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. 2024 Oct 15;7(11):3518–3526. doi: 10.1021/acsptsci.4c00421

Targeted Degradation of Receptor-Interacting Protein Kinase 1 to Modulate the Necroptosis Pathway

Hiroyuki Inuzuka †, Chao Qian ‡, Yihang Qi †, Yan Xiong ‡, Chaoyu Wang †, Zhen Wang †, Dingpeng Zhang †, Can Zhang §, Jian Jin ‡,*, Wenyi Wei †,*
PMCID: PMC11555510  PMID: 39539258

Abstract

graphic file with name pt4c00421_0006.jpg

Necroptosis is a highly regulated form of necrotic cell death that plays an essential role in pathogen defense and tissue homeostasis. Abnormal regulation of the necroptotic pathway has been implicated in the pathogenesis of various human diseases, including cancer, inflammatory, and neurodegenerative diseases. Receptor-interacting protein kinase 1 (RIPK1) serves as a crucial regulator of the necroptotic signaling pathway and has been identified as a potential therapeutic target. Mechanistically, RIPK1 serves as both a protein kinase and a scaffolding protein, fulfilling its dual function through a combination of kinase activity-dependent and kinase activity-independent mechanisms. Thus, employing a targeted RIPK1 knockdown strategy is a highly effective means of inhibiting RIPK1 functions. To achieve a targeted RIPK1 knockdown, we generated a RIPK1-PROTAC, MS2031, by connecting the ZB-R-55 RIPK1 binder to the VHL ligand, thereby recruiting the CUL2-RING-VHL (CRL2VHL) E3 ubiquitin ligase complex for targeted degradation of RIPK1 through the 26S proteasome. Notably, MS2031 treatment effectively reduced the abundance of RIPK1 protein in the nanomolar range in various cell lines we examined, including HT-29 and T47D cells, and modulated the necroptosis signaling pathway. These results suggest that MS2031 may hold potential for the treatment of human diseases resulting from aberrant regulation of RIPK1.

Keywords: RIPK1, PROTAC, Necroptosis, Protein degradation, Proteasome, VHL

Necroptosis is a crucial cellular mechanism that maintains tissue homeostasis and regulates pathogen defenses.1−6 Necroptosis is regulated by intricate signaling pathways that are tightly controlled, and receptor-interacting protein kinase 1 (RIPK1) plays a pivotal role in the modulation of the necroptosis signaling pathway. Dysregulation of RIPK1 has been linked to several pathological conditions such as inflammation, neurodegenerative diseases, and cancer.7−16 Therefore, targeting RIPK1 is a promising therapeutic strategy to combat human diseases.17−21 RIPK1 kinase activity is precisely controlled by various types of protein post-translational modifications (PTMs), including phosphorylation and ubiquitination, which play critical roles in modulating RIPK1 function to determine various cellular processes such as necroptosis, apoptosis, and inflammation (Figure 1). RIPK1 kinase activity is suppressed in membrane-associated complex I, where RIPK1 is subjected to M1/K63-linked polyubiquitination, thereby providing scaffolding for the IKKs/NEMO and TAK1/TABs complexes to activate the NF-κB signaling22−25 (Figure 1). In contrast, RIPK1 kinase activity increases through RIPK1 autophosphorylation at S166, leading to the subsequent activation of the necroptosis and RIPK1-dependent apoptosis (RDA) cell death pathways.12,19,26 Considering the key roles of RIPK1 in regulating cell death induction and NF-κB activation, RIPK1 operates as both a protein kinase-dependent and kinase-independent function.17 These findings suggest that inhibition of RIPK1 kinase activity by RIPK1 inhibitors may not be adequate for RIPK1 targeted therapy in certain diseases where RIPK1 kinase-independent function is involved. While the research remains ongoing to discover effective approaches to reduce RIPK1 kinase-independent function, it is imperative to reduce total RIPