
- Restoration of Synaptic Function in P301S Tau Mice: Whole-cell patch-clamp recordings in PFC slices from P301S Tau mice showed that WDR5-0103 treatment markedly restored the reduced AMPAR-EPSC and NMDAR-EPSC input/output curves, as well as spontaneous EPSC (sEPSC) amplitude and frequency. Biochemical analysis confirmed that WDR5-0103 treatment restored the reduced synaptic levels of GluR1, NR1, and NR2A in PFC of P301S Tau mice. [1]

- Fluorescence Polarization (FP)-based Peptide Displacement Assay: To measure binding affinity to WDR5, an FP assay was used. A fluorescein-labeled MLL WIN peptide (GSARAEVHLRKS) was used at a constant concentration of 40 nM with 5 μM WDR5 protein in assay buffer (100 mM potassium phosphate pH 8.0, 150 mM NaCl, 0.01% Triton X-100). Compounds were titrated over a range of concentrations. The decrease in FP signal upon compound binding was measured using a microplate reader with excitation at 485 nm and emission at 528 nm. The Kdisplacement value for WDR5-0103 was determined to be 3.0 ± 0.1 μM. [3]
- Isothermal Titration Calorimetry (ITC): To confirm direct binding and determine the dissociation constant (Kd), ITC experiments were performed. Purified WDR5 protein (25 μM) in ITC buffer (100 mM Hepes, pH 7.5, 150 mM NaCl) was placed in the sample cell. WDR5-0103 at 0.2 mM was injected in 10 μL increments for a total of 25 injections at 25°C. Data were fitted with a one-binding site model. The Kd value for WDR5-0103 was 0.45 ± 0.02 μM. [3]
- Differential Scanning Fluorimetry (DSF): To assess binding-induced protein stabilization, DSF measurements were performed. WDR5 protein at 0.1 mg/mL in buffer (0.1 M Hepes pH 7.5, 0.15 M NaCl) was mixed with SYPRO Orange dye. Compounds were added at various concentrations. The temperature was increased from 20°C to 95°C at a rate of 1°C/min, and fluorescence was monitored. The thermal melting temperature (Tm) was calculated from the midpoint of the transition. WDR5-0103 induced a ΔTm of 8.4 ± 0.1 °C. [3]
- In Vitro MLL Histone Methyltransferase (HMT) Activity Assay: The effect of WDR5-0103 on MLL complex activity was tested using a radioactive assay. Reactions (20 μL) contained 20 mM Tris/HCl (pH 8.0), 5 mM DTT, 0.01% Triton X-100, 2 μM ³H-SAM, 5 μM biotinylated H3 (1-25) peptide, and various concentrations of WDR5-0103 (1 μM to 1 mM). The reaction was initiated by adding the MLL complex (trimeric or tetrameric) at specified concentrations (e.g., 125, 500, 1000 nM for trimeric; 10, 50, 125 nM for tetrameric). After 1 hour at room temperature, the reaction was quenched with 7.5 M guanidinium chloride. The mixture was transferred to a streptavidin-coated FlashPlate, incubated for ≥1 hour, and counted using a Topcount plate reader. IC50 values were calculated using SigmaPlot software. [3]

- Cytotoxicity Assay (MTT/CCK-8): Cells (e.g., KB-3-1, KB-V1, S1, S1-MI-80, NCI-H460, NCI-H460/MX20, HEK293 transfectants) were seeded in 96-well plates and treated with various concentrations of WDR5-0103 (or in combination with cytotoxic drugs) for 72 hours. Cell viability was determined using MTT or CCK-8 reagents. The highest non-toxic concentration of WDR5-0103 was determined to be 10 μM for subsequent experiments. [2]
- Drug Accumulation Assay (Flow Cytometry): To measure the drug efflux function of ABCB1 and ABCG2, cells were incubated with the fluorescent substrate calcein-AM (for ABCB1) or pheophorbide A (PhA, for ABCG2) in the presence or absence of WDR5-0103 (20 μM), positive control inhibitors (5 μM tariquidar for ABCB1; 5 μM Ko143 for ABCG2), or DMSO control. After incubation, cells were washed and analyzed by flow cytometry to determine the intracellular fluorescence intensity. [2]
- Apoptosis Assay (Annexin V-FITC/PI Staining): Cells (KB-3-1, KB-V1, S1, S1-MI-80) were treated for 48 hours with DMSO (control), WDR5-0103 (10 μM), a cytotoxic drug (0.5 μM colchicine for KB cells; 5 μM topotecan for S1 cells), or a combination of WDR5-0103 and the cytotoxic drug. Cells were then stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry to quantify the percentage of cells undergoing apoptosis (early and late). [2]

