Tubulin Resists Degradation by Cereblon-Recruiting PROTACs

Dysregulation of microtubules and tubulin homeostasis has been linked to developmental disorders, neurodegenerative diseases, and cancer. In general, both microtubule-stabilizing and destabilizing agents have been powerful tools for studies of microtubule cytoskeleton and as clinical agents in oncology. However, many cancers develop resistance to these agents, limiting their utility. We sought to address this by developing a different kind of agent: tubulin-targeted small molecule degraders. Degraders (also known as proteolysis-targeting chimeras (PROTACs)) are compounds that recruit endogenous E3 ligases to a target of interest, resulting in the target’s degradation. We developed and examined several series of α- and β-tubulin degraders, based on microtubule-destabilizing agents. Our results indicate, that although previously reported covalent tubulin binders led to tubulin degradation, in our hands, cereblon-recruiting PROTACs were not efficient. In summary, while we consider tubulin degraders to be valuable tools for studying the biology of tubulin homeostasis, it remains to be seen whether the PROTAC strategy can be applied to this target of high clinical relevance.

Quantitative assessment of intracellular CRBN engagement using a BRD4 BRD2 -eGFP-mCherry reporter assay. CRBN engagement was assessed by monitoring rescue of dBET6-mediated degradation of BRD4 BRD2 -eGFP. Cells stably expressing BRD4 BRD2 -eGFP and mCherry were treated with 100 nM of dBET6, a specific degrader of BRD4 BRD2 , and increasing concentrations of the candidate tubulin degraders. The eGFP and mCherry signals were quantified by laser scanning cytometry and the concentration of compound that rescued 50% of BRD4 BRD2 -eGFP fluorescence (EC 50 ) was determined by nonlinear regression. Data from n=2 biological replicates. Error bars represent standard deviation of the mean. CPD -compound with log base 10. Quantitative assessment of intracellular CRBN engagement using a BRD4 BRD2 -eGFP-mCherry reporter assay. CRBN engagement was assessed by monitoring rescue of dBET6-mediated degradation of BRD4 BRD2 -eGFP. Cells stably expressing BRD4 BRD2 -eGFP and mCherry were treated with 100 nM of dBET6, a specific degrader of BRD4 BRD2 , and increasing concentrations of the candidate tubulin degraders. The eGFP and mCherry signals were quantified by laser scanning cytometry and the concentration of compound that rescued 50% of BRD4 BRD2 -eGFP fluorescence (EC 50 ) was determined by nonlinear regression. Data from n=2 biological replicates. Error bars represent standard deviation of the mean. CPD -compound with log base 10.

Competitive displacement assay for cellular CRBN engagement
Cells stably expressing the BRD4BD2-GFP with mCherry reporter [1] were seeded at 30-50% confluency in 384-well plates with 50 µL FluoroBrite DMEM media (Thermo Fisher Scientific A18967) containing 10% FBS per well a day before compound treatment. Compounds and 100 nM dBET6 were dispensed using a D300e Digital Dispenser (HP), normalized to 0.5% DMSO, and incubated with cells for 5 hours. The assay plate was imaged immediately using an Acumen High Content Imager (TTP Labtech) with 488 nm and 561 nm lasers in 2 µm x 1 µm grid per well format. The resulting images were analyzed using CellProfiler [2]. A series of image analysis steps ('image analysis pipeline') was constructed. First, the red and green channels were aligned and cropped to target the middle of each well (to avoid analysis of heavily clumped cells at the edges), and a background illumination function was calculated for both red and green channels of each well individually and subtracted to correct for illumination variations across the 384-well plate from various sources of error. An additional step was then applied to the green channel to suppress the analysis of large auto fluorescent artifacts and enhance the analysis of cell specific fluorescence by way of selecting for objects under a given size, 30 A.U., and with a given shape, speckles. mCherry-positive cells were then identified in the red channel filtering for objects between 8-60 pixels in diameter and using intensity to distinguish between clumped objects. The green channel was then segmented into GFP positive and negative areas and objects were labeled as GFP positive if at least 40% of it overlapped with a GFP positive area. The fraction of GFP-positive cells/mCherry-positive cells in each well was then calculated, and the green and red images were rescaled for visualization. The values for the concentrations that lead to a 50% increase in BRD4BD2-eGFP accumulation (EC50) were calculated using the nonlinear fit variable slope model (GraphPad Software).

Experimental procedures and characterizations
General procedure A. SNAr reaction of primary amines with S1. A solution of aryl fluoride S1 (1.0 equiv), primary amine (1.0-1.3 equiv), and DIPEA (2.0-4.0 equiv) in DMSO (0.1-0.3 M) was heated to 130 °C overnight. The reaction was then cooled to room temperature, diluted with water, and extracted with ethyl acetate. The combined organic layers were washed three times with water and then with brine, dried over magnesium sulfate, filtered, and concentrated.

General procedure B. Amide coupling with MMAE.
To a solution of thal-linker-CO2H (1.5 equiv) and monomethyl auristatin E (1.0 equiv) in DMSO (0.05 M) were added HATU (1.2 equiv) and DIPEA (2.5-3.5 equiv). The reaction was stirred at room temperature for 4 hours, and then diluted with DMSO and purified directly by reverse-phase preparative HPLC.

General procedure C. Reductive amination with MMAE.
To solution of thal-linker-OH (2.0 equiv relative to MMAE) in DCM (0.05 M) was added Dess-Martin periodinane (3.0-4.0 equiv relative to MMAE). The mixture was stirred at room temperature overnight, and then filtered and concentrated with a stream of nitrogen. The crude aldehyde intermediate was dissolved in methanol (0.05 M). Monomethyl auristatin E (1.0 equiv) was added, followed by sodium cyanoborohydride (NaBH3CN; 3.3 equiv) and 2 drops of glacial acetic acid. The reaction was stirred at room temperature overnight. Methanol was removed with a stream of N2, and then the crude reaction was diluted with DMSO and purified by reverse-phase preparative HPLC.

General procedure D. Mitsunobu reaction with CA4.
To a solution of CA4 (1.0 equiv), triphenylphosphine (1.0 equiv), and N-Boc-linker-alcohol (1.0 equiv) in DMF (0.4 M), DIAD (1.0 equiv) was added at room temperature. The reaction was stirred overnight, then diluted with water and extracted three times with DCM. The combined organic layers were washed with 1 M hydrochloric acid, saturated aqueous sodium bicarbonate, and brine, then dried over magnesium sulfate, filtered, and concentrated.