MDA-MB-231 and HeLa cells were obtained from the American Type Culture Collection (ATCC), cultured in RPMI-1640 medium (1 × ) and Dulbecco’s modified Eagle’s medium (DMEM; 1 × ), respectively, containing 10% fetal bovine serum and 1% penicillin–streptomycin and grown in a humidified incubator at 37 °C and 5% CO2. For extended dose-response experiments of MDA-MB-231 cells (Supplementary Figure 3), all incubation medium was supplemented with 0.025% Tween 80 and DMSO68 to enhance PROTAC solubility at the higher concentrations.
Lysates from MDA-MB-231 cells were washed once with ice-cold PBS (1 × ), followed by lysis in buffer containing 25 mM Tris [pH 7.5], 0.25% sodium deoxycholate, 1% Triton X-100, supplemented with 1X protease inhibitor cocktail (Roche), and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, and 20 mM β-glycerophosphate), unless otherwise noted. Lysates were spun at 14,000 × g for 10 min at 4 °C and supernatant was evaluated for protein content using a Pierce BCA Protein Assay (ThermoFisher Scientific). In all experiments, 25–50 μg of protein was loaded onto 10% SDS-PAGE gels or 4–20% Criterion TGX precast gradient gels (Bio-Rad), transferred to nitrocellulose membranes, and probed with the specified antibodies overnight at 4 °C in 1X TBS-Tween (Tris-buffered saline plus 0.02% Tween 20) containing 5% non-fat milk. Immunoblots were visualized using a Bio-Rad ChemiDoc imaging instrument and subsequently processed and quantified using the accompanying Bio-Rad ImageLab software. See Supplementary Figure 8 for uncropped scans of western blots shown in the main text figures.
Rabbit antibodies purchased from Cell Signaling Technologies (CST) with their respective antibody dilutions are as follows: p38α (#9218) 1:2000, p38δ (#2308) 1:1000, p38β (#2339) 1:1000, p38γ (#2307) 1:500, ERK2 (#9108) 1:2000, JNK2 (#9258) 1:1000, and VHL (#68547) 1:5000. Additional antibodies purchased from CST with their respective dilutions are as follows: mouse HA-tag (#2367) 1:1000, mouse JNK1 (#3708) 1:1000, and FLAG (DYKDDDDK) Tag Sepharose bead conjugate (#70569). Mouse antibodies purchased from Sigma-Aldrich with their respective antibody dilutions are as follows: alpha-tubulin (#T9026) 1:5000 and FLAG M2 (#F1804) 1:1000. Rabbit Cullin 2 (CUL2) was purchased from ThermoFisher Scientific (#700179) 1:5000 and rabbit ERK1 (C-16) 1:2000 from Santa Cruz Biotechnology (#sc-93).
Wild-type human p38alpha-MAPK (MAPK14) was received as a gift from Dr. D. Martin Watterson (Northwestern University, under MTA) and was described previously69. The plasmid contains an N-terminal His6 tag and encodes a region spanning amino acids 2–360 of the human p38α kinase (NCBI Reference Sequence: NM_139012). BL21-CodonPlus(DE3)-RIPL E. coli cells (Agilent Technologies) were transformed with pMCSG7-His6-p38α and were selected in Luria-Bertani (LB) medium containing carbenicillin (100 µg mL−1), chloramphenicol (15 µg mL−1), and spectinomycin (50 µg mL−1) at 37 °C until OD600 = 0.6–0.8. At this point, cells were induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and grown at 25 °C for 14–16 h. Cell pellets were collected by centrifugation (5000 rpm, 10 min, 4 °C) and homogenized in lysis buffer (10 mM Tris pH 8.3, 500 mM NaCl, 5 mM β-mercaptoethanol, 10 mM imidazole, and 10% glycerol) containing a 1X protease inhibitor cocktail tablet (Roche). The homogenized cells were subsequently passed through a microfluidizer three times at 15k PSI and lysate was clarified by centrifugation (16,000 rpm, 45 min, 4 °C). The resultant supernatant was then applied to Ni-NTA agarose beads (QIAGEN) with gentle rotation for 1 h at 4 °C, washed once with lysis buffer with 10 mM imidazole (pH 8.3), twice with lysis buffer containing 20 mM imidazole (pH 8.3), and eluted off of the nickel resin