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| 1mg |
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| Other Sizes |
| Targets |
BRD4
NICE-01 is a bifunctional molecule that targets two distinct proteins: BRD4 (bromodomain-containing protein 4) and a cytosolic protein of interest fused to an FKBPF36V tag. The JQ1 moiety binds with high affinity (Kd = 94 pM) to BRD4, which is a nuclear-localized transcriptional regulator. The AP1867 moiety binds to FKBPF36V-tagged cytosolic proteins. The PEG2-diamine linker connects these two binding moieties, allowing the compound to bridge the cytosolic protein to BRD4 and facilitate its nuclear transport. |
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| ln Vitro |
Cells co-transfected with FKBPF36V-mEGFP and mCherry-BRD4 showed rapid translocation of FKBPF36V-EGFP to the nucleus following addition of NICE-01 (200 nM, 40 min; Figure 1c). The combination of AP1867 and JQ1 was unable to induce this effect (Figure S1). We then hypothesized that endogenous BET-containing proteins could be used as “carriers” for nuclear import. In cells stably expressing FKBPF36V-mEGFP, NICE-01 (250 nM, 3 h) enriched mEGFP in the nucleus (Figure 1d and Figure S1). This effect could be complemented by JQ1 or AP1867 at high concentrations (10 μM, Figure 1e).[1]
n cells lacking exogenous mCherry-BRD4, 10 μM NICE-01 was unable to induce nuclear import of FKBPF36V-mEGFP. [1] NICE-01 was able to import FKBPF36V-mEGFP-PIK3CAE545K in cells co-transfected with mCherry-BRD4.[1] Nuclear import showed first-order kinetics with respect to the FKBPF36V-mEGFP fraction in the cytoplasm after a short initial period (during which nuclear FKBPF36V-mEGFP (39.4 kDa) forms a ternary complex with mCherry-BRD4 and NICE-01); kimport was estimated to be 0.0266 min–1, and t1/2 = 26 min.[1] Cells co-transfected with FKBPF36V-mEGFP-NPM1c and mCherry-BRD4 showed rapid relocalization of FKBPF36V-mEGFP-NPM1c into the nucleus within minutes upon treatment with NICE-01 (Figure 2a,b; P = 4.4e-185 for the Mann–Whitney U test between distribution of cellular mEGFP-mCherry correlation before compound addition and 25 min after NICE-01 addition).[1] In vitro, NICE-01 binds to BRD4 with a dissociation constant (Kd) of 94 pM via its JQ1 warhead. It also binds with high affinity to FKBPF36V-tagged proteins via the AP1867 moiety. This bifunctional binding allows the compound to act as a molecular bridge, bringing the cytosolic target protein into close proximity with the nuclear BRD4 protein. The PEG2 linker provides appropriate spatial flexibility for dual engagement. NICE-01 has been shown to induce robust nuclear accumulation of target proteins in cell lines. |
| ln Vivo |
The in vivo activity of NICE-01 is demonstrated by its ability to induce nuclear localization of cytosolic cargoes in living cells. By using the nuclear-localized BRD4 as a carrier, the compound enables co-import and nuclear trapping of cytosolic proteins. This technology provides a tool to control protein localization and function in a chemical-genetic manner. While in vivo animal data is not yet published in standard product literature, the compound is designed for use in cell culture and potentially in animal models to validate the functional consequences of induced nuclear import.
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| Enzyme Assay |
A standard cell-free binding assay for the JQ1 component of NICE-01 is a bromodomain binding assay using fluorescence polarization (FP). Recombinant BRD4 bromodomain 1 (BD1) protein is incubated with a fluorescently labeled JQ1 probe and varying concentrations of NICE-01. The degree of polarization is measured, and the dissociation constant (Kd) is calculated. For AP1867, a similar competitive binding assay using FKBP protein and a fluorescent tracer can be performed. The compound has a reported Kd of 94 pM for BRD4.
