WDR5 (WD40 repeat domain protein 5) - degrader that recruits VHL E3 ligase; DC50 = 3.7 ± 1.4 nM
Binding affinity: Kd (to WDR5) = 63 ± 10 nM; Kd (to VCB) = 140 ± 7.2 nM. Ternary complex Kd = 52 ± 8.3 nM; cooperativity (α = Kd(binary)/Kd(ternary)) = 2.74. [1]

- In MV4;11 human MLL-r AML cells, MS67 induced WDR5 degradation at concentrations as low as 1 nM with a DC50 of 3.7 ± 1.4 nM and achieved near-complete depletion of WDR5 at 0.5 μM with Dmax of 94 ± 1%. OICR-9429 and the negative control MS67N did not degrade WDR5. [1]
- In MIA PaCa-2 PDAC cells, MS67 had a DC50 of 45 ± 16 nM and Dmax of 85 ± 6%. [1]
- MS67 induced WDR5 degradation in a time-dependent manner in both MV4;11 cells (as early as 2 hours, maximal at ~4 hours) and MIA PaCa-2 cells (maximal at ~24 hours). MS67N and OICR-9429 did not degrade WDR5. [1]
- In a panel of six MLL-r AML cell lines (MV4;11, EOL-1, RS4;11, THP1, MOLM13, KOPN8) and four PDAC cell lines (MIA PaCa-2, HPAF-II, BxPC-3, Panc 10.05), MS67 effectively degraded WDR5 without a hook effect, in a concentration-dependent manner in PDAC cells. MS67N and OICR-9429 were ineffective. [1]
- Pretreatment of MIA PaCa-2 cells with OICR-9429, carfilzomib (proteasome inhibitor), MLN4924 (neddylation inhibitor), or VHL ligands Ac-VHL-Me and Ac-VHL suppressed MS67-induced WDR5 degradation. Ac-VHL-Me was more effective than Ac-VHL. VHL knockout in 293FT cells abrogated MS67-mediated WDR5 degradation. [1]
- Washout studies showed that WDR5 protein expression rebounded at ~48 hours and was nearly fully recovered at ~72 hours after MS67 treatment in MV4;11 cells. In MIA PaCa-2 cells, rebound occurred at ~24 hours with near-full recovery at ~36 hours. [1]
- MS67 also degraded WDR5 in three murine AML cell lines (Hoxa9+Meis1, MLL-AF9, MLL-ENL), albeit with lower potency than in human AML lines. [1]
- MS-based global proteomic profiling in MIA PaCa-2 cells treated with 1.5 μM MS67 for 2.5 hours detected over 4000 proteins; WDR5 was the sole protein showing significant decrease (cutoff: P < 0.01, fold change > 1.5). [1]
- MS67 did not effectively inhibit or bind over 100 targets including 22 protein methyltransferases, 45 kinases, and 44 GPCRs/ion channels/transporters, except Sigma 2 receptor (67 ± 10% binding at 1 μM, Ki = 1.0 ± 0.8 μM). [1]
- Overexpression of WDR5 D172A mutant (but not Y191A) abrogated MS67-mediated degradation, indicating the importance of specific WDR5-VHL interactions. [1]
- MS67, but not OICR-9429 or MS67N, decreased chromatin-bound fractions of MLL complex components (MLL, RBBP5, Menin) and c-MYC, and reduced H3K4me2/3 levels without affecting H3K9me3, H3K27me3, or H3K36me3. [1]
- MS67 showed potent antiproliferative effects in MLL-r AML cells: GI50 values were 15 ± 8 nM (MV4;11), 38 ± 1 nM (EOL-1), 200 ± 37 nM (MOLM13), 320 ± 110 nM (KOPN8), 460 ± 58 nM (RS4;11), and 530 ± 23 nM (THP-1). Non-MLL-r leukemia cells (K562, HL-60) were insensitive (GI50 > 30 μM). [1]
- In PDAC cells, MS67 GI50 values were 2.6 ± 0.2 μM (HPAF-II), 7.0 ± 1.1 μM (BxPC-3), 8.1 ± 2.6 μM (MIA PaCa-2), and 10 ± 2.2 μM (Panc 10.05). MS67 was less effective in other cancer cell lines (MCF7, NCI-H2009, PC3, SK-ES-1) with GI50 > 30 μM. [1]
- MS67 induced cell cycle arrest (increased G0/G1 phase, decreased S phase) and apoptosis in sensitive AML and PDAC cells, while OICR-9429 and MS67N had minimal effects. [1]
- In primary AML patient samples, MS67 (0.05-1 μM) effectively reduced WDR5 protein expression and suppressed cell growth in a concentration-dependent manner, while MS67N had no effect. [1]