in lysis buffer containing 50 mM imidazole (pH 8.3). Eluted protein was assessed for identity and purity via Coomassie staining of sample run on an SDS-PAGE gel and pure elutions were pooled, concentrated, and diluted in ion-exchange buffer A (10 mM Tris pH 8.3, 5 mM β-mercaptoethanol) until the salt concentration was 50 mM, before loading onto a Mono Q 5/50 GL column (GE Life Sciences). The protein was subjected to a step-wise wash protocol, followed by a linear gradient from 200 to 500 mM NaCl using ion-exchange buffer B (10 mM Tris 8.3, 1 M NaCl, 5 mM β-mercaptoethanol). Fractions were then assessed for purity via Coomassie, pooled, concentrated, and run on a HiLoad 16/600 Superdex-200 column (GE Healthcare Life Sciences) using size-exclusion buffer (10 mM Tris pH 8.3, 150 mM NaCl, 5 mM β-mercaptoethanol). Pure fractions of p38α were pooled, concentrated to ~5 mg mL−1, aliquoted, and flash-frozen before storing at −80 °C.
Wild-type human p38δ kinase (NCBI Reference Sequence: NM_002754.4) encoding a region spanning amino acids 1–365 was PCR amplified from a pcDNA3.3-p38delta-MAPK (MAPK13) template that we received as a gift from Dr. Romeo Ricci (IGBMC), described previously70. This p38δ region was cloned into a pET28a vector containing an N-terminal His6 tag using NheI and BamHI restriction sites. As before, BL21-CodonPlus(DE3)-RIPL E. coli cells (Agilent Technologies) were transformed with pET28a-His6-p38delta and were selected in LB medium containing kanamycin (50 µg mL−1), chloramphenicol (15 µg mL−1), and spectinomycin (50 µg mL−1) at 37 °C until OD600 = 0.8. Purification of His6-p38δ was performed identical to His6-p38α above, except Ni-NTA agarose beads were washed twice with lysis buffer with 20 mM imidazole (pH 8.3), once with lysis buffer containing 50 mM imidazole (pH 8.3), and eluted off of the nickel-conjugated resin in lysis buffer containing 150 mM imidazole (pH 8.3). Pure fractions of p38δ were pooled, concentrated to ~ 3 mg mL−1, aliquoted, and flash-frozen before storing at −80 °C.
For the expression of GST-tagged VHL:Elongin B:Elongin C (herein referred to as GST-VHL), wild-type human VHL, Elongin B, and Elongin C were co-expressed in E. coli. BL21(DE3) cells were co-transformed with pBB75-Elongin C and pGEX4T-2-VHL-rbs-Elongin B and selected in LB medium containing carbenicillin (100 µg mL−1) and kanamycin (25 µg mL−1) at 37 °C until OD600 = 0.8, at which point the culture was chilled to 16 °C and induced with 0.4 mM IPTG for 16 h. Cells were homogenized and lysed, as described above, except the lysis buffer was composed of 30 mM Tris [pH 8.0], 200 mM NaCl, 5% glycerol, 5 mM DTT containing a 1X protease inhibitor cocktail tablet (Roche). Clarified cell lysate was applied to Glutathione Sepharose 4B beads (GE Life Science) and gently rotated for 2 h at 4 °C. Beads were washed with four column volumes of lysis buffer, followed by four column volumes of elution buffer (50 mM Tris pH 8.0, 200 mM NaCl, 10 mM Glutathione). Eluted protein was assessed for identity and purity via Coomassie staining of sample run on an SDS-PAGE gel and pure elutions were pooled, concentrated, and diluted in ion-exchange buffer A (30 mM Tris pH 8.0, 5% glycerol, 1 mM DTT) until the salt concentration was 50 mM, before loading onto a Mono Q 5/50 GL column (GE Life Sciences). The protein was subjected to a linear gradient of NaCl (0–500 mM NaCl) using ion-exchange buffer B (30 mM Tris 8.0, 1 M NaCl, 5% glycerol, 1 mM DTT). Fractions were then assessed for purity via Coomassie, pooled, concentrated, and run on a Superdex-200 column (GE Life Sciences) using size-exclusion buffer (30 mM Tris pH 8.0, 100 mM NaCl, 10% glycerol, 1 mM DTT). Pure fractions of GST-VHL were pooled, concentrated to ~5 mg mL−1, aliquoted, and flash-frozen before storing at −80 °C.