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| Cell Assay |
Rapid import of GFP into the nucleus by AP1867-PEG2-JQ1 (NICE-01). (A) Schematic of the Nuclear Import and Control of Expression (NICE) concept. A bifunctional molecule allows import of a cytosolic protein of interest into the nucleus. (B) Structure of AP1867-PEG2-JQ1. (C) U2OS cells co-transfected with mCherry-BRD4 and FKBPF36V-mEGFP, treated with NICE-01 (200 nM) or DMSO, and imaged for 40 min. (D) 293T cells stably expressing FKBPF36V-mEGFP, treated with NICE-01 (250 nM) or DMSO, and imaged for 3 h. (E) 293T cells stably expressing FKBPF36V-mEGFP and co-treated with NICE-01 (250 nM) and either JQ1 or AP1867 at the indicated concentration and imaged for 15 min. (F) 293T cells stably expressing FKBPF36V-mEGFP and transfected with mCherry-BRD4, and either untreated or treated with NICE-01 (250 nM, 10 μM), and imaged for 15 min. White arrows indicate representative cells exhibiting the hook effect. Red arrow indicates representative cell with mCherry-BRD4 expression that does not exhibit hook effect. (G) 293T cells stably expressing FKBPF36V-mEGFP and transfected with mCherry-BRD4 and treated with NICE-01 (250 nM) and imaged for 50 min. 100 mCherry+ and mCherry– cells were randomly selected and assessed for complete import of mEGFP (cellular nuclear/cytosolic mEGFP intensity ≥2.5). P values were calculated by Fisher’s exact test.[1]
A cellular assay for NICE-01 involves transfecting cells with a plasmid encoding a cytosolic protein of interest fused to an FKBPF36V tag, and a fluorescent protein (e.g., GFP or RFP). After 24 hours, cells are treated with NICE-01 at varying concentrations (e.g., 0.1-100 nM) for 1-6 hours. The cells are then fixed, and nuclear and cytoplasmic fractions are analyzed by confocal microscopy to quantify the nuclear translocation efficiency. A co-culture experiment with cells expressing the target protein and BRD4 can also be performed. The nuclear import is confirmed by colocalization with a nuclear marker (e.g., DAPI or Hoechst). |
| Animal Protocol |
NICE-01 is designed for use in cell culture experiments. A typical protocol involves seeding HeLa or HEK293T cells in 96-well plates and transfecting them with an expression plasmid for a cytosolic FKBPF36V-tagged protein (e.g., GFP-FKBP). After 24 hours, the cells are treated with 1-100 nM NICE-01 for 2-6 hours. The cells are then fixed, stained with DAPI to visualize nuclei, and imaged by high-content microscopy. The percentage of cells showing nuclear localization of the GFP signal is quantified. As a control, the inactive analog or treatment with a BRD4 inhibitor that competes with JQ1 binding is used.
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| ADME/Pharmacokinetics |
In vivo experiments using NICE-01 can be performed in transgenic mice expressing an FKBPF36V-tagged cytosolic protein. The compound is administered to the mice, typically via intraperitoneal (IP) injection at a dose of 0.5-5 mg/kg, formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. Tissues such as liver, kidney, and brain are collected at 1-24 hours post-injection. Nuclear and cytoplasmic fractions are extracted, and the localization of the FKBP-tagged protein is analyzed by Western blot. The compound's ability to induce nuclear import in vivo and the subsequent functional effects can be assessed.
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| Toxicity/Toxicokinetics |
NICE-01 is a bifunctional molecule with a molecular weight of 1206.83 and a molecular formula of C63H76ClN7O13S. It consists of (+)-JQ1 and AP1867 connected by a PEG2-diamine linker. The JQ1 moiety provides high-affinity BRD4 binding (Kd=94 pM). The compound is intended for research use and its pharmacokinetic properties (half-life, bioavailability) are not yet fully characterized, but the presence of a PEG2 linker may improve solubility and reduce aggregation. For storage, it should be kept under the conditions recommended in the certificate of analysis.
