In vitro, homo-PROTAC pVHL30 degrader 1 dimerizes von Hippel-Lindau (VHL) with great affinity and promotes strong, quick, and proteasome-dependent autoregulation of VHL in several cell lines in a highly isotype-selective manner. without causing a hypoxic reaction to occur [1].
The most active Homo-PROTAC, CM11, induces complete depletion of pVHL30 after 4 h already at 10 nM. Potent and selective degradation of pVHL30 was long-lasting, with a half-degrading concentration (DC50) of <100 nM, a remarkable increase in cellular activity of >1000-fold compared to the parent inhibitor VH032. Mechanistically, we show that CM11 activity is strictly dependent on proteasome activity, Cul2 neddylation, and on VHL binding, and specifically on the formation of an avid 2:1 complex with VHL. Our data therefore supports a model in which a highly cooperative ternary complex (VHL)2:CM11 functions as the key species responsible for the induced degradation of VHL itself (Fig. 8). Future structural studies of this ternary species are warranted. Interestingly, CM11 also led to a decrease in cellular levels of Cullin2, which we hypothesize to be the result of direct ubiquitination and degradation of Cullin2 as part of the CRL2VHL complex. To our knowledge, it is unprecedented that a PROTAC can induce the degradation of a protein forming part of the same complex with the protein targeted directly[1].

Isothermal titration calorimetry (ITC). [1]
Titrations were performed on an ITC200 micro-calorimeter. Homo-PROTACs (CM11, CMP98 or CMP99) were diluted from a 100 mM DMSO stock solution to 150 µM in a buffer containing 20 mM Bis-tris propane, 150 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.4. The final DMSO concentration was 0.15% v/v. VCB protein experiments were carried out in a buffer containing 20 mM Bis-tris propane, 150 mM NaCl, 1 mM TCEP, 0.15% v/v DMSO, pH 7.4. The titrations consisted of 19 injections of 2 µL compounds solution (150 µM, in the syringe) at a rate of 0.5 µl s−1 at 120 s time intervals into the VCB protein solution (20 µM, in the cell). An initial injection of compound solution (0.4 µL) was made and discarded during data analysis. All experiments were performed at 25 °C, whilst stirring the syringe at 600 rpm. The data were fitted to a single binding site model to obtain the stoichiometry n, the dissociation constant K d and the enthalpy of binding ΔH using the Microcal LLC ITC200 Origin software provided by the manufacturer.

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Size-exclusion chromatography (SEC). [1]
SEC experiments were carried out in a ÄKTA pure system at room temperature. The oligomeric state of the VCB complex in solution was analyzed by gel filtration in a buffer containing 20 mM Bis-Tris (pH 7), 150 mM NaCl and 1 mM 1,4-dithiothreitol (DTT) using a Superdex 200 Increase 10/300 GL column calibrated with globular proteins of known molecular weight. VCB protein (50 µM) was incubated with CM11 (30 µM), CMP98 (30 µM), CMP99 (30 µM), VH032 (30 µM) or DMSO (0.5 %) for 20 min at room temperature prior to injection. Sample volume for each injection was 200 µl, and the flow rate was 0.5 ml min−1. Peak elution was monitored using ultraviolet absorbance at 280 nm.

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Biotinylation of VCB. [1]
The VCB complex was mixed with EZ-link NHS-PEG4-biotin in a 1:1 molar ratio and incubated at room temperature for 1 h. The reaction was quenched using 1 M Tris-HCl, pH 7.5, and unreacted NHS-biotin was removed with a PD-10 MiniTrap desalting column equilibrated with 20 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM DTT.

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AlphaLISA assay. [1]
All assays were performed at room temperature in 384-well plates with a final assay volume of 25 µL per well; plates were sealed with transparent film between addition of reagents. All reagents were prepared as 5× stocks diluted in 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.1% (w/v) bovine serum albumin and 0.02% (w/v) 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Biotinylated VCB (20 nM final) and His6-VCB (20 nM final) were incubated with a range of Homo-PROTAC concentrations (0.5–200 nM; three-in-five serial dilution) for 1 h. Anti-His acceptor beads (PerkinElmer, 10 µg ml−1 final) were added and plates were incubated for another hour. Streptavidin-coated donor beads (10 µg ml−1 final) were added and plates were incubated for a final 1 h. Plates were read on a PHERAstar FS using an optic module with an excitation wavelength of 680 nm and emission wavelength of 615 nm. Intensity values were plotted against PROTAC concentration on a log10 scale.

Cell culture. [1]
Human cell lines HeLa, U2OS and HEK 293, purchased from ATCC, were propagated in DMEM supplemented with 10% fetal bovine serum (FBS), L- glutamine, 100 μg ml−1 of penicillin/streptomycin at 37 °C and 5% CO2. Cells were maintained for no more than 30 passages. All cell lines were routinely tested for mycoplasma contamination using MycoAlert kit from Lonza. For compound treatment experiments, cells were transferred in six-well plates with either 3 × 105 or 5 × 105 cells per well in 2 ml of media. At 80% confluence, cells were treated with compounds at the desired concentration, reaching final DMSO concentration of 0.1% v/v. Cells were incubated at 37 °C and 5% CO2 for the desired time before harvesting.