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WDR5 degradation is induced by MS67 (0.001-1 μM) at a dose of 1 nM. In all six mixed lineage leukemia (MLL)-r acute myeloid leukemia (AML) and four pancreatic ductal adenocarcinoma (PDAC) cell lines, MS67 significantly more effectively induces WDR5 depletion without a hook effect and in a concentration-dependent manner in PDAC cells[1]. In MV4;11 and MIA PaCa-2 cells, MS67 reduces H3K4me2/3, but has no effect on other histone methylation marks that have been studied, including H3K9me3, H3K27me3, and H3K36me3. WDR5/MLL-induced H3K4 methylations on chromatin and WDR5-related gene expression programs are both effectively suppressed by MS67[1]. The two most sensitive AML lines, MV4;11 and EOL-1, have GI50 values of MS67 of 15 nM and 38 nM, respectively. Leukemia cell lines that did not harbor MLL-r (such as K562, HL60, and a murine AML line transformed by Hoxa9 plus Meis1) are insensitive to MS67, whereas MLL-r acute leukemia cell lines, such as MV4;11, EOL-1, MOLM13, KOPN8, RS4;11, and THP-1, are sensitive to it[1]... With a Kd of 140 nM, MS67 binds to the VHL-Elongin C-Elongin B ternary complex (VCB)[1].

- MV4;11 xenograft model: NSG mice bearing subcutaneous MV4;11 xenografts were treated with MS67 (75 mg/kg, i.p., twice daily, 5 days/week) starting at day 26 after inoculation. MS67 significantly inhibited tumor growth compared to vehicle (P = 0.028 at day 38). No body weight loss was observed. WDR5 was substantially degraded in tumor samples from MS67-treated mice. MS67 concentrations in plasma and tumor samples at 2 hours after the last dose were ~5 μM and 0.6 μM, respectively. RT-qPCR analysis showed down-regulation of WDR5 target genes (ribosome subunits, BCL2, CSNK1E). [1]
- AML PDX model: NSG-SGM3 mice bearing MLL-AF9+ AML PDX tumors were treated with MS67 (150 mg/kg, i.p., BID on Mon/Wed/Fri, SID on Tue/Thu) starting at day 13 after inoculation. MS67 significantly suppressed tumor growth (P = 9.69 × 10⁻⁰⁵ at day 15) and prolonged survival (P = 0.0013) compared to vehicle, while OICR-9429 (37.5 mg/kg, same schedule) showed no significant effect. No body weight changes were observed. MS67 concentration in tumor samples was 0.47 μM (vs. 20 μM for OICR-9429), yet MS67 effectively degraded WDR5 while OICR-9429 did not. [1]

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MS67 (ip; twice daily, five days a week; 75 mg/kg) dramatically suppresses tumor growth in vivo and increases the length of time that treated mice survive[1]. Following a single 75 mg/kg intraperitoneal (ip) injection, the Cmax was approximately 4.2 μM, and the concentration of MS67 remained above 0.5 μM for a duration of 12 hours[1].

- Isothermal Titration Calorimetry (ITC) for MS67 binding: ITC experiments were performed to measure binding affinities. For MS67 binding to WDR5, WDR5 was titrated into MS67. For MS67 binding to VCB (VHL-Elongin C-Elongin B complex), VCB was titrated into MS67. To assess ternary complex cooperativity, WDR5 was first titrated into MS67 to saturation, then VCB was titrated into the saturated WDR5-MS67 complex. The dissociation constant (Kd) for MS67 binding to VCB was 140 ± 7.2 nM, and to WDR5 was 63 ± 10 nM. The ternary complex Kd was 52 ± 8.3 nM, with cooperativity α = 2.74. [1]
- X-ray crystallography of WDR5-MS67-VCB ternary complex: The crystal structure of the WDR5-MS67-VCB complex was solved at 2.1-Å resolution (PDB ID: 7JTP). MS67 had VHL and WDR5 closer together than in the MS33 ternary complex, resulting in a more extensive protein-protein interface. The trajectory of the MS67 linker was roughly orthogonal to that of MS33. New hydrogen bonds were present, including between WDR5 Asp172 and VHL Arg107/Arg108, and nonpolar contacts between WDR5 Tyr191/Leu234 and VHL His110/Pro71. Cross protein-ligand interactions were observed: WDR5 made contacts with the VHL-binding portion of MS67, and VHL made contacts with the WDR5-binding portion. The methyl groups at positions 2 and 4 of the piperazine ring filled the hydrophobic cavity, and the fluoro group on the phenyl ring interacted with Phe133 and Tyr191 of WDR5. [1]

