Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase

Abstract

PROteolysis-TArgeting Chimeras (PROTACs) are hetero-bifunctional molecules that recruit an E3 ubiquitin ligase to a given substrate protein resulting in its targeted degradation. Many potent PROTACs with specificity for dissimilar targets have been developed; however, the factors governing degradation selectivity within closely-related protein families remain elusive. Here, we generate isoform-selective PROTACs for the p38 MAPK family using a single warhead (foretinib) and recruited E3 ligase (von Hippel-Lindau). Based on their distinct linker attachments and lengths, these two PROTACs differentially recruit VHL, resulting in degradation of p38α or p38δ. We characterize the role of ternary complex formation in driving selectivity, showing that it is necessary, but insufficient, for PROTAC-induced substrate ubiquitination. Lastly, we explore the p38δ:PROTAC:VHL complex to explain the different selectivity profiles of these PROTACs. Our work attributes the selective degradation of two closely-related proteins using the same warhead and E3 ligase to heretofore underappreciated aspects of the ternary complex model.

Fig. 1

Foretinib-based PROTACs recruit VHL using two different linkage vectors resulting in isoform-selective degradation. a Crystal structure (PDB: 4W9H) of VHL-recruiting ligand in the HIF-1α-binding pocket. VHL is rendered as a tan surface with the small molecule ligand visualized in stick representation and colored by atom (gray carbon atoms, red oxygen atoms, blue nitrogen atoms, and yellow sulfur atoms), with the exception of two carbon atoms colored in purple. These purple carbons (and the corresponding purple arrows) represent the structure-guided linker attachment points used in subsequent PROTAC design (see Supplementary Figure 1). b Structures of the two VHL-recruiting ligands used in this study. Top: VHL-recruiting ligand with an amide attachment vector (shown in red). Bottom: VHL-recruiting ligand with a phenyl attachment vector (shown in red). c, d SJFα PROTAC (13-atom linker, amide attachment) selectively degrades p38α, whereas SJFδ PROTAC (10-atom linker, phenyl attachment) selectively degrades p38δ in MDA-MB-231 cells. See Supplementary Figure 1 for the structures of all of the PROTACs screened in this study with their corresponding western blots (Supplementary Figures 2-3) and summary table (Supplementary Table 1). See Supplementary Figure 4 for further investigation into the activity of the lead PROTACs on p38, ERK, and JNK MAPK families

Fig. 2

SJFα and SJFδ degradation of p38 isoforms is rapid, sustained, and proteasome dependent. a MDA-MB-231 cells were either pre-treated or not with the proteasome inhibitor epoxomicin (1 µM) or the neddylation inhibitor MLN4924 (1 µM) for 30 min and subsequently treated with either vehicle (DMSO), SJFα (250 nM), or SJFδ (250 nM) for 6 h. b Quantitative real-time PCR was performed after 24 h of treatment with either vehicle (DMSO), SJFα (250 nM), or SJFδ (250 nM) in MDA-MB-231 cells. The dotted line represents DMSO-treated conditions standardized to a value of 1.0 and mRNA abundance (per treatment group) is represented as a fold-change relative to that value. Data are based on biological duplicates and are normalized to beta-tubulin. Error bars denote s.d. c Cycloheximide (CHX) chase assay. MDA-MB-231 cells were pre-treated with 100 µg mL⁻¹ CHX for 1 h prior to treating for the indicated times with either vehicle (DMSO), SJFα (250 nM), or SJFδ (250 nM). Tubulin-normalized p38α and p38δ abundance values are reported beneath individual lanes. d MDA-MB-231 cells were treated with either vehicle (DMSO), SJFα (250 nM), or SJFδ (250 nM) for 24 h before re-plating onto new plastic in fresh medium for an additional 24, 48, or 72 h (“washout” conditions)

Fig. 3

p38α selectivity is discriminated by ternary complex formation. a Only p38α incubated in the presence of SJFα can be immunoprecipitated with GST-tagged VHL/EloB/EloC (VBC). Immobilized VBC was used as a “bait” to trap purified p38α in the presence of vehicle (DMSO), SJFα, or SJFδ (represented in micromolar concentrations). The rightmost lane represents a 1:25 dilution of initial input protein used in each pull-down. b Proximity-based AlphaLISA assay detects significant p38α:SJFα:VHL ternary complex, but no p38α:SJFδ:VHL ternary complex. His-p38α and GST-VBC were incubated in the presence of increasing concentrations of SJFα and SJFδ and the extent of ternary complex formation was assessed by excitation with incident light with λ = 680 nm and capture of the emission light at λ = 615 nm. Error bars represent the s.d from quadruplicate experiments. c Only SJFα ubiquitinates FLAG-p38α in HeLa cells. HeLa cells co-transfected with HA-Ubiquitin (HA-Ub) and FLAG-p38α were subsequently treated with either vehicle (DMSO), 500 nM SJFα, or 500 nM SJFδ for 1 h. FLAG-immunoprecipitated lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and assessed via western blots detecting HA (Ub). Smears represent ubiquitin-conjugated FLAG-p38α and numerical markers to the left of the western blot refer to kilodalton (kDa) masses. WCL refers to “whole-cell lysate” input

