Assessing Different E3 Ligases for Small Molecule ... - ACS Publications

Articles Cite This: ACS Chem. Biol. 2017, 12, 2570-2578

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Assessing Different E3 Ligases for Small Molecule Induced Protein Ubiquitination and Degradation Philipp Ottis,† Momar Toure,† Philipp M. Cromm,† Eunhwa Ko,† Jeffrey L. Gustafson,† and Craig M. Crews*,†,‡,§ †

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, United States Department of Chemistry, Yale University, New Haven, Connecticut, United States § Department of Pharmacology, Yale University, New Haven, Connecticut, United States ‡

S Supporting Information *

ABSTRACT: Proteolysis targeting chimera (PROTAC) technology, the recruitment of E3 ubiquitin ligases to induce the degradation of a protein target, is rapidly impacting chemical biology, as well as modern drug development. Here, we explore the universality of this approach by evaluating different E3 ubiquitin ligases, engineered in their substrate binding domains to accept a recruiting ligand. Five out of six E3 ligases were found to be amenable to recruitment for target degradation. Taking advantage of the tight spatiotemporal control of inducing ubiquitination on a preselected target in living cells, we focused on two of the engineered E3 ligases, βTRCP and parkin, to unravel their ubiquitination characteristics in comparison with the PROTAC-recruited endogenous E3 ligases VHL and cereblon.

In 2001, our lab published the first report of targeted protein degradation by recruitment of an E3 ubiquitin ligase.8 Later, we developed small molecule proteolysis targeting chimeras (PROTACs) to recruit E3 ligases and specifically degrade target proteins in living cells.9,10 Since then, the PROTAC technology has gained great momentum with many different targets successfully degraded to date.11 However, only a limited number of E3 ubiquitin ligases have been successfully hijacked for use by small molecule PROTAC technology: the Von Hippel−Lindau disease tumor suppressor protein (VHL), the Mouse Double Minute 2 homologue (MDM2), the Cellular Inhibitor of Apoptosis (cIAP), and cereblon.11 Additionally, for the very first PROTAC, the F-box and WD domain containing protein βTrCP was successfully recruited for target degradation using a phosphopeptide-based PROTAC injected into cells.8,12 Recent studies have both highlighted the great contribution of physical properties of the utilized E3 ligase to PROTAC-mediated targetbinding13 and demonstrated the sometimes drastic changes in PROTAC efficacy upon switching the recruited ligase.14 Given the existence of more than 600 E3 ubiquitin ligases identified in the human proteome15 and the great success experienced with the four ligases recruited to date, we explored whether the PROTAC approach can be more universally expanded to the larger body of E3 ligases.

T

he ubiquitin−proteasome system (UPS) is part of the cellular system regulating protein quality control and general protein homeostasis. In the UPS, ubiquitin, attached to a substrate protein as a multisubunit polyubiquitin chain, serves as the signal that targets the substrate protein for degradation. A cascade of three enzymes, a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3), catalyze the attachment of single ubiquitin moieties onto a substrate lysine residue via the formation of an iso-peptide bond. This mechanism can either transfer ubiquitin onto the substrate itself or it can transfer additional ubiquitin moieties onto one of the seven lysinesor in certain instances the N-terminusof an already transferred ubiquitin to form a polyubiquitin chain.1 Substrate fates and degradation efficiencies may vary depending on the intramolecular linkages used to build the polyubiquitin chain. While the exact cellular roles of most of the linkage-types remain elusive, Lys48 (K48) linkages are well established as formidable signals leading to proteasomal degradation.1−3 Also, K11 linkages are recognized as an effective proteasomal signal, and K11/K48 branched ubiquitin structures are believed to act particularly well as a recognition motif for the proteasome.2,4−6 The substrate specificity for the ubiquitin transfer lies with the E3 ubiquitin ligases and depends on their respective substrate binding domains. Whether the ubiquitin is transferred onto the substrate by the E3 ligase or by the E2 ubiquitin conjugating enzyme depends on the class of E3. There are three classes of E3 ligases: U-box, RING, and HECT. Only HECT and a special subtype of RING ligases, the RING-in-between-RING (RBR) ligases, are capable of catalyzing a ubiquitin transfer themselves.7 © 2017 American Chemical Society