- Western blot analysis for WDR5 degradation: Cells were treated with MS67 at indicated concentrations for specified times. Cells were lysed, protein lysates resolved by SDS-PAGE, transferred to membranes, and probed with anti-WDR5 and anti-tubulin antibodies. Band intensities were quantified using ImageJ software to determine DC50 and Dmax values. [1]
- Cell proliferation assays (GI50 determination): Cells were seeded in 96-well plates and treated with serial dilutions of MS67, MS67N, or OICR-9429 for 6 days (AML cells) or 8 days (PDAC cells). Relative cell numbers were measured using CellTiter-Glo or similar viability assays. GI50 values were calculated using nonlinear regression. [1]
- Cell cycle analysis: Cells were treated with DMSO or compounds for 48-96 hours, fixed with ethanol, stained with propidium iodide, and analyzed by flow cytometry. Cell cycle distribution was determined using FlowJo software. [1]
- Apoptosis analysis: Cells were treated with DMSO or compounds for 96 hours, stained with Annexin V and propidium iodide, and analyzed by flow cytometry. [1]
- Chromatin fractionation: Cells were treated with compounds, lysed, and fractionated into cytoplasmic, nuclear-soluble, and chromatin-bound fractions. Chromatin-bound proteins were analyzed by immunoblotting. [1]
- RNA-seq and data analysis: Total RNA was isolated from cells treated with DMSO, OICR-9429, MS67N, or MS67. RNA-seq libraries were prepared and sequenced. Differentially expressed genes were identified with cutoff of |log2FC| > 0.58 and FDR < 0.05. Gene set enrichment analysis (GSEA) was performed. [1]
- ChIP-seq for H3K4me2: Cells were treated with DMSO, OICR-9429, MS67N, or MS67, cross-linked, and chromatin immunoprecipitated with anti-H3K4me2 antibody. Libraries were prepared and sequenced. Spike-in normalization was used to assess genome-wide changes. [1]
- Primary AML cell culture and growth inhibition: Primary AML cells from deidentified patients were cultured and treated with DMSO, MS67, or MS67N at indicated concentrations for 24-96 hours. WDR5 degradation was assessed by immunoblotting, and cell growth was measured using viability assays. [1]

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Western Blot Analysis[1]
Cell Types: MV4;11 cells
Tested Concentrations: 0.001 μM, 0.005 μM, 0.01 μM, 0.05 μM, 0.1 μM, 0.5 μM, 1 μM
Incubation Duration: 18 hrs (hours)
Experimental Results: Induced WDR5 degradation at a concentration as low as 1 nM with DC50 of 3.7 nM.

- Mouse PK study: Female Balb/c mice were administered a single intraperitoneal (i.p.) injection of MS67 at 75 mg/kg. Blood was collected at various time points (0, 0.5, 1, 2, 4, 6, 8, 12 hours) and plasma concentrations of MS67 were measured by LC/MS. [1]
- MV4;11 xenograft efficacy study: NOD/SCID/gamma(c)(null) (NSG) mice were inoculated subcutaneously with MV4;11 cells. When tumors reached approximately 200-300 mm³ (day 26), mice were randomized and treated with either vehicle or MS67 (75 mg/kg, i.p., twice daily, 5 days/week). Tumor volumes were measured every 2-3 days with calipers. Body weights were monitored. At study termination, tumor and plasma samples were collected for WDR5 immunoblotting, drug concentration measurement, and RT-qPCR analysis. [1]
- AML PDX efficacy study: NSG-SGM3 mice were inoculated subcutaneously with MLL-AF9+ AML PDX tumor fragments. When tumors reached approximately 100-200 mm³ (day 13), mice were randomized and treated with vehicle, MS67 (150 mg/kg, i.p., BID on Monday/Wednesday/Friday, once daily on Tuesday/Thursday), or OICR-9429 (37.5 mg/kg, same schedule). Tumor volumes were measured, body weights monitored, and survival recorded. At study termination, tumor and plasma samples were collected for drug concentration measurement and WDR5 immunoblotting. [1]

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Animal/Disease Models: MV4;11 MLL-r AML xenograft mouse[1]
Doses: 75 mg /kg
Route of Administration: ip; twice (two times) daily; 5 days a week; for 20 days
Experimental Results: Inhibited tumor growth in vivo.