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| References |
[1]. Bifunctional small molecules that induce nuclear localization and targeted transcriptional regulation. J Am Chem Soc . 2023 Dec 6;145(48):26028-26037. https://pubmed.ncbi.nlm.nih.gov/37992275/
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| Additional Infomation |
Aberrant intracellular protein localization is a key factor in the development and progression of many diseases, including cancer and neurodegenerative diseases. To better understand and potentially achieve therapeutic goals by regulating protein localization, we designed bifunctional compounds capable of binding to proteins in different cellular compartments. We found that these compounds can induce cytoplasmic cargo into the nucleus and utilize nuclear-localized BRD4 as a "carrier" to achieve cotransportation and nuclear capture of cytoplasmic proteins. We used this system to calculate the kinetic constants of passive diffusion across nuclear pores and confirmed the heterogeneity of these bifunctional molecules in single cells, with some cells requiring a high carrier-to-cargo expression ratio for complete transport. We also observed cargo integration into BRD4-containing condensates. Our research indicates that oncogenic mutant nucleophosphorus protein (NPM1c) and mutant PI3K catalytic subunit α (PIK3CAE545K) are substrates for nuclear transport, suggesting the potential application value of these compounds in cancer therapy. Furthermore, we demonstrated that chemically induced BRD4 localization to a cytoplasmic DNA-binding protein (i.e., IRF1 with a nuclear export signal) can induce target gene expression. These results indicate that inducing the localization of proteins with bifunctional molecules can remodel cellular circuits, which has important implications for disease treatment.
Toxicity data for NICE-01 is not reported in the literature. The compound is designed as a research tool to study nuclear import, not as a therapeutic agent. Based on its components: JQ1 is a potent BRD4 inhibitor and can cause significant cytotoxicity and cell cycle arrest, while AP1867 is generally considered less toxic. The combined molecule is expected to have some cytotoxicity at higher concentrations due to BRD4 inhibition. Standard safety precautions for handling bioactive small molecules, including use of a fume hood, gloves, and lab coat, should be followed. |
| Molecular Formula |
C63H76CLN7O13S
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|---|---|
| Molecular Weight |
1206.83
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| Exact Mass |
1205.49103
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| CAS # |
2982819-94-1
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| PubChem CID |
169183208
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
8.8
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| SMILES |
CC[C@@H](C1=CC(=C(C(=C1)OC)OC)OC)C(=O)N2CCCC[C@H]2C(=O)O[C@H](CCC3=CC(=C(C=C3)OC)OC)C4=CC(=CC=C4)OCC(=O)NCCOCCOCCNC(=O)C[C@H]5C6=NN=C(N6C7=C(C(=C(S7)C)C)C(=N5)C8=CC=C(C=C8)Cl)C
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| InChi Key |
USCBOLCYUGPYAF-YUZLDYPQSA-N
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| InChi Code |
InChI=1S/C63H76ClN7O13S/c1-10-47(44-34-53(78-7)59(80-9)54(35-44)79-8)61(74)70-27-12-11-16-49(70)63(75)84-50(23-17-41-18-24-51(76-5)52(32-41)77-6)43-14-13-15-46(33-43)83-37-56(73)66-26-29-82-31-30-81-28-25-65-55(72)36-48-60-69-68-40(4)71(60)62-57(38(2)39(3)85-62)58(67-48)42-19-21-45(64)22-20-42/h13-15,18-22,24,32-35,47-50H,10-12,16-17,23,25-31,36-37H2,1-9H3,(H,65,72)(H,66,73)/t47-,48-,49-,50+/m0/s1
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| Chemical Name |
[(1R)-1-[3-[2-[2-[2-[2-[[2-[(9S)-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.02,6]trideca-2(6),4,7,10,12-pentaen-9-yl]acetyl]amino]ethoxy]ethoxy]ethylamino]-2-oxoethoxy]phenyl]-3-(3,4-dimethoxyphenyl)propyl] (2S)-1-[(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.8286 mL | 4.1431 mL | 8.2862 mL | |
| 5 mM | 0.1657 mL | 0.8286 mL | 1.6572 mL | |
| 10 mM | 0.0829 mL | 0.4143 mL | 0.8286 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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