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ML4924 and MG132 co-treatment. [1]
Cells were transferred in six-well plates with 5 × 105 cells per well in 2 ml media in order to achieve 80% confluence the day after. At t = 0, MLN4924 was added into the desired wells at 3 μM final concentration and 0.1% v/v of DMSO. DMSO (0.1% v/v final conc.) was added to the remaining wells in order to match identical conc. of vehicle in all wells. At t = 3 h, MG 132 was added into the desired wells at 50 μM final conc. and 0.1% v/v of DMSO. DMSO (0.1% v/v final conc.) was added to the remaining wells in order to achieve the same conc. of vehicle in all the wells. At t = 3.5 h, the desired wells were treated with 1 μM of CM11 in 0.1% v/v DMSO final concentration. DMSO (0.1% v/v final conc.) was added to the remaining wells to obtain the same conc. of vehicle in all the wells. The total final concentration of DMSO was therefore 0.3% v/v. Plates were incubated for 4 h at 37 °C and 5% CO2 before harvesting. For competition experiments with VH032, cells were treated with VH032 at a final concentration of 150 μM (or IOX4 at 50 μM) for 30 min before treatment with CM11 at 1 μM final concentration for 4 h. Plates were incubated for the desired time at 37 °C and 5% CO2 before harvesting.

[1]. Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nat Commun. 2017 Oct 10;8(1):830.

In summary, we present CM11, a chemical probe for rapid and selective pVHL30 knockdown. CM11 provides a chemical tool alternative to conventional knockdown RNAi approaches and gene editing knockout technologies such as CRISPR-Cas9. Relevant information to the use of CM11 will be made available in the newly established ‘Chemical Probes Portal’ (http://www.chemicalprobes.org/)72. We anticipate CM11 will find wide use amongst chemical and cell biologists alike interested in investigating and dissecting the pleiotropic biological functions of pVHL. More generally, we provide proof-of-concept that bivalent molecules can be designed to induce an E3 ligase to destroy itself. This strategy opens powerful new avenues to drugging E3 ligases in ways that are not possible with inhibitors alone.[1]

C58H82N8O14S2

1179.44689321518

1178.539

C, 59.06; H, 7.01; N, 9.50; O, 18.99; S, 5.44

2244684-49-7

2244684-49-7

134160242

White to off-white solid powder

3.6

6

18

33

82

1870

6

S1C=NC(C)=C1C1C=CC(=CC=1)CNC([C@@H]1C[C@H](CN1C([C@H](C(C)(C)C)NC(COCCOCCOCCOCCOCCOCC(N[C@H](C(N1C[C@@H](C[C@H]1C(NCC1C=CC(C2=C(C)N=CS2)=CC=1)=O)O)=O)C(C)(C)C)=O)=O)=O)O)=O

WGJCHHJGGFCCRS-DEYDLUNASA-N

InChI=1S/C58H82N8O14S2/c1-37-49(81-35-61-37)41-13-9-39(10-14-41)29-59-53(71)45-27-43(67)31-65(45)55(73)51(57(3,4)5)63-47(69)33-79-25-23-77-21-19-75-17-18-76-20-22-78-24-26-80-34-48(70)64-52(58(6,7)8)56(74)66-32-44(68)28-46(66)54(72)60-30-40-11-15-42(16-12-40)50-38(2)62-36-82-50/h9-16,35-36,43-46,51-52,67-68H,17-34H2,1-8H3,(H,59,71)(H,60,72)(H,63,69)(H,64,70)/t43-,44-,45+,46+,51-,52-/m1/s1

N1,N20-Bis((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide

CM-11; 2244684-49-7; CM 11; Homo-PROTAC pVHL30 degrader 1; N1,N20-bis((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)-3,6,9,12,15,18-hexaoxaicosanediamide; CHEMBL4740486; (2S,4R)-4-hydroxy-1-[(2S)-2-[[2-[2-[2-[2-[2-[2-[2-[[(2S)-1-[(2S,4R)-4-hydroxy-2-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methylcarbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]amino]-2-oxoethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]acetyl]amino]-3,3-dimethylbutanoyl]-N-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide; N,N'-bis[(2S)-1-[(2S,4R)-4-hydroxy-2-({[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl}carbamoyl)pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]-3,6,9,12,15,18-hexaoxaicosanediamide; CM 11; Homo-PROTACCM11

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 : ≥ 150 mg/mL (~127.18 mM)
H2O : ~25 mg/mL (~21.20 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.12 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 (2.12 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 (2.12 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.)