Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

Our homodimeric PROTACs are the first chemical inhibitors of Cereblon and may help to further elucidate the molecular mechanism of thalidomide analogs, as well as to investigate the physiological function of Cereblon. This new PROTAC platform technology can be highly helpful for specifically targeting proteins of interest that are otherwise considered to be untrackable. CRBN inhibition alone has no anti-tumor activity.

However, CRBN degraders may have clinical implications in obesity, infectious diseases, and other disorders. The synthesis is challenging and it's hard to find optimal linkers and attachment to generate effective protein degraders when designing a PROTAC compound. To begin, place 2.46 grams of Boc-protected L-glutamine and 50 milliliters of tetrahydrofuran in a 100 milliliter round-bottomed flask, and stir at 800 rpm for one minute.

Then, add 1.95 grams of 1, 1'carbonyldiimidazole and a catalytic amount of 4-pyridine. Continue stirring for 10 hours at reflux to obtain a clear colorless solution of glutarimide compound. Let the solution cool to room temperature, and remove the solvent with a rotary evaporator.

Redissolve the residue in 200 milliliters of ethyl acetate and pour it into a separatory funnel. Wash the organic layer with 50 milliliters each of deionized water and brine, and dry the organic layer over sodium sulfate. Gravity filter the solution through silica gel to remove the desiccant, and then rinse the silica gel with 200 milliliters of ethyl acetate.

Remove the solvent from the filtrate under vacuum to obtain Compound 1 as a colorless solid. Dry the product under vacuum overnight before using it. Next, dissolve 0.50 grams of sodium acetate in 20 milliliters of glacial acetic acid in a 100 milliliter round-bottom flask equipped with a stir bar.

Add 1.25 grams of 3-fluorophthalic anhydride and 1.14 grams of glutarimide and stir at reflux for six hours to obtain a purple solution of Compound 3. Let the mixture cool to room temperature, and then pour it into 100 milliliters of deionized water and stir for 10 minutes to precipitate Compound 3. Collect the solid by filtration, wash it with water and petroleum ether, and dry it under vacuum for two days.

Next, place 0.83 grams of Compound Three and 20 milliliters of dry dimethyl sulfoxide in a 50 milliliter Schlenk flask and stir for two minutes. Add 0.22 grams of alpha omega diamine linker Compound 6, and 1.05 millilitres of N, N-diisopropylethylamine and stir the mixture under argon at 90 degrees celsius for 18 hours to obtain Homodimer 8 in a dark green mixture. Let the mixture cool to room temperature and pour it into 100 millilitres of deionized water.

Extract the product into three 50 milliliter portions of ethyl acetate, combine the organic layers, and wash them with 50 milliliters each of water and brine. Dry the washed organic layer over sodium sulfate, filter out the desiccant and remove the solvent under vacuum. Redissolve the residue in a minimal amount of chromatography solvent and apply to a silica gel column for purification.

Collect the fluorescent product fractions, combine them, and remove the solvent to obtain Homodimer 8 as a yellow solid. To begin, dissolve 2.28 grams of Glutarimide 1 in 25 milliliters of dimethylformamide in a 100 milliliter round-bottom flask. Suspend 2.76 grams of milled potassium carbonate in this solution.

Then, draw 0.62 milliliters of iodomethane into a syringe equipped with a 20-gauge needle. Add the iodomethane drop-wise over the course of 10 seconds. Stopper the flask with a rubber septum, and add a vent needle.

Sonicate the mixture for two hours at room temperature to obtain Glutarimide Compound 2. Dilute the mixture with 50 milliliters of ethyl acetate and pour it into a separatory funnel. Rinse the flask into the funnel with another 50 milliliters of ethyl acetate.

Work up the product and purify it with silica gel column chromatography to obtain Glutarimide 2 as a colorless solid. Compare the retention times of Glutarimide Compounds 1 and 2 to confirm that N-methylation was successful. To begin the validation, treat multiple myeloma cells with the compounds of interest.

Isolate the relevant proteins, and prepare samples for a Western Blot analysis of protein degradation. Then, prepare a gel sandwich for SDS-PAGE and fill the anode buffer tank with a pH 8.9 tris-HCl solution. Load the protein samples into the gel and run it at 70 Volts for 20 minutes, followed by 115 volts for 150 minutes.

Next, prepare 1x transfer buffer for immunoblotting and carefully move the gel from its cassette to the transfer buffer to equilibrate. You have to be very careful when you remove the gel from the cassette for equilibration, because it can burst and crumple very quickly. Immerse a 0.45 micrometer polyvinylidene difluoride membrane in pure methanol for at least 20 seconds to activate it, and then immerse it in 1x transfer buffer.

Let the gel and the membrane equilibrate for 10 minutes. Then, assemble the Western Blotting cassette and apply 180 milliamperes for 90 minutes to transfer the proteins to the membrane. Afterwards, visualize the proteins by immunostaining.

To evaluate the effects on cell viability, first, seed 50, 000 multiple myeloma cells per well of a 96 well plate in biological triplicate. Add to each well 100 microliters of media containing DMSO, or a 0.1, one, or 10 micromolar of Compound 8, Compound 9, or pomalidomide. To perform cell viability rescue experiment, instead treat the cells with a 100 nanomolar solution of Compound 8 for three hours, either before or after adding a one micromolar solution of pomalidomide.

Incubate the cells for 24, 48, or 96 hours at 37 degrees celsius in a 5%carbon dioxide atmosphere, and measure cell viability with a luminescent assay. The structure of homodimeric PROTAC 8 was confirmed with proton and carbon NMR. Heterodimeric PROTAC 9 was synthesized as a negative control, as N-methylation within the glutarimide portion results in a loss of Cereblon, or CRBN, binding capability.

The homo PROTAC 8 induced near complete proteosomal CRBN degradation with minimal effects on the lymphoid transcription factors IKZF1 and IKZF3. In contrast, the hetero PROTAC 9 showed behavior similar to pomalidomides. Compound 8 induced CRBN degradation could be blocked by a direct inhibitor of the proteasome, or indirectly blocked by netylation inhibition.

Cells pre-treated with Compound 8 significantly resisted the effects of pomalidomide on IKZF1 and IKZF3. Compound 8 induced CRBN degradation had much less of an effect on multiple myeloma cell viability after 96 hours than the IKZF1 and IKZF3 degradation from Compound 9 and pomalidomide did. Multiple myeloma cells pre-treated with Compound 8 had significantly greater viability when exposed to pomalidomide than untreated cells, suggesting that Compound 8 could be used to mimic immunomodulatory IMiD drug-resistance.

We found that Compound 8 induces complete proteasomal degradation of Cereblon, has no side effects on IKZF1 and IKZF3 cell viability and proliferation. Extending the project technology to other targets has the potential for inactivating and tracking of proteins related to cancer and other diseases. When adapting this technique for other drugs and proteins, one should carefully investigate the molecular structures of the drug and the protein target to choose the optimal linkers and attachments.

The IMiDs will not work in mice because of a point mutation in Cereblon, but a transgenic knock-in mouse model exists which can be used for further animal studies. As pomalidomide is an analog thalidomide, which is highly teratogenic, we do not recommend pregnant women working with these drugs.