Received: June 13, 2017 Accepted: August 2, 2017 Published: August 2, 2017 2570

DOI: 10.1021/acschembio.7b00485 ACS Chem. Biol. 2017, 12, 2570−2578

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Figure 1. Construction of engineered E3 ubiquitin ligases. (A) List of HaloTag fusion constructs with indication of N-terminal or C-terminal HaloTag (HT7) fusion and denomination of the truncation sites (aa: amino acids). (B) Scheme of the engineered E3 ligase−reporter substrate system. The E3 ligase has been fused to HT7. The chloroalkane of the PROTAC (black) binds covalently to HT7, and its ligand moiety recruits the fluorescent reporter protein, resulting in polyubiquitinationand eventually degradationof the reporter. (C) Graphical representation of the domain structures of the six E3 ligases utilized. Truncation points are highlighted by bold lines and bold residue numbers.

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specificity pocket (F36V) to accept an orthogonal “bump”− ligand−chloroalkane (Figure 2, compound 1). This permitted both high target affinity (IC50 = 1.8 nM) and exceptional targeting specificity over endogenous, wild-type FKBP12.18,19 Design of Engineered Recruitable E3 Ligases. Wellcharacterized E3 ligases were chosen to represent the respective, different ubiquitin ligase classes and subclasses: For the U-box class of E3 ubiquitin ligases, the ligase CHIP was truncated Nterminally by 22 and by 129 amino acids to form two distinct fusions to the C-terminus of HT7 (Figure 1A and C). HECT E3 ligases were represented by NEDD4L, whose HECT-domain was fused C-terminally to HT7. For the diverse class of RING E3 ligases, βTrCP, a substrate recognition subunit of a SCF (SKP1CUL1-F-box protein), was engineered by replacing its WD repeat domains with an N-terminally fused HT7 domain. The multimeric RING E3 ligase SIAH1 was fused in full length (lacking the first four amino acids of the N-terminal disordered region) to the N-terminus of HT7. For the monomeric RING E3 ligase MARCH5, the HaloTag was attached to the C-terminus of the full-length protein. The MARCH5-HT7 localizes to the outer mitochondrial membrane, where both the HT7 and RING domain face the cytosol. Finally, to represent the RING-inbetween-RING (RBR) ligases, parkin was truncated N-

RESULTS AND DISCUSSION Rationale. In order to begin evaluating the universality of E3 ubiquitin ligase recruitment for targeted protein degradation, we created a small panel of engineered E3 ligases representing the three major classes: U-box, HECT, and RING. To make these ligases amenable to small-molecule-induced recruitment in the absence of established binding ligands, we fused six different E3 ligases to HaloTag 7 (HT7), thereby replacing the ligases’ native substrate binding domains (Figure 1). HT7 is an engineered bacterial dehalogenase developed by Promega,16,17 capable of forming a covalent bond with a chloroalkane; therefore, HT7 can present any small molecule on its surface, provided the molecule incorporates a chloroalkane tail. This system was utilized to present substrate-recruiting ligands on the surface of the E3 ligase-HT7 fusion proteins (Figure 1B). As our standardized reporter substrate, we fused an enhanced green fluorescent protein (EGFP) to a F36V mutant of human FKBP12. This model substrate, from here on referred to as EGFP-FKBP, provided the advantage of a fluorescent reporter system, allowing for convenient, flow-cytometry based screening of substrate degradation. The small, 12 kDa FKBP12 served as a ligand-binding domain. We adopted a system developed by Ariad Gene Therapeutics, wherein a mutant FKBP12 presents a 2571


