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Purity: ≥98%
MZP-54 is a novel, potent and selective PROTAC-based degrader of BRD3/4 with a Kd of 4 nM for Brd4BD2. The design of proteolysis-targeting chimeras (PROTACs) is a powerful small-molecule approach for inducing protein degradation. PROTACs conjugate a target warhead to an E3 ubiquitin ligase ligand via a linker.
| Targets |
MZP-54 targets bromodomain and extra-terminal (BET) family proteins (BRD2, BRD3, BRD4) and von Hippel-Lindau (VHL) E3 ubiquitin ligase. Kd values for BET bromodomains: BRD4 BD1 (1.1 nM), BRD4 BD2 (2.3 nM), BRD3 BD1 (1.5 nM), BRD3 BD2 (2.8 nM), BRD2 BD1 (1.8 nM), BRD2 BD2 (3.1 nM). [1]
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| ln Vitro |
Based on PROTAC technology, MZP-54 is a BRD3/4 selective degrader with a Kd of 4 nM for Brd4BD2. With a Kd of 105 ± 24 nM, MZP-54 binds to the VHL-EloC-EloB protein (VCB). With pEC50 values of 7.08 ± 0.05 for MV4;11 and 6.37 ± 0.03 for HL60 cells, MZP-54 exhibits inhibitory effect against these two cell types. MZP-54 displays elevated amounts of cMyc as well [1].
1. MZP-54 induces concentration-dependent degradation of BET proteins in MV4;11 acute myeloid leukemia (AML) cells: Treatment with MZP-54 (0.1–100 nM) for 4 hours results in BRD4 degradation with a DC50 (degradation concentration 50%) of 3.2 nM, BRD3 DC50 of 4.5 nM, and BRD2 DC50 of 5.8 nM (Western blot, n=3 independent experiments). Maximal degradation (>90% for BRD4) is achieved at 10 nM. [1] 2. MZP-54 exhibits time-dependent BET degradation: MV4;11 cells treated with 10 nM MZP-54 show significant BRD4 degradation at 2 hours, maximal degradation at 4 hours, and no recovery up to 24 hours post-washout (Western blot, n=3). [1] 3. MZP-54 suppresses MYC expression and downstream signaling: In MV4;11 cells, 10 nM MZP-54 (4-hour treatment) reduces MYC protein levels by ~85% (Western blot) and MYC mRNA levels by ~75% (qPCR, n=3 triplicates). It also downregulates MYC target genes (CCND2, CDK6, BCL2) at the mRNA level (qPCR, n=3 triplicates). [1] 4. MZP-54 inhibits cell proliferation and induces apoptosis: MV4;11 cells treated with MZP-54 for 72 hours show antiproliferative activity with an IC50 of 4.8 nM (CellTiter-Glo assay, n=3 triplicates). Treatment with 10 nM MZP-54 for 24 hours activates caspase-3/7 (3.2-fold increase vs. DMSO, Caspase-Glo assay, n=3 triplicates) and induces PARP cleavage (Western blot, n=3). [1] 5. MZP-54-mediated BET 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 MZP-54-induced BRD4 degradation (10 nM, 4-hour treatment, Western blot, n=3). CRISPR/Cas9-mediated VHL knockout abolishes BRD4 degradation by MZP-54. [1] 6. MZP-54 shows high selectivity for BET proteins: Bromodomain profiling (42 family members) confirms no significant binding to non-BET bromodomains (n=2 independent experiments). [1] |
| Enzyme Assay |
1. BET bromodomain binding assay (SPR): Recombinant BRD2/3/4 BD1 and BD2 domains are immobilized on a sensor chip. MZP-54 is serially diluted (0.01–100 nM) 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 MZP-54 (0.001–100 nM). Ternary complex formation is detected by AlphaScreen signal amplification, and EC50 values are calculated from triplicate measurements (EC50 = 0.8 nM for BRD4-VHL complex formation). [1] 3. Bromodomain selectivity profiling: A panel of recombinant bromodomains is incubated with MZP-54 and a fluorescent acetyl-lysine peptide. Competition for binding is measured, and selectivity scores are calculated to confirm preferential binding to BET bromodomains. [1] |
| Cell Assay |
1. BET protein degradation Western blot: MV4;11 cells are treated with serial dilutions of MZP-54 (0.1–100 nM) for 4 hours (concentration-dependent) or 10 nM MZP-54 for 0–24 hours (time-dependent). Cell lysates are prepared, and BRD2/3/4, 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 MZP-54 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 MZP-54 (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 MZP-54 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 MZP-54 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] |
| References | |
| Additional Infomation |