Mutant p38δ was generated by QuikChange Lightning site-directed mutagenesis kit (Agilent). Transfections were carried out using Lipofectamine 2000 reagent (Invitrogen) in HeLa cells seeded at 3 × 105 cells per six well. One microgram of FLAG-containing pcDNA5-p38delta-WT or pcDNA5-p38delta-K220ET221E was used per transfection. Opti-MEM media were changed after 6 h to DMEM (1 × ), at which point the indicated compounds were added for 24 h before harvesting.
Glutathione Sepharose 4B (Glutathione-conjugated beads in a slurry containing 20% ethanol) were washed twice with 1X wash buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 0.01% NP40, 5 mM MgCl2, 10% Glycerol) and then blocked for 1 h at room temperature with 10% bovine serum albumin (BSA) in wash buffer. The beads were then washed again three times with wash buffer and then purified GST-VBC (stable form of VHL complexed with EloB/EloC, described above) was immobilized for 1 h at 4 °C at 3.6 pmole µL–1 of beads. The beads were then washed three times with wash buffer, resuspended, divided into two equal volumes, and p38α or p38δ protein was added. The bead:p38 mixture was then aliquoted to separate tubes and PROTAC was added at the indicated concentration (PROTACs were intermediately diluted in 10% DMSO and 0.25% CHAPS). This mixture was incubated at 4 °C for 2 h. The beads were washed three times with 10 column volumes of wash buffer and then eluted with SDS loading buffer at 75 °C for 10 min.
For experiments in which the input substrate is a WCL (Fig. 4b), the sample was prepared as follows. Five 150 mm dishes of confluent MDA-MB-231 cells were washed with 1X PBS, pH 7.4, and then dissociated using enzyme-free PBS-based cell dissociation buffer (ThermoFisher Scientific) for 10 min at 37 °C. Cells were then pelleted, resuspended in wash buffer (same as above, but supplemented with 1X protease inhibitor cocktail (Roche) and 5 µM epoxomicin), and then lysed by sonication (Branson sonicator microtip, power = 6, 50% duty cycle for three cycles of 30 s on and 1 min off at 4 °C). The lysate was cleared by centrifugation and then added to the GST-VBC-conjugated beads as an input substrate, as above.
Assays were performed at room temperature and plates sealed with transparent film between addition of reagents to prevent well contamination. All reagents were diluted in 50 mM HEPES pH 7.5, 50 mM NaCl, 69 µM Brij-35, and 0.1 mg mL−1 BSA. Recombinant GST-VBC was mixed with His6-p38α and PROTAC (diluted one-in-three from 6× stock) to a final volume of 15 μL per well in a OptiPlate-384 well microplate (PerkinElmer) and incubated for 30 min. VBC and p38α were kept at a constant final concentration of 150 nM. In all, 7.5 μL of Anti-6xHis AlphaLISA Acceptor beads (PerkinElmer) were added to each well and plates were incubated for 15 min in the dark. In total, 7.5 μL of Alpha Glutathione Donor beads (PerkinElmer) were added to each well and plates were incubated for 45 min in the dark. Plates were read on a Synergy 2 microplate reader using the Gen5 imager software (BioTek Instruments) with an excitation wavelength of 680 nm and emission wavelength of 615 nm. Intensity values were plotted in Graphpad Prism with PROTAC concentration values represented on a log10 scale.