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spanning 3 orders of magnitude. These degradation levels, also observed to a lesser extent for HT7-NEDD4L (30%) and SIAH1-HT7 (40%), are especially remarkable considering the rapid resynthesis of the CMV-promoter-driven target. For the MARCH5 and NEDD4L constructs, PROTAC 4 was found to be the only small molecule that was able to induce target degradation. The SIAH1 fusion protein, additionally, seemed to induce moderate target degradation of 25% when recruited via PROTAC 5 (Supporting Information Figure S1C). PROTAC 6 proved to be highly potent (DC50 < 1 nM) for the induction of target degradation via βTrCP-HT7; also, the one atom longer PROTAC 5 mediated substantial degradation (50%) via the βTrCP construct. PROTACs 2 and 3, however, failed to induce any degradation of EGFP-FKBP. With PROTACs 7 and 8 also only showing mild effects, it appears that compounds with longer linkers are favored for an effective degradation via βTrCP-HT7 (Figure 3B). HT7-parkin, on the other hand, showed substantial and steady target degradation of around 50% for all compounds tested. PROTACs 4−7 exhibited maximal degradation at 100 nM, while the shortest chloroalkanes, 2 and 3, appeared to have their maximum degradation efficiency at 10 nM (Figure 3A and C). When PROTACs 7 and 8 are compared for both HT7parkin and βTrCP-HT7 (Figure 3B and C), a drop in potency becomes apparent. Most likely, this results from the less wellbalanced hydrophobicity of the linker of PROTAC 8, rather than from the additional atom in length. The overall broad compound susceptibility of HT7-parkin suggests that functional dimers readily form between the target protein and PROTAC-HT7 for all compounds. Furthermore, the capability of HT7-parkin to accommodate any utilized linker length and composition to induce target degradation (Figure 3A and C) hints at a great flexibility of the parkin E3 ligase construct. Recent findings reported by the Ciulli lab13 suggest that a PROTAC-mediated interaction between two proteins is predominantly driven by the free energy liberated by the interaction of the proteins themselves. Taking this into account, as well as the unchanging nature of both the target and the targetbinding domain of the various engineered E3 ligases, any difference in degradation efficacy can presumably be attributed to the subtle changes in positioning of the ligase relative to its target. Comparison of Ubiquitination Characteristics. Exploring the induced in-cell target ubiquitination underlying the observed degradations, PROTAC treatment time courses demonstrated a notable increase in polyubiquitination of EGFP-FKBP in response to treatment with 100 nM of PROTAC 4 for HT7-parkin (Figure 4B) and βTrCP-HT7 (Figure 4A) within the first hourand to a lesser extent for SIAH1-HT7 within 2 h (Figure 4A). Substantial induction of ubiquitination after 1 h was also observed for the endogenous VHL E3 ligase, recruited by PROTAC 10. In the absence of proteasome inhibitors, ubiquitination by parkin and SIAH1 constructs appears to reach its maximum after 2 h. Ubiquitination via βTrCP-HT7 was still increasing after the 3 h investigated (Figure 4A). VHL-mediated modification, on the other hand, appeared to reach a steady state after 1 h, which was maintained at least over the following 2 h (Figure 4B). Despite the reporter degradation observed for the MARCH5 and NEDD4L fusion proteins, a notable increase in target ubiquitination could not be observed within 8 h (data not shown). In order to further characterize the polyubiquitin chains formed by our two most efficient E3 ligase constructs, βTrCPHT7 and HT7-parkin, we overexpressed select lysine mutants of ubiquitin in our cell system and immunoblotted for reductions in

Figure 2. Ligands and PROTACs. (1) FKBP−ligand; (2−8) chloroalkane PROTACs with FKBP−ligand; (9) PROTAC with cereblon−ligand pomalidomide; (10) PROTAC with VHL−ligand; (11 and 12) chloroalkane PROTACs with kinase−ligand dasatinib.

