OTUB1

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.[1]

Gel-Based ABPP[1]
Recombinant OTUB1 (0.1μg/sample) was pre-treated with either DMSO vehicle or covalent ligand or DUBTACs at 37 °C for 30 min in 25 μL of PBS, and subsequently treated with of IA-Rhodamine (concentrations designated in figure legends) at room temperature for 1 h. The reaction was stopped by addition of 4×reducing Laemmli SDS sample loading buffer. After boiling at 95 °C for 5 min, the samples were separated on precast 4–20% Criterion TGX gels. Probe-labeled proteins were analyzed by in-gel fluorescence using a ChemiDoc MP.

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NJH-2-057 Probe Labeling of Recombinant OTUB1[1]
Recombinant and pure OTUB1 protein (0.5 μg) per sample per replicate was suspended in 50 μL total PBS. 1 μL of either DMSO or NJH-2-075 (to give final concentrations of 50, 10, 1, and 0.1 μM) was added, followed by a 1.5 h incubation at 37 °C. Next, 7.8 μL of a solution composed of 9.4 μL of 5mM Azide-Fluor 545 (in DMSO), 112 μL of TBTA ligand (Stock 1.7 mM in 4 parts t-butanol + 1 part DMSO), 37.5 μL of 50 mM TCEP (in water), and 37.5 μL of 50 mM Copper (II) sulfate was added to each sample and the samples were incubated for 1 hour at room temperature. Following CuAAC, 30 μL of Laemmli Sample Buffer (4 x) was added to each sample, vortexed and boiled for 6 min at 95 °C. Samples were loaded on an SDS/PAGE gel and analyzed for in-gel fluorescence.

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Deubiquitinase Activity Assay[1]
Previously described methods were used to assess EN523 effects on OTUB1 activity 23. Recombinant OTUB1 (500 nM) was pre-incubated with DMSO or EN523 (50 μM) for 1 hr. To initiate assay pre-treated OTUB1 enzyme was mixed 1:1 with di-Ub reaction mix for final concentrations of 250 nM OTUB1, 1.5 μM di-Ub, 12.5 μM UBE2D1 and 5 mM DTT. The appearance of mono-Ub was monitored by Western blotting over time by removing a portion of the reaction mix and adding Laemmli’s buffer to terminaye reaction. Blot shown is a representative gel from n=3 biologically independent experiments/group.

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Bio-NMR Analysis of EN523-OTUB1 Interactions[1]
We recorded all NMR spectra on a Bruker 600 MHz spectrometer, equipped with a 5 mm QCI-F cryo probe with z-gradient, and kept the temperature constant at 298K during all experiments. To probe compound and E2 ligase binding to OTUB1, we recorded 1H-1D and 13C-SOFAST-HMQC experiments. We used 3 mm NMR tubes filled with 160 μL of 50 μM {U}-2H,1H/13C-methyl-Ile/Leu/Val/Ala(ILVA),{U}-15N labeled OTUB1, 25 mM d-Tris, pH 7.5, 150 mM NaCl, 5% D2O (to lock), 100 μM DSS (internal standard), 75 μM EN-523 (dissolved in 100% d6-DMSO; for compound binding study) and/or 100 μM E2 D2 / Ub-E2 D2 (for ligase binding studies). To allow for complete binding of the compound to OTUB1, we chose an incubation period of ~40 hours. We also recorded reference spectra with the adequate volumes of pure d6-DMSO and/or E2 buffer to compensate for solvent induced effects, and repeated experiments after 40 hours to make sure that any spectral changes were not related to protein oxidation.