CETSA protocol was adapted from60. In all, 3 × 107 MDA-MB-231 cells (1 × 107 cells per condition) were collected and resuspended in ice-cold 1X PBS supplemented with 1X protease inhibitor cocktail (Roche) and lysed by three cycles of liquid nitrogen snap freeze, followed by 50% thaw in a room temperature water bath and an additional 50% thaw at 4 °C. After each freeze–thaw cycle, lysate was vortexed briefly to ensure homogenous thawing. Lysate was then cleared for 20 min by centrifugation (14,000 × g at 4 °C) and the soluble fraction was divided into three equal aliquots. The aliquots were treated with vehicle (2.5% DMSO), 100 µM SJFα, or 100 µM SJFδ and incubated at room temperature for 30 min with gentle rotation. Each aliquot was then divided (50 µL) into eight PCR tubes and individually heated at the indicated temperature for 3 min followed by cooling at room temperature for 3 min. After cooling, samples were spun down for 20 min (14,000 × g at 4 °C) and the supernatant (soluble fraction) was analyzed by SDS-PAGE and immunoblotted for p38δ and alpha-tubulin (negative control).
SPR experiments were conducted on a Biacore 3000 instrument (GE Healthcare) at room temperature. GST antibody was immobilized through direct amination onto a carboxymethylated dextran surface (CM5) and GST-VBC was captured on the CM5 surface. This prepared surface was equilibrated over 3 h in running buffer (10 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.4 mg mL−1 BSA, 0.005% P20, 2% DMSO). All compounds were prepared in 100% DMSO stock plates with a top concentration of 500 µM in a 3× serial dilution. Compounds were transferred from the stock plate to the assay plate and diluted into running buffer without DMSO. Equimolar concentration of target protein was added to corresponding compound wells. All compounds were run as a 12-point concentration series with a final assay top concentration of 50 µM to measure p38δ:PROTAC and VHL:PROTAC binding and top concentration of 5 µM to measure p38δ:PROTAC:VHL binding. All compounds were injected for 60 s and allowed to dissociate for 300 s. Data analysis was performed in Scrubber 2 (BioLogic Software). Blanks from reference flow cell containing immobilized GST-VBC were subtracted to correct for noise and data were solvent-corrected against a standard DMSO curve. All reported Kd values represent an average of at least two replicates and were obtained by fitting to a minimum of five concentrations using a 1:1 fitting algorithm.
HeLa cells (2 × 106) were seeded into 10 cm dishes and allowed to adhere overnight. On the following day, cells were transfected with 3 µg pcDNA5-FLAG-p38α or pcDNA5-FLAG-p38δ and 1 µg pRK5-HA-Ubiquitin-WT (Addgene plasmid #17608) in Opti-MEM media using Lipofectamine 2000 (Invitrogen). After 6 h, Opti-MEM media were replaced with DMEM (1 × ) and cells were grown for an additional 24 h. After this time, cells were treated with indicated concentrations of either vehicle (DMSO) or PROTAC for the indicated amount of time at 37 °C. Cells were then placed on ice, rinsed twice with ice-cold 1X PBS and lysed in 500 µL modified 1X RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing 5 mM 1,10-phenanthroline monohydrate, 10 mM N-ethylmaleimide, 20 µM PR-619, and 1X protease inhibitor cocktail (Roche). Lysates were spun down at 14,000 × g at 4 °C for 20 min and protein content was measured by Pierce BCA Protein Assay (ThermoFisher Scientific). Protein lysate was normalized and 1 mg of lysate was aliquoted onto 20 µL (bed volume) of DYKDDDDK-sepharose beads (CST: #70659). FLAG-containing proteins were immunoprecipitated from HeLa lysates overnight at 4 °C with gentle rotation, after which samples were spun down at 3000 × g at 4 °C for 2 min and the beads were washed once with ice-cold lysis buffer and three times with ice-cold 1X TBS-T (137 mM NaCl, 2.7 mM KCl, 19 mM Tris-HCl pH 7.5, 0.02% Tween 20). Beads were resuspended in 1X lithium dodecyl sulfate (LDS) sample buffer containing 5% 2-mercaptoethanol (BME). Immunoprecipitated protein was eluted off of the beads by heating at 95 °C for 5 min and the supernatant was run on an SDS-PAGE gel and evaluated for the presence of immunoprecipitated FLAG-tagged proteins (anti-FLAG M2, Sigma #F1804), as well as ubiquitinated FLAG-tagged proteins (anti-HA-tag (6E2), CST #2367). WCL refers to the normalized input lysate loaded onto DYKDDDDK-sepharose beads.