- Alzheimer's Disease Mouse Model Study: Male and female P301S Tau transgenic mice (PS19 line, 5-6 months old) and 5xFAD mice (5-6 months old) were used. WDR5-0103 was dissolved in DMSO to make a stock solution (100 mM) and then diluted with saline to working concentrations. The DMSO concentration in the working solution was <0.2%. Mice received intraperitoneal (i.p.) injections of WDR5-0103 (1, 2.5, or 5 mg/kg) or saline control once daily for 3 days. The injection volume was 10 mL/kg body weight. Behavioral tests (NORT, Barnes maze, open-field, rotarod), tissue collection, and electrophysiology recordings were conducted 24 hours after the last drug administration. [1]

In animal studies, no significant changes in locomotion, open-field, or rotarod tests were observed in WDR5-0103-treated mice, suggesting normal locomotor activity and motor coordination. [1]

[1]. Targeting histone K4 trimethylation for treatment of cognitive and synaptic deficits in mouse models of Alzheimer's disease. Sci Adv. 2020 Dec 9;6(50):eabc8096.
[2]. The WD repeat-containing protein 5 (WDR5) antagonist WDR5-0103 restores the efficacy of cytotoxic drugs in multidrug-resistant cancer cells overexpressing ABCB1 or ABCG2. Biomed Pharmacother. 2022 Oct;154:113663.
[3]. Small-molecule inhibition of MLL activity by disruption of its interaction with WDR5. Biochem J. 2013 Jan 1;449(1):151-159.

- The cocrystal structure of WDR5-0103 in complex with WDR5 (PDB code 3UR4) was determined. The methoxy substituent induces a 2.5 Å shift of the benzamide ring in the upper shallow region of the pocket compared to WDR5-0102. The increased potency of WDR5-0103 is in agreement with improved occupancy of the shallow pocket by the methoxy group, and of the hydrophobic groove between Tyr and Phe by the ester moiety. [3]
- In the context of Alzheimer's disease, WDR5-0103 was used to inhibit H3K4me3-catalyzing enzymes (SET1/MLL family HMTs). This treatment reversed the up-regulated expression of harmful genes involved in neurodegeneration (e.g., apoptosis, oxidative stress) and rescued the down-regulated expression of neuroprotective genes involved in cell communications in the PFC of P301S Tau mice. One of the top-ranking target genes identified was Sgk1 (serum and glucocorticoid-regulated kinase 1). [1]
- In the context of multidrug resistance, molecular docking analysis predicted that WDR5-0103 interacts with hydrophobic residues within the substrate-binding pockets of ABCB1 (e.g., Met68, Met69, Phe72, Phe336, Tyr953, Met986) and ABCG2 (e.g., Phe439, Val442, Val546), outcompeting the binding of substrate drugs and attenuating their efflux function. [2]

C21H25N3O4

383.440905332565

383.184

C, 65.78; H, 6.57; N, 10.96; O, 16.69

890190-22-4

890190-22-4;

6457069

Light yellow to yellow solid powder

1.2±0.1 g/cm3

495.2±45.0 °C at 760 mmHg

253.3±28.7 °C

0.0±1.3 mmHg at 25°C

1.603

2.63

1

6

6

28

536

0

O=C(C1C=C(NC(C2C=C(OC)C=CC=2)=O)C(N2CCN(C)CC2)=CC=1)OC

ZPLBXOVTSNRBFB-UHFFFAOYSA-N

InChI=1S/C21H25N3O4/c1-23-9-11-24(12-10-23)19-8-7-16(21(26)28-3)14-18(19)22-20(25)15-5-4-6-17(13-15)27-2/h4-8,13-14H,9-12H2,1-3H3,(H,22,25)

methyl 3-(3-methoxybenzamido)-4-(4-methylpiperazin-1-yl)benzoate

WDR5-0103; WDR 5-0103; WDR-5-0103; 890190-22-4; WDR5-01,03; Methyl 3-(3-methoxybenzamido)-4-(4-methylpiperazin-1-yl)benzoate; WDR5 0103; 3-[(3-Methoxybenzoyl)amino]-4-(4-methyl-1-piperazinyl)benzoic acid methyl ester; WDR50103; WDR-50103; WDR 50103; WD-Repeat Protein 5-0103.

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 : ~50 mg/mL (~130.40 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.52 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.

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Solubility in Formulation 2: 2.5 mg/mL (6.52 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.52 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 25.0 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.)