- After a single intraperitoneal (i.p.) injection of MS67 at 75 mg/kg in mice, the maximum plasma concentration (Cmax) reached approximately 4.2 μM, and the concentration remained above 0.5 μM over 12 hours. [1]
- In MV4;11 xenograft tumor-bearing mice treated with MS67 (75 mg/kg, i.p., BID for 5 days), the average MS67 concentration in plasma was approximately 5 μM, and in tumor samples was approximately 0.6 μM at 2 hours after the last dose. [1]
- In the AML PDX model, the concentration of MS67 in tumor samples was 0.47 μM, which was substantially lower than that of OICR-9429 (20 μM) under similar dosing conditions, yet MS67 effectively degraded WDR5 while OICR-9429 did not. [1]

- In MV4;11 xenograft mice treated with MS67 (75 mg/kg, i.p., BID, 5 days/week) for up to 20 days, no body weight loss was observed compared to vehicle-treated controls. [1]
- In the AML PDX model, MS67-treated mice (150 mg/kg, i.p., intermittent dosing) showed no body weight loss compared to vehicle-treated controls. [1]
- MS67 did not effectively inhibit or bind over 100 off-targets including 45 kinases and 44 GPCRs/ion channels/transporters, with the exception of Sigma 2 receptor (Ki = 1.0 ± 0.8 μM). [1]

[1]. A selective WDR5 degrader inhibits acute myeloid leukemia in patient-derived mouse models. Sci Transl Med. 2021 Sep 29;13(613):eabj1578.

- MS67 is a PROTAC (proteolysis-targeting chimera) that simultaneously binds WDR5 and the VHL E3 ligase, leading to ubiquitination and proteasomal degradation of WDR5. It was developed through structure-based optimization from an initial degrader MS33, with key improvements including a shorter linker, methyl substituents at positions 2 and 4 of the piperazine ring, a fluoro group on the phenyl ring, and replacement of VHL-1 with methylated VHL-1 (VHL-1-Me) for enhanced VHL binding. [1]
- The crystal structure of the WDR5-MS67-VCB ternary complex (PDB: 7JTP) revealed a more extensive VHL-WDR5 interface and cross protein-ligand interactions compared to MS33, explaining the enhanced cooperativity and degradation efficiency. [1]
- MS67 suppresses transcription of WDR5-regulated genes, including ribosomal components and oncogenesis-related transcripts such as BCL2 and CSNK1E, and reduces H3K4me2 at cancer-associated gene loci. [1]
- MS67 is more effective than the WDR5 PPI inhibitor OICR-9429 in suppressing tumor growth in vivo and prolonging survival in a MLL-r AML PDX model, despite achieving lower tumor concentrations, demonstrating the catalytic advantage of PROTACs. [1]
- MLL-r AML cell lines show variable sensitivity to MS67, with MV4;11 and EOL-1 being the most sensitive, while RS4;11 and THP-1 are less sensitive. The reason for this variation warrants further investigation. [1]

C52H59F4N9O7S

1030.14

1029.419

C, 60.63; H, 5.77; F, 7.38; N, 12.24; O, 10.87; S, 3.11

2407452-77-9

153379992

White to off-white solid powder

5.5

6

15

14

73

2090

6

N(C1C=C(C2C(=CC=C(C(=O)NCC(=O)N[C@@H](C(C)(C)C)C(N3C[C@H](O)C[C@H]3C(=O)N[C@H](C3C=CC(C4SC=NC=4C)=CC=3)C)=O)C=2)F)C=CC=1N1C[C@H](C)N(C)[C@H](C)C1)C(C1=CNC(=O)C=C1C(F)(F)F)=O

HBHSDSLZXDASLT-FLKKWHKHSA-N

InChI=1S/C52H59F4N9O7S/c1-27-23-64(24-28(2)63(27)8)41-16-14-33(18-40(41)61-48(70)37-21-57-43(67)20-38(37)52(54,55)56)36-17-34(13-15-39(36)53)47(69)58-22-44(68)62-46(51(5,6)7)50(72)65-25-35(66)19-42(65)49(71)60-29(3)31-9-11-32(12-10-31)45-30(4)59-26-73-45/h9-18,20-21,26-29,35,42,46,66H,19,22-25H2,1-8H3,(H,57,67)(H,58,69)(H,60,71)(H,61,70)(H,62,68)/t27-,28+,29-,35+,42-,46+/m0/s1

N-[5-[2-fluoro-5-[[2-[[(2S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]amino]-2-oxoethyl]carbamoyl]phenyl]-2-[(3R,5S)-3,4,5-trimethylpiperazin-1-yl]phenyl]-6-oxo-4-(trifluoromethyl)-1H-pyridine-3-carboxamide

MS67; MS-67; 2407452-77-9; MS 67; CHEMBL5415680; N-{2'-fluoro-5'-[({[(2S)-1-[(2S,4R)-4-hydroxy-2-{[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]carbamoyl}pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl}methyl)carbamoyl]-4-[(3R,5S)-3,4,5-trimethylpiperazin-1-yl]-[1,1'-biphenyl]-3-yl}-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide; N-(2'-fluoro-5'-((2-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-2-oxoethyl)carbamoyl)-4-((3R,5S)-3,4,5-trimethylpiperazin-1-yl)-[1,1'-biphenyl]-3-yl)-6-oxo-4-(trifluoromethyl)-1,6-dihydropyridine-3-carboxamide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~97.07 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)