TUBE1 immunoprecipitation experiments were carried out exactly as described above, except for the fact that 1 mg of normalized HeLa lysate was loaded onto 20 µL TUBE1 agarose (LifeSensors) resin per sample.
MDA-MB-231 cells were plated at 3 × 105 cells per well in a six-well dish, allowed to adhere, and were treated with either vehicle (DMSO) or PROTAC (250 nM) for 24 h. Cell were lysed in 1 mL of TRIzol reagent (ThermoFisher Scientific) per six well. Chloroform was added (200 µL) per sample, after which samples were vortexed vigorously for 15 s, and centrifuged at 12,000 × g for 15 min at 4 °C. Total RNA was then precipitated from the aqueous phase by addition of isopropanol (500 µL), followed by centrifugation at 12,000 × g for 10 min at 4 °C. RNA pellet was washed twice with 75% EtOH and allowed to air dry for 15 min, after which RNA was dissolved in 25 µL of nuclease-free H2O. Complementary DNA was synthesized from 4 µg of total RNA per condition according to the manufacturer’s protocol (Applied Biosystems) and real-time PCR was performed with 800 nM primers, diluted with 4 µL SYBR Green Reaction Mix (Applied Biosystems). RT-PCR experiments were performed with the following protocol on a LightCycler 480 Instrument II (Roche): 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 45 s. qRT-PCR samples were performed and analyzed in triplicate, from two independent experiments. Beta-tubulin was used for normalization. Primers used in this study are as follows:
Beta_Tub_F: 5ʹ-TGGACTCTGTTCGCTCAGGT-3ʹ
Beta_Tub_R: 5ʹ-TGCCTCCTTCCGTACCACAT-3ʹ
p38α_F: 5ʹ-TCGCATGAATGATGGACTGAAAT-3ʹ
p38α_R: 5ʹ-CCCGAGCGTTACCAGAACC-3ʹ
p38δ_F: 5ʹ-TGAGCCGACCCTTTCAGTC-3ʹ
p38δ_R: 5ʹ-AGCCCAATGACGTTCTCATGC-3ʹ.
MDA-MB-231 cells were plated at 3 × 105 cells per well in a six-well dish, allowed to adhere overnight, and switched to serum-free RPMI-1640 (1 × ) media for 16 h. Cells were then pre-treated with CHX (Sigma) at 100 µg mL−1 for 1 h prior to adding either vehicle (DMSO) or PROTAC (250 nM). At the indicated timepoints, cells were immediately placed on ice, rinsed with 1X PBS, lysed, and boiled.
MDA-MB-231 cells were plated at 1.5 × 105 cells per well in a six-well dish and allowed to adhere overnight. On the following day, all cells were treated with either vehicle (DMSO) or PROTAC (250 nM). After 24 h, cells were either harvested or washed with 1X PBS, trypsinized, and re-plated onto new six-well dishes in fresh RPMI-1640 (1 × ) media without additional compound. For these washout conditions, cells were collected every 24 h until final harvest (72 h washout).
The starting coordinates for p38δ came from the crystal structure downloaded from Protein Data Bank (PDB) entry . In order to replace the ligand in this structure with foretinib, the PDB entry 5IA4 was used by overlaying its protein backbone with that of 4EYJ, transferring foretinib to the 4EYJ structure and replacing the original ligand. The obtained p38δ–foretinib complex was subject to the Protein Preparation Wizard of Maestro 2016-3 program available from Schrodinger Inc. (New York City, NY), with which the hydrogen atoms were added, the missing side chains were built, and the protonation states were assigned assuming a pH of 7.0 for the ionizable groups. An energy minimization of the complex was performed for 500 steps.
The starting coordinates for VHL came from the PDB entry 4W9H. The starting coordinates for the p38δ:PROTAC:VHL trimer were prepared as follows. (1) The electrostatic surface was generated for p38δ–ligand complex and VHL ligand complex, respectively. (2) The VHL ligand complex was set to have different relative dispositions with respect to the p38δ–ligand complex in a way that the hydrophobic patch of the VHL ligand surface opposed different hydrophobic patches and grooves of the p38δ–ligand surface, thus producing different starting modes in terms of the relative dispositions between p38δ and VHL. (3) For each starting mode, a linker was built to connect foretinib and VHL ligand and form the full PROTAC. And (4) an energy minimization of 500 steps was performed for each starting point of trimer.
OPLS3 force-field was used throughout the calculation steps. The torsional angle parameters were examined with Force Field Builder program and found that the torsional angles between the amide and cyclopropyl group and between the fluorophenyl group and the oxygen ether atom attached to the quinoline group in foretinib needed corrections; and thus the new torsional profiles were generated to match the profiles given by Jaguar quantum mechanical calculations.
Each starting point of the p38δ:PROTAC:VHL trimer was subject to MD simulation. The system setup was done using System Builder of Maestro program, in which the periodic boundary condition was used; the box shape was cubic with absolute size of each side greater than the largest dimension of the system by 5 Å. The explicit waters were added. The system was neutralized using sodium and chloride ions and salted into 0.15 M ionic strength. The MD was done using Desmond Multisim version 3.8.5.19, which was an eight-stage process: (1) task; (2) simulation of 100 picosecond with Brownian dynamics NVT with T at 10 K, small time-steps and restraints on solute heavy atoms; (3) simulation of 12 picosecond, NVT ensemble, T at 10 K, small time-steps and restraints on solute heavy atoms; (4) simulation of 12 picosecond, NPT ensemble, T at 10 K and restraints on solute heavy atoms; (5) solvation of unfilled pockets; (6) simulation of 12 picosecond up to the target temperature of 310 K, NPT ensemble and restraints on solute heavy atoms; (7) simulation of 24 picosecond, NPT ensemble without restraints at T of 310 K; and finally, (8) production run of 100 nanoseconds. During the production run, coordinate frames were saved at every 10 picoseconds. The target pressure was set to 1.01325 bar in the related steps.
The post-simulation analysis after each run was done as follows. The last 20 nanoseconds of trajectory frames were extracted. A clustering analysis using hierarchical clustering method was performed. The distance between any two members (frames) was the root-mean-square deviation of the solute heavy atoms between the members after overlaying them. The cutoff distance was 2 Å. Every frame was used. The structure closest to the centroid of each cluster was written out as the representative structure of that cluster. The representative structure of the largest cluster for each MD simulation was considered as the representative structure of that simulation run. Such a structure can be considered as the most-visited conformation of that run.
The MD simulation was performed using the g2.2 × large instances of Amazon Web Service cloud machines. The Desmond GPU-enabled code was used and mainly run using GPU.
Details of PROTAC chemical syntheses can be found in the Supplementary Information as Supplementary Note 1.
Western blot data in Figs. 2, 6, and Supplementary Figures 2, 3 were quantified by using the band feature in Image Lab (Bio-Rad). The p38 values were normalized to the α-tubulin signal to account for differences in protein load, and PROTAC-treated normalized values were expressed as a % of the vehicle-treated (no PROTAC) value, which itself was defined as 100% p38 protein level. These data were then graphed and fit to a sigmoidal curve by non-linear regression using Prism data-fitting software (Graphpad Prism). The sigmoidal fit established both the full extent of degradation, or Dmax at the bottom asymptote of the curve, as well as the DC50, which is the inflection point at which 50% of the total degradation observed was reached.
Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.
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