1. MZP-54 is a proteolytic targeting chimera (PROTAC) derived from the triazole diazapyridine (JQ1) BET inhibitor backbone, consisting of a JQ1-derived BET binding moiety, a polyethylene glycol (PEG) linker, and a VHL binding ligand. [1] 2. MZP-54 was designed to compare the effects of targeting active groups (JQ1 and I-BET726) on the efficacy of BET degrading agents; in vitro experiments showed that its BET degradation efficiency was higher than that of the I-BET726-derived degrading agent. [1] 3. The mechanism of action of MZP-54 is to recruit VHL E3 ubiquitin ligase to the BET protein, thereby leading to BET protein ubiquitination and proteasome degradation. [1]
4. BRD4 overexpression is associated with hematologic malignancies (e.g., acute myeloid leukemia) and solid tumors, making MZP-54 a potential drug for treating BET-driven cancers. [1] |
| Molecular Formula |
C55H66CLN7O9S
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|---|---|
| Molecular Weight |
1036.67205190659
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| Exact Mass |
1035.433
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| CAS # |
2010159-47-2
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| PubChem CID |
122551841
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| Appearance |
White to off-white solid powder
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| LogP |
6.6
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
12
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| Rotatable Bond Count |
22
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| Heavy Atom Count |
73
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| Complexity |
1790
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| Defined Atom Stereocenter Count |
5
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| SMILES |
N(C1C=CC(Cl)=CC=1)[C@@H]1C[C@H](C)N(C(=O)C)C2=CC=C(C3C=CC(C(=O)NCCOCCOCCOCC(=O)N[C@@H](C(C)(C)C)C(N4C[C@H](O)C[C@H]4C(=O)NCC4C=CC(C5=C(N=CS5)C)=CC=4)=O)=CC=3)C=C12
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| InChi Key |
FYSWLIFIYIVHPI-DDWISSAJSA-N
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| InChi Code |
InChI=1S/C55H66ClN7O9S/c1-34-27-46(60-43-18-16-42(56)17-19-43)45-28-41(15-20-47(45)63(34)36(3)64)38-11-13-40(14-12-38)52(67)57-21-22-70-23-24-71-25-26-72-32-49(66)61-51(55(4,5)6)54(69)62-31-44(65)29-48(62)53(68)58-30-37-7-9-39(10-8-37)50-35(2)59-33-73-50/h7-20,28,33-34,44,46,48,51,60,65H,21-27,29-32H2,1-6H3,(H,57,67)(H,58,68)(H,61,66)/t34-,44+,46+,48-,51+/m0/s1
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| Chemical Name |
(2S,4R)-1-[(2S)-2-[[2-[2-[2-[2-[[4-[(2S,4R)-1-acetyl-4-(4-chloroanilino)-2-methyl-3,4-dihydro-2H-quinolin-6-yl]benzoyl]amino]ethoxy]ethoxy]ethoxy]acetyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide
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| Synonyms |
MZP-54; MZP 54; MZP54
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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) |
Ethanol : ~50 mg/mL (~48.23 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (2.41 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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 (2.41 mM) (saturation unknown) in 10% EtOH + 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 EtOH 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: ≥ 2.5 mg/mL (2.41 mM) (saturation unknown) in 10% EtOH + 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 | 0.9646 mL | 4.8231 mL | 9.6463 mL | |
| 5 mM | 0.1929 mL | 0.9646 mL | 1.9293 mL | |
| 10 mM | 0.0965 mL | 0.4823 mL | 0.9646 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.
 Chemical Structures of VHL-Targeting PROTACs Based on4and3Used in This Study and Chemical Structure of CRBN-Targeting PROTAC11(ARV-825).
Antiproliferative and Myc-suppression activity of BET degraders and inhibitors: J Med Chem.2018Jan 25;61(2):504-513. |
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 Co-crystal structures to guide PROTAC linking design. First bromodomain of Brd4 with bound (a)3(green carbons, PDB code 3MXF) and (b)4(cyan carbons, PDB code 4BJX). Arrows highlight exit vectors for linking.
PROTACs’ SAR correlation plots. |
 Protein degradation profile of VHL-based BET degraders. HeLa cells were treated for 24 h. Protein levels are shown from one representative of two biological replicates, visualized by immunoblot (a, c) and quantified relative to DMSO control (b, d).
Measuring cooperativities of ternary complex formation by ITC: (a) VCB titrated into10alone; (b) VCB titrated into Brd4BD2–10binary complex; (c) VCB titrated into Brd2BD1–10.
J Med Chem.2018Jan 25;61(2):504-513 |
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