| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
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| 10mg |
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| Other Sizes |
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| Targets |
OTUB1 (covalent binding to non-catalytic cysteine C23); F508-CFTR (via lumacaftor moiety) [1]
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|---|---|
| ln Vitro |
NJH-2-057 engaged recombinant OTUB1 in gel-based ABPP assays, competing with IA-rhodamine labeling of OTUB1 (Figure 3c-d). Dose-dependent stabilization of F508-CFTR protein was observed in CFBE41o-4.7 human cystic fibrosis bronchial epithelial cells (8 μM, 16 h), with maximal stabilization at 10 μM (Extended Data Figure 5). The stabilized CFTR band (>225 kDa) corresponded to the mature glycosylated form, confirmed by three independent antibodies and attenuated upon CFTR knockdown (Extended Data Figures 6-7).
Quantitative TMT proteomics revealed NJH-2-057 selectively stabilized CFTR (7.8-fold vs. vehicle) among 4,552 quantified proteins, with only 21 proteins showing >5-fold stabilization (adjusted p<0.01). Companion proteins (e.g., HSPA6, DNAJB1) were upregulated, likely due to compensatory chaperone responses (Figure 3g; Table S3). Native mass spectrometry confirmed ternary complex formation: NJH-2-057 induced OTUB1-CFTR interactions, undetected with DMSO or EN523 alone (Figure 3h-i). Stabilization was abolished by OTUB1 knockdown or pretreatment with lumacaftor/EN523, confirming target dependence (Figure 4a-d). In transepithelial conductance assays using primary human bronchial epithelial cells, NJH-2-057 (10 μM, 24 h) combined with VX770 potentiated CFTR-dependent chloride transport, significantly outperforming lumacaftor (p<0.05) (Figure 4e-f). |
| Enzyme Assay |
OTUB1 engagement was assessed via gel-based ABPP: Recombinant OTUB1 (0.1 μg) was pre-incubated with NJH-2-057 or DMSO (37°C, 30 min), then labeled with IA-rhodamine probe (room temperature, 1 h). Reactions were stopped with Laemmli buffer, resolved by SDS-PAGE, and visualized via in-gel fluorescence (Page 12).
Deubiquitinase activity assays: OTUB1 (500 nM) ± EN523 (50 μM) was incubated with di-ubiquitin, UBE2D1, and DTT. Mono-ubiquitin release was monitored by western blotting over time (Page 12). |
| References | |
| Additional Infomation |
NJH-2-057 is a heterobifunctional DUBTAC comprising an OTUB1-targeting acrylamide (EN523) linked to lumacaftor (F508-CFTR corrector) via a C5 alkyl spacer. It stabilized F508-CFTR in an OTUB1-dependent manner by promoting deubiquitination, reducing proteasomal degradation (Pages 6-9).
Linker optimization showed C5/C6 alkyl chains enabled CFTR stabilization, while C3/C4 alkyl or PEG linkers failed (Extended Data Figure 8). A non-reactive propiolamide analog (NJH-2-106) lacked activity, confirming covalent mechanism (Extended Data Figure 10). IsoTOP-ABPP chemoproteomics in CFBE41o-4.7 cells indicated high proteome-wide selectivity: Only 5/1,270 cysteines showed >4-fold reduced IA-alkyne labeling by NJH-2-057 (adjusted p<0.05), none in ubiquitin-proteasome pathways (Extended Data Figure 14; Table S4). |
| Molecular Formula |
C43H44F2N6O8
|
|---|---|
| Molecular Weight |
810.84
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| Exact Mass |
810.318
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| CAS # |
2858812-70-9
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| PubChem CID |
162624827
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| Appearance |
White to off-white solid powder
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| Density |
1.40±0.1 g/cm3(Temp: 20 °C; Press: 760 Torr)(Predicted)
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| Boiling Point |
1071.9±65.0 °C(Predicted)
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| LogP |
5.1
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
16
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| Heavy Atom Count |
59
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| Complexity |
1550
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| Defined Atom Stereocenter Count |
0
|
| SMILES |
FC1(OC2=CC=C(C=C2O1)C1(C(NC2C=CC(C)=C(C3C=CC=C(C(NCCCCCNC(CCC4=CC=C(N5C(CN(C(C=C)=O)CC5)=O)O4)=O)=O)C=3)N=2)=O)CC1)F
|
| InChi Key |
GBRJFUUBXKOHMN-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C43H44F2N6O8/c1-3-36(53)50-22-23-51(37(54)26-50)38-17-13-31(57-38)12-16-35(52)46-20-5-4-6-21-47-40(55)29-9-7-8-28(24-29)39-27(2)10-15-34(48-39)49-41(56)42(18-19-42)30-11-14-32-33(25-30)59-43(44,45)58-32/h3,7-11,13-15,17,24-25H,1,4-6,12,16,18-23,26H2,2H3,(H,46,52)(H,47,55)(H,48,49,56)
|
| Chemical Name |
3-[6-[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino]-3-methylpyridin-2-yl]-N-[5-[3-[5-(2-oxo-4-prop-2-enoylpiperazin-1-yl)furan-2-yl]propanoylamino]pentyl]benzamide
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| Synonyms |
NJH-2-057; 2858812-70-9; CHEMBL5208354; SCHEMBL27163877; 3-[6-[[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropanecarbonyl]amino]-3-methylpyridin-2-yl]-N-[5-[3-[5-(2-oxo-4-prop-2-enoylpiperazin-1-yl)furan-2-yl]propanoylamino]pentyl]benzamide;
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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) |
DMSO: 100 mg/mL (123.33 mM)
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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 | 1.2333 mL | 6.1664 mL | 12.3329 mL | |
| 5 mM | 0.2467 mL | 1.2333 mL | 2.4666 mL | |
| 10 mM | 0.1233 mL | 0.6166 mL | 1.2333 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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