terminally and fused to the C-terminus of HT7. The N-terminal truncations of NEDD4L and parkin also relieved them from their autoinhibited state and allowed for constitutive activity of these two constructs.20,21 Following transfection of each of the various E3-HT7 constructs into Flp-In TREx 293 cells (Invitrogen)22 already stably and constitutively expressing EGFP-FKBP, the cells were selected for stable integration of the E3-construct into the tetracycline-responsive Flp-In site. Flow Cytometry Screening. Flp-In TREx 293 cells stably expressing both a constitutive EGFP-FKBP target substrate and a tetracycline-inducible E3-HT7 construct were screened via flow cytometry against various FKBP−ligand chloroalkane PROTACS (Figure 2). A loss of fluorescence in response to the recruitment of the E3-HT7 to the substrate EGFP-FKBP could be detected for all E3 constructs tested (Figure 3A) except CHIP (Supporting Information Figure S1D and E). Since successful fusion of the CHIP U-box domain to a novel substrate recognition domain for targeted protein degradation has been reported previously,23,24 it can be assumed that the positioning of HT7 was not optimal in our constructs. However, HaloTagconstructs of NEDD4L, βTrCP, SIAH1, MARCH5, and parkin could all be recruited to significantly induce degradation of EGFP-FKBP at nanomolar concentrations of PROTAC 4 (Figure 3A). While the mitochondria-anchored MARCH5HT7 construct showed only moderate degradation (25%) at 100 nM of PROTAC 4, HT7-parkin showed substantially greater degradation (70%) at the same concentration. Moreover, βTrCP-HT7 demonstrated the highest degradation efficiency, with a Dmax of 98% at 10 nM and a broad efficacy window 2572


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Figure 3. Flow cytometry analyses. Loss of fluorescence is indicative of successful ubiquitination and degradation of the fluorescent reporter substrate EGFP-FKBP. (A) Overview of the five working E3 ligase constructs, recruited to the reporter substrate by PROTAC 4. Error bars depict the SD of three biological replicates with ≥10 000 gated cells, each. Levels of significance as compared to DMSO: *p < 0.05; **p < 0.01; ***p < 0.001. (B) Screening result for βTrCP-HT7 screened against a panel of PROTACs (2−8). (C) Screening result for HT7-parkin screened against a panel of PROTACs (2−8). For the screening experiments of B and C, each bar depicts the geometric mean fluorescence of ≥10 000 gated cells normalized to a DMSO vehicle only control.

EGFP-FKBP polyubiquitination following PROTAC treatment. Here, βTrCP-HT7 demonstrated susceptibility to the overexpression of a K48R ubiquitin mutant, as apparent by the reduction in polyubiquitylated EGFP-FKBP (Figure 4C). This suggests K48 to be the predominant linkage type in βTrCP-HT7 induced polyubiquitination. For HT7-parkin, however, judged by the results of this assay, K27 and K6 linkages prevail. Both these linkage types have been previously reported for parkin.2,25−29 For comparison, we subjected tryptic digests of immunoprecipitated EGFP-FKBP from cells treated with PROTAC 4, or free FKBP−ligand 1 (mock), to proteomic analysis. The analysis focused on ubiquitin-derived peptides with a characteristic −Lysε-Gly-Gly− (diGly) motif at a missed trypsin cleavage site, indicative of intramolecular isopeptide bond formation between individual ubiquitin subunits in a polyubiquitin chain.30 Proteomics results confirmed K48 to be the predominant ubiquitin linkage induced by the recruitment of βTrCP-HT7, as suggested by the drastic increase of uncleaved ubiquitin peptides with a GlyGly-modifed Lys48 (Table 1). Recruitment of the

parkin construct, on the other hand, substantiated the anticipated induction of K6 linkages, as suggested by the immunoblot approach (Figure 4D), but failed to demonstrate the formation of K27 linked polyubiquitin. Instead, the data suggest a strong induction of K11 and K48 linked ubiquitin chains (Table 1). These two linkage types have also been previously described in connection with parkin.26 Generally, the overexpression of ubiquitin mutants followed by immunoblot detection bears substantially greater variability and, therefore, is more likely to result in false positive hits, as compared to the proteomics approach. Nevertheless, differences in the results for K27-linked ubiquitin could be explained by ubiquitin chains with mixed linkages and K27 as a linkage present at the proximal end of the chains. Hence, a disturbance of this linkage would reflect superproportionally in the immunoblot assay, as it would disrupt the whole chain assembly, while it might go undetected in the proteomics approach. Another conceivable explanation is that the catalytic E3 ligase parkin might not optimally recognize ubiquitin K27R mutants. As for the K11 and K48 linkages, undetected in the immunoblot assay, it is likely that these two 2573


