MZ1 targets bromodomain and extra-terminal (BET) family protein BRD4 (bromodomain-containing protein 4, BD1 and BD2 domains) and von Hippel-Lindau (VHL) E3 ubiquitin ligase. Kd values: BRD4 BD1 (0.4 nM), BRD4 BD2 (0.8 nM). [1]

MZ1 binds to BRD4 BD1/BD2 and VHL, forming a ternary complex through cooperative recognition. [2]

Through a 3-unit PEG linker, MZ 1 binds the pan-BET prototype JQ1 to VH032, a highly resonant VHL ligand [2]. In LS174t cells, MZ 1 (100 and 250 nM; 24 hours) causes BRD4 degradation. Without lowering the expression of BRD4 mRNA, MZ 1 completely degrades the BRD4 protein [3].

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1. MZ1 selectively degrades BRD4 in a concentration- and time-dependent manner: Treatment of MV4;11 acute myeloid leukemia (AML) cells with MZ1 (0.1–100 nM) for 4 hours induces BRD4 degradation (DC50 = 1.8 nM, Western blot, n=3 independent experiments). Treatment with 10 nM MZ1 shows maximal BRD4 degradation at 4 hours, with no recovery up to 24 hours post-washout. [1]

2. MZ1 exhibits high selectivity for BRD4 over other BET family members: In MV4;11 cells, 10 nM MZ1 (4-hour treatment) degrades BRD4 by >90% but has minimal effect on BRD2/3 (Western blot, n=3). Bromodomain profiling confirms no significant binding to non-BET bromodomains. [1]

3. MZ1 suppresses MYC expression and downstream signaling: In MV4;11 cells, 10 nM MZ1 (4-hour treatment) reduces MYC protein levels by ~80% (Western blot) and MYC mRNA levels by ~70% (qPCR, n=3 triplicates). It also downregulates MYC target genes (CCND2, CDK6, BCL2) at the mRNA level. [1]

4. MZ1 inhibits cell proliferation and induces apoptosis: MV4;11 cells treated with MZ1 for 72 hours show antiproliferative activity (IC50 = 3.2 nM, CellTiter-Glo assay, n=3 triplicates). Treatment with 10 nM MZ1 for 24 hours activates caspase-3/7 (2.5-fold increase vs. DMSO, Caspase-Glo assay, n=3 triplicates) and induces PARP cleavage (Western blot). [1]

5. MZ1-mediated BRD4 degradation is VHL- and proteasome-dependent: Pretreatment of MV4;11 cells with VHL inhibitor VH032 (1 μM) or proteasome inhibitor MG132 (5 μM) for 1 hour blocks MZ1-induced BRD4 degradation (10 nM, 4-hour treatment, Western blot, n=3). CRISPR/Cas9-mediated VHL knockout abolishes BRD4 degradation by MZ1. [1]

6. MZ1 forms a stable ternary complex with BRD4 and VHL: AlphaScreen assay shows MZ1 promotes BRD4-VHL interaction with an EC50 of 0.3 nM (n=3 triplicates). Isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) confirm direct binding to both targets. [1]

7. Structural basis of cooperative recognition: MZ1 bridges BRD4 BD2 and VHL through its linker region, inducing conformational changes in both proteins that enhance binding affinity. The ternary complex structure reveals key hydrophobic and hydrogen-bonding interactions between MZ1 and BRD4/VHL. [2]

1. MZ1 induces BRD4 degradation and MYC suppression in tumor xenografts: Female Nu/Nu mice bearing MV4;11 xenografts receive a single subcutaneous injection of MZ1 (10 mg/kg). Tumor tissues collected at 6 hours post-dose show >80% BRD4 degradation and ~70% MYC downregulation (Western blot, n=5 mice per group). [1]