Labeling of Endogenous OTUB1 in HEK293T Cells with NJH-2-075 Probe[1]
One plate of 70% confluent HEK293T cells per condition per replicate were treated with either DMSO vehicle or NJH-02-075 (50 μM) for 2 hours. Cells were harvested by scraping, suspended in 600 μL of PBS, lysed by probe sonication, and centrifuged for 10 min at 5000 rpm to remove debris. Lysate was normalized to 3.1 mg/mL and 85 μL removed for Western blotting analysis of input. 500 μL of lysate was then incubated for 1 hour at room temperature with 10 μL of 5 mM biotin picolyl azide (in water), 10 μL of 50mM TCEP (in water), 30 μL TBTA ligand (Stock 1.7 mM in 4 parts t-butanol + 1 part DMSO), and 10 μL of 50 mM Copper (II) sulfate. Following CuAAC, precipitated proteins were washed 3 x with cold methanol and resolubilized in 200 μL 1.2% SDS/PBS. To ensure solubility, proteins were heated to 90 °C for 5 min following resuspension. 1 mL of PBS was then added to each sample, followed by 50 μL of high-capacity streptavidin beads. Samples were then incubated overnight on a rocker at 4 °C. The following morning the samples were warmed to room temperature, and non-specific binding proteins were washed away with 3 x PBS washes followed by 3 x water washes. Beads were then resuspended in 100 μL PBS and 30 μL Laemmli Sample Buffer (4 x) and boiled for 13 min at 95 °C. Samples were vortexed and loaded onto an SDS/PAGE gel along with saved input samples for Western blotting analysis.

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Western Blotting[1]
Proteins were resolved by SDS/PAGE and transferred to nitrocellulose membranes using the Trans-Blot Turbo transfer system. Membranes were blocked with 5% BSA in Tris-buffered saline containing Tween 20 (TBS-T) solution for 30 min at RT, washed in TBS-T, and probed with primary antibody diluted in recommended diluent per manufacturer overnight at 4°C. After 3 washes with TBS-T, the membranes were incubated in the dark with IR680- or IR800-conjugated secondary antibodies at 1:10,000 dilution in 5 % BSA in TBS-T at RT for 1 h. After 3 additional washes with TBST, blots were visualized using an Odyssey Li-Cor fluorescent scanner. The membranes were stripped using ReBlot Plus Strong Antibody Stripping Solution when additional primary antibody incubations were performed.

[1]. Deubiquitinase-targeting chimeras for targeted protein stabilization. Nat Chem Biol. 2022;18(4):412-421.

In this study, we discovered a covalent small-molecule recruiter EN523 for the K48-ubiquitin chain-specific DUB OTUB1. We demonstrated that this recruiter can be used incorporated into fully synthetic heterobifunctional DUBTACs by linking a DUB recruiter to protein targeting ligands to enable TPS of actively degraded target proteins in cells. We showed two successful examples of TPS with ΔF508-CFTR and WEE1. For ΔF508-CFTR, we also demonstrated that we not only heightened the levels of the mutant protein, but also improved cell surface chloride channel conductance of CFTR with our DUBTAC in combination with the potentiator ivacaftor, compared to lumacaftor and ivacaftor treatments. While we showed early validation of the DUBTAC platform here, there are many avenues for future exploration. These include further optimization of DUB recruiters against OTUB1 to improve their potency and proteome-wide selectivity, as well as the discovery of new recruiters against other candidate DUBs. For exploring optimization of CFTR DUBTACs, further improvement of the linker between lumacaftor and the DUB recruiter could improve potency and degree of CFTR stabilization. In addition, elucidating the mechanism, structural underpinnings, and kinetics in the formation of the ternary complex formed between CFTR and OTUB1 and understanding how CFTR is deubiquitinated by the DUBTAC will be important. Furthermore, better understanding of whether we are disrupting endogenous OTUB1 function would be important to understanding the mechanism and safety of DUBTACs.[1]

C12H14N2O3

234.25

234.1

2094893-05-5

126817009

Typically exists as White to off-white solids at room temperature

1

0

3

2

17

343

0

KYXPDOMFYBFXKO-UHFFFAOYSA-N

InChI=1S/C12H14N2O3/c1-3-10(15)13-6-7-14(11(16)8-13)12-5-4-9(2)17-12/h3-5H,1,6-8H2,2H3

1-(5-methylfuran-2-yl)-4-prop-2-enoylpiperazin-2-one

1-(5-methylfuran-2-yl)-4-(prop-2-enoyl)piperazin-2-one; 4-Acryloyl-1-(5-methyl-2-furyl)piperazin-2-one; EX-A6269; MFCD32859220; AKOS040760003;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~250 mg/mL (~1067.24 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.)