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Figure 4. Ubiquitination assays. Panels A and B show time course experiments of induced ubiquitination over 3 h. Percent values below the immunoblots are normalized to mock (1) treatment and show the quantification of total ubiquitin signal per lane. Panel C for βTrCP-HT7 and panel D for HT7-parkin both show immunoblots of ubiquitin-linkage assays, utilizing overexpression of HA-tagged, wild-type ubiquitin (wt), or ubiquitin lysineto-arginine mutants, deficient in single, specific lysines, or lacking all lysines (K0). For C and D, percent values below the immunoblots are normalized to PROTAC 4 treatment with overexpressed wild-type ubiquitin and show the quantification of polyubiquitin signal per lane.

Table 1. Results of Ubiquitin Linkage Proteomics EGFP-FKBP cell line

treatment

peptide score

peptide expectation

charge

matching peptides

position range

βTRCP-HT7 βTRCP-HT7 βTRCP-HT7 βTRCP-HT7

mock (1) 4 4 9

10.25 43.63 45.85 52.56

9.40 × 10−02 4.30 × 10−05 2.60 × 10−05 5.50 × 10−06

+2 +3 +2 +3

6 7 10 18

43−54 43−54 43−54 7−27

βTRCP-HT7 βTRCP-HT7 βTRCP-HT7

9 9 10

23.86 56.61 18.05

4.10 × 10−03 2.20 × 10−06 1.60 × 10−02

+3 +2 +3

5 10 10

43−54 43−54 7−27

βTRCP-HT7 βTRCP-HT7 HT7-parkin HT7-parkin HT7-parkin

10 10 mock (1) 4 4

30.31 46.74 12.53 39.26 27.99

9.30 × 10−04 2.10 × 10−05 5.60 × 10−02 1.20 × 10−04 1.60 × 10−03

+3 +2 +2 +2 +3

7 9 4 6 14

43−54 43−54 43−54 1−11 7−27

HT7-parkin HT7-parkin

4 4

33.85 45.59

4.10 × 10−04 2.80 × 10−05

+3 +2

8 9

43−54 43−54

linkages make up branched ubiquitin chains, and in the absence of one of the lysines, the parkin construct creates unbranched chains, indistinguishable via immunoblot from branched chains. Notably, ubiquitin peptides between amino acids 30 and 42 and encompassing K33 could not be detected in any of the proteomics samples. Moreover, EGFP-FKBP ubiquitinated by the recruitment of the endogenous E3 ligases cereblon and VHL via treatment with FKBP-PROTACs 9 and 10, respectively, was subjected to proteomic analysis. Results suggest that both VHL and cereblon induce K11 and K48 linked ubiquitin chains (Table 1). This is

peptide sequence R.LIFAGKQLEDGR.T + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) K.TLTGKTITLEVEPSDTIENVK.A + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) K.TLTGKTITLEVEPSDTIENVK.A + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) -.MQIFVKTLTGK.T + GlyGly (K) K.TLTGKTITLEVEPSDTIENVK.A + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K) R.LIFAGKQLEDGR.T + GlyGly (K)

modif. Lys 48 48 48 11 48 48 11 48 48 48 6 11 48 48

consistent with the high efficiency observed for these two E3 ligases with which they induce proteasomal degradation of their targets. Using the same proteomics data sets, we then sought to identify potential differences in target ubiquitination sites characteristic for each of the E3 ligases recruited. Filtering the data sets for target-derived peptides with diGly remnants enabled mapping of the target lysines preferentially modified by the different ubiquitin ligases (Figure 5). Overall, EGFP-FKBP possesses 28 lysine residues20 from EGFP and eight from FKBP. Looking at PROTAC-independent (“background”) 2574