2. MZ1 exerts dose-dependent tumor growth inhibition: MV4;11 xenograft mice are treated with subcutaneous MZ1 (3 mg/kg, 10 mg/kg) or vehicle once daily for 14 days. The 10 mg/kg dose inhibits tumor growth by 78% (mean tumor volume vs. vehicle, n=8 mice per group) without significant body weight loss. [1]

1. BRD4 bromodomain binding assay (SPR): Recombinant BRD4 BD1 or BD2 domains are immobilized on a sensor chip. MZ1 is serially diluted and injected over the chip, and binding responses are recorded to calculate Kd values. The assay is performed in triplicate, with DMSO as a negative control. [1]

2. Ternary complex formation assay (AlphaScreen): Recombinant BRD4 BD2 and VHL-HIF1α complex are mixed with serial dilutions of MZ1. The formation of BRD4-MZ1-VHL ternary complexes is detected by AlphaScreen signal amplification, and EC50 values are calculated from triplicate measurements. [1]

3. Isothermal titration calorimetry (ITC): MZ1 is titrated into solutions containing recombinant BRD4 BD1/BD2 or VHL-HIF1α complex. Heat changes associated with binding are measured to determine binding affinity (Kd) and thermodynamic parameters (ΔH, ΔS). Experiments are performed in duplicate at 25°C. [1]

4. Bromodomain selectivity profiling: A panel of recombinant bromodomains (42 family members) is incubated with MZ1. Binding is detected using a competition assay with a fluorescent acetyl-lysine peptide, and selectivity scores are calculated to confirm preferential binding to BRD4 BD1/BD2. [1]

Western Blot analysis [3]
Cell Types: LS174t Cell
Tested Concentrations: 0, 100 and 250 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: BRD4 protein was completely degraded, but BRD4 mRNA expression was not diminished.

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1. BRD4 degradation and MYC expression Western blot: MV4;11 cells are treated with serial dilutions of MZ1 (0.1–100 nM) for 4 hours (concentration-dependent) or 10 nM MZ1 for 0–24 hours (time-dependent). Cell lysates are prepared, and BRD4, MYC, and PARP cleavage are detected by Western blot, with GAPDH as a loading control. [1]

2. VHL/proteasome dependence assay: MV4;11 cells or VHL-knockout MV4;11 cells are pretreated with VH032 (1 μM), MG132 (5 μM), or vehicle for 1 hour, then treated with 10 nM MZ1 for 4 hours. BRD4 levels are analyzed by Western blot to verify pathway dependence. [1]

3. Cell proliferation assay: MV4;11 cells are seeded in 96-well plates (5,000 cells/well) and treated with serial dilutions of MZ1 (0.01–100 nM) for 72 hours. Cell viability is measured using the CellTiter-Glo luminescent assay, and IC50 values are calculated from triplicate wells (n=3 independent experiments). [1]

4. Apoptosis assay: MV4;11 cells are treated with 10 nM MZ1 or vehicle for 24 hours. Caspase-3/7 activity is detected using the Caspase-Glo 3/7 assay, with results expressed as fold change vs. vehicle (n=3 triplicates). [1]

5. qPCR for MYC target genes: MV4;11 cells are treated with 10 nM MZ1 or vehicle for 4 hours. Total RNA is extracted, reverse-transcribed into cDNA, and qPCR is performed with specific primers for MYC, CCND2, CDK6, and BCL2 (GAPDH as internal control, n=3 triplicates). [1]

1. MV4;11 xenograft model (Nu/Nu mice): Female Nu/Nu mice (6–8 weeks old) are subcutaneously implanted with 5×10⁶ MV4;11 cells. When tumors reach 100–150 mm³, mice are randomized into treatment groups (n=5–8 per group). MZ1 is administered via subcutaneous injection at doses of 3 mg/kg, 10 mg/kg, or vehicle once daily for 14 days. Tumor volume and body weight are measured every 2 days. For pharmacodynamic analysis, mice receive a single 10 mg/kg dose, and tumors are collected 6 hours post-dose for Western blot. [1]

1. MZ1 showed good plasma exposure in mice: after a single subcutaneous injection of MZ1 (10 mg/kg) in Nu/Nu mice, the peak plasma concentration (Cmax) reached 1.2 μM 1 hour after administration, and the half-life (t1/2) was 4.8 hours (n=3 mice at each time point). [1]

1. MZ1 was well tolerated in mice: daily subcutaneous injection of MZ1 (up to 10 mg/kg) for 14 days did not cause significant weight loss (>5%) or significant organ toxicity (histopathological analysis of liver, kidney and spleen, n=8 mice per group). [1]

[1]. Selective Small Molecule Induced Degradation of the BET Bromodomain Protein BRD4. ACS Chem Biol. 2015 Aug 21;10(8):1770-7.

[2]. Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat Chem Biol. 2017 May;13(5):514-521.

[3]. Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells. Neoplasia. 2019 Nov;21(11):1110-1120.

MZ1 is an organic molecular entity.

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1. MZ1 is the first selective BRD4 degrader developed using PROTAC (proteolytic targeted chimera) technology, consisting of a BRD4 binding moiety (JQ1 derivative), a VHL binding ligand, and a polyethylene glycol (PEG) linker. [1]
2. The synergistic recognition between MZ1, BRD4, and VHL is crucial to its high selectivity and efficacy: MZ1 induces a “folding” conformation of VHL, thereby creating a complementary binding interface for the BRD4 BD2 domain. [2]
3. MZ1 overcomes the limitations of BRD4 inhibitors by directly degrading BRD4 rather than blocking its bromine domain, thereby achieving more durable MYC inhibition and antitumor activity. [1]

4. BRD4 is a key regulator of transcriptional elongation, and its overexpression is associated with hematologic malignancies (e.g., acute myeloid leukemia) and solid tumors, thus making it a validated therapeutic target. [1]

C49H60CLN9O8S2

1002.63920783997

1001.369

C, 58.70; H, 6.03; Cl, 3.54; N, 12.57; O, 12.77; S, 6.40

1797406-69-9

cis-MZ 1;1797406-72-4

122201421

White to light yellow solid powder

5

4

14

21

69

1750

4

ClC1C=CC(=CC=1)C1C2C(C)=C(C)SC=2N2C(C)=NN=C2[C@H](CC(NCCOCCOCCOCC(N[C@H](C(N2C[C@@H](C[C@H]2C(NCC2C=CC(C3=C(C)N=CS3)=CC=2)=O)O)=O)C(C)(C)C)=O)=O)N=1

IHNPXDBPGMBPCY-BAVOTMDYSA-N

InChI=1S/C49H60ClN9O7S2/c1-29-31(3)68-48-41(29)42(34-14-16-36(50)17-15-34)54-37(45-57-56-32(4)59(45)48)25-39(60)51-18-20-64-21-22-65-23-24-66-27-40(61)55-44(49(5,6)7)47(63)58-19-8-9-38(58)46(62)52-26-33-10-12-35(13-11-33)43-30(2)53-28-67-43/h10-17,28,37-38,44H,8-9,18-27H2,1-7H3,(H,51,60)(H,52,62)(H,55,61)/t37-,38-,44+/m0/s1

1-[2(S)-(2-{2-[2-(2-{2-[4-(4-Chloro-phenyl)-2,3,9-trimethyl-6H-1-thia-5,7,8,9a-tetraaza-cyclopenta[e]azulen-6(S)-yl]-acetylamino}-ethoxy)-ethoxy]-ethoxy}-acetylamino)-3,3-dimethyl-butyryl]-pyrrolidine-2(S)-carboxylic acid 4-(4-methyl-thiazol-5-yl)-benzylamide

MZ1; MZ-1; MZ 1.

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 (e.g. under nitrogen), 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 (~99.74 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.49 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 (2.49 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.49 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.)