Fig. 4

Differential p38δ:PROTAC:VHL complex affinities result in distinct cellular ubiquitination. a Both SJFα and SJFδ pull-down p38δ in vitro, however, SJFδ maintains ternary complex at lower concentrations. As in Fig. 3a, GST-tagged VBC was used as a “bait” to trap recombinant p38δ in the presence of vehicle (DMSO) or the indicated concentrations of SJFα or SJFδ. The rightmost lane represents a 1:25 dilution of initial input protein used in each pull-down. b SJFδ forms a more stable ternary complex with endogenous p38δ and VHL than does SJFα. GST-VBC was immobilized on glutathione sepharose beads and incubated with MDA-MB-231 whole-cell lysate (WCL) in the presence of vehicle (DMSO) or the indicated concentrations of SJFα or SJFδ. Beads were washed and “trapped” proteins (i.e., those that engage in a ternary complex) were eluted with SDS sample buffer and separated by SDS-PAGE. Samples were assessed by western blot and CUL2 and α-tubulin serve as positive and negative GST-VBC immunoprecipitation controls, respectively. c Only SJFδ ubiquitinates FLAG-p38δ in HeLa cells. As in Fig. 3c, HeLa cells were co-transfected with HA-Ub and FLAG-p38δ and were either treated with vehicle (DMSO), 1 µM SJFα, or 1 µM SJFδ for 2 h. FLAG-immunoprecipitated lysates were separated by SDS-PAGE and assessed via western blots detecting HA (Ub). Smears represent ubiquitin-conjugated FLAG-p38δ and numerical markers to the left of the western blot refer to kilodalton (kDa) masses. See Supplementary Figures 5 and 6 for additional p38δ:PROTAC:VHL characterization

Fig. 5

MD simulations reveal PPI interfaces between p38δ and VHL that vary between SJFδ and SJFα recruitment. a The SJFδ-recruited VHL interacts with p38δ in a different mode than the SJFα-recruited VHL. p38δ, VHL, and either SJFδ or SJFα were docked and a 100-ns MD simulation relaxed the ternary structure. The p38δ:SJFδ:VHL ternary complex is colored in tan and the p38δ:SJFα:VHL ternary complex is colored in dark blue. The p38δ structures from both ternary complex MD simulations were aligned and the resulting divergent VHL structures can be visualized. b SJFδ and SJFα PROTACs deviate at the exit of the p38δ kinase pocket to interact with the p38δ:VHL PPI interface in distinct ways. Using the same p38δ alignment as in (a), the p38δ and VHL structures were hidden such that individual PROTACs could be visualized in stick representation. SJFδ carbons are colored in tan and SJFα carbons are colored in dark blue with oxygen atoms colored in red, nitrogen atoms in blue, sulfur atoms in yellow, and fluorine atoms in pale blue for both PROTACs. SJFδ or SJFα (which are composed of the same foretinib warhead) show profound alignment within the p38δ kinase, but deviate greatly upon exiting the pocket. Linker length differences between SJFδ (10 atoms) and SJFα (13-atoms) reveal the limits in their respective ability to dock along the p38δ:VHL PPI interface. In addition, the orientation of attachment of the VHL-recruiting ligands result in hydroxyproline (Hyp) moieties that differ by ~147° between the two PROTAC structures

Fig. 6

Mutation of MD simulation-identified amino acids on p38δ permits SJFα-dependent degradation by VHL. a MD simulation identifies amino acid residues of p38δ and VHL that stabilize ternary complex in the presence of SJFδ. p38δ is colored by electrostatic potential, from negative (red) to neutral (white) to positive (blue); interacting residue Arg108 of VHL is shown as sticks against the surface of p38δ interacting with key residues Glu49, Glu160. b MD simulation identifies amino acids of p38δ and VHL predicted to disfavor ternary complex in the presence of SJFα. p38δ is colored by electrostatic potential, from negative (red) to neutral (white) to positive (blue); interacting residue Arg108 of VHL is shown as sticks against the surface of p38δ interacting with charged and polar residues K220, T221, respectively. c SJFα potently degrades mutated (K220E/T221E) p38δ, but not wild-type p38δ expressed in HeLa cells. d Quantitation of wild-type or mutant (K220E/T221E) p38δ levels in transfected HeLa cells treated with either SJFα or SJFδ. Levels of p38δ are normalized to α-tubulin and values expressed relative to those from cells treated with DMSO (vehicle). Error bars display the s.d. of duplicate experiments