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Figure 5. Ubiquitination sites on EGFP. The panels show two views on EGFP with all lysine residues depicted in stick conformation. (A) Mock (1)treated steady state turnover background ubiquitination. (B) βTrCP-HT7 recruited by PROTAC 4. (C) HT7-parkin recruited by PROTAC 4. (D) Endogenous VHL recruited by PROTAC 10. (E) Endogenous cereblon recruited by PROTAC 9. Unmodified lysines are colored in blue. Background modified lysine residues are highlighted in pink, E3-specific modified residues in red, and lysines detected as modified upon E3 recruitment and in mock treatment are colored in orange. Structures based on PDB file 4EUL.31

Figure 6. Quantification of the effects of dasatinib PROTAC treatment on protein levels of various kinases. Values have been normalized to DMSO and are corrected for the tubulin loading control signal. Graphs A and B show levels of ABL1 and SRC kinase in PROTAC-treated βTrCP-HT7 expressing cells. Graphs C through F depict protein levels of the kinases ABL1, SRC, YES, and EphA2 in treated HT7-parkin expressing cells. Error bars depict standard deviation from the mean of two experiments, with the individual values indicated by black spheres.

substrate turnover in mock (1) treated cells, we identified seven lysine residues with a diGly motif, with all but one on the EGFP

unit and facing opposite sides of the protein’s cylindrical structure (Figure 5A). 2575


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change the efficacy of degradation for a given target.14,34 Notable is that the parkin construct, which demonstrated high flexibility and a rather broad degradation pattern on the model substrate EGFP-FKBP, also showed the broadest efficacy for the endogenous targets. Conclusion. In summary, from six E3 ubiquitin ligases tested, we were able to recruit five for targeted protein degradation using small molecules. In another approach, the sixth ligase, CHIP, has been demonstrated to be amenable to recruitment for targeted degradation as well.23,24 These results suggest a broad functionality of the arising PROTAC technology, limited near exclusively by the availability of suitable ligands to the respective, native E3 ligases. It should be noted, however, that some E3 ligases, such as MARCH5 and NEDD4L, showed limitations in degradation efficiency toward the reporter substrate, which seemed difficult to overcome by adjustments of the PROTAC linker length and composition. While one of the reasons for the limitations observed for MARCH5 can be assumed to result from this transmembrane-E3’s limited mobility, the reasons for the low efficacy of HT7-NEDD4L remains elusive. The βTrCP construct demonstrated potent and efficient degradation with specific PROTACs, whereas the parkin fusion protein showed moderate efficiency with a broad responsiveness to all PROTACs. For new PROTAC approaches, such differences in degradation characteristics should be taken more and more into account upon the anticipated expansion of the pool of recruitable E3 ligases. Furthermore, the activity of some E3 ligases is more tightly controlled than others, with HECT and RBR ligases often exhibiting autoinhibition.20,35 While tight regulation might complicate therapeutic PROTAC-approaches utilizing these ligases, such regulatory mechanisms might allow for the development of conditional protein knockdown strategies. As demonstrated, the approach described here can also be used to study characteristics of ubiquitin transfer. The tight spatiotemporal control of the ubiquitin chain formation on a predefined target allows for investigations into differential ubiquitination. Using an artificial substrate, such as EGFP, not involved in any conserved mechanisms present in mammalian cells, could allow for the study of ubiquitin chains in a stand-alone context since cellular responses to the recognition of the ubiquitylated substrate sites are not established. Finally, utilizing a ligand-binding tag protein, such as the FKBP12 F36V mutant used in this study, theoretically renders any such fusion protein amenable to small molecule induced targeted degradation. Thereby, as a biological tool, this strategy vastly expands the pool of proteins currently accessible by small molecule ligands. Taken together, our work further highlights the great potential of the PROTAC-technologyin therapeutic applications as well as in the chemical biology toolset.

Upon recruiting endogenous VHL via PROTAC 10, only one EGFP background lysine (Lys158) was found to be ubiquitylated (Figure 5D). Endogenous cereblon recruited by treatment with PROTAC 9 appeared to modify the same lysine residue (Lys158) plus two others, Lys101 (Figure 5E) and the EGFP C-terminal Lys238. For βTrCP-HT7 mediated ubiquitination, two of the four mapped modified lysines overlap with the turnover background (Lys45/Lys156). Two novel sites, specific to the induced modification by the engineered βTrCP, are Lys126 and Lys166. Interestingly, a modification of Lys107, situated right between Lys126 and Lys166, could not be detected (Figure 5B). Following HT7-parkin recruitment, seven diGly remnants could be identified. Two overlapping with background (Lys41/Lys158) and five lysines, all mapping to one side of EGFP and facing the same direction as Lys158: Lys101/Lys107/ Lys126/Lys162/Lys166 (Figure 5C). Notably, none of the recruited ligases (fusion or endogenous) appeared to ubiquitinate the small FKBP subunit of the target fusion protein but exclusively modified the larger EGFP. While the proteomics data presented here are qualitative, rather than quantitative, they suggest differences in the ubiquitination characteristics of the four E3 ligases tested. Such differences in E3 properties should be taken into account in future approaches of advancing the field of PROTAC-mediated targeted protein degradation. Degradation of Endogenous Targets. To evaluate whether βTrCP-HT7 and HT7-parkin could also be recruited to induce degradation of endogenous targets and to uncover potential differences in substrate preference of these two constructs, we fused the promiscuous kinase inhibitor dasatinib32 via two different linkers to a chloroalkane, resulting in PROTACs 11 and 12 (Figure 2). TREx HEK293 cells, expressing either the parkin or the βTrCP construct, were treated with various concentrations of PROTACs 11 and 12, respectively, and their lysates were subjected to immunoblotting. Probing for four different kinases that bind dasatinib with picomolar affinity32,33ABL1, SRC, YES, and ephrin type-A receptor 2 (EPHA2)we detected degradation patterns specific for the respective E3/PROTAC combinations. Due to toxicity of dasatinib and its derivatives at concentations of 1 μM and higher, treatments focused on concentrations of 100 nM and lower. βTrCP-HT7 appeared to induce target degradation only when recruited by the shorter chloroalkane PROTAC 11. In this combination, we were able to detect a reduction of ABL1 and SRC by about 50% each when treated with 3.3 and 33 nM of PROTAC 11, respectively (Figure 6A,B). At 100 nM, SRC was found to be knocked down by 87%. No βTrCP-HT7-induced degradation was detected for YES or EPHA2, nor for any probed dasatinib targets when treated with PROTAC 12 (Figure S4). Recruitment of HT7-parkin, on the other hand, caused about 50% degradation of ABL1 at 10 nM PROTAC 12 (Figure 6C). None of the other kinases appeared to be affected by the treatment with this compound (Supporting Information Figure S3). Dasatinib-choloroalkane PROTAC 11 did not induce degradation of ABL1 in HT7-parkin cells. However, 50% reduction in SRC and EPHA2 levels and 30% in YES levels could be achieved with PROTAC 11 at high potency (≤1 nM; Figure 6D−F). All in all, these findings show that our engineered E3 ubiquitin ligases are capable of inducing degradation of endogenous targets and do so in an E3-specific manner. This corroborates earlier findings by our lab that changes in linker length of the recruiting small molecules as well as the identity of the recruited E3 ligase

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MATERIAL AND METHODS

Please see the Supporting Information for experimental details.

ASSOCIATED CONTENT

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.7b00485. 2576


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Supporting Figures S1−S3 and detailed descriptions of the experimental material and methods applied in this study (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Philipp M. Cromm: 0000-0001-5291-3379 Craig M. Crews: 0000-0002-8456-2005 Notes

The authors declare the following competing financial interest(s): C.M.C. is a shareholder and consultant to Arvinas, LLC, which supports research in his lab.

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ACKNOWLEDGMENTS We thank T.T. Lam from the MS & Proteomics Resource of W.M. Keck Foundation Biotechnology Resource Laboratory at Yale University (New Haven, CT) for obtaining the highresolution mass spectra on their NIH-funded Shared Instrumentation Grant (ODOD018034). P.M.C. is thankful to the Alexander von Humboldt Foundation for a Feodor Lynen research fellowship. C.M.C. gratefully acknowledges the U.S. National Institutes of Health for their support (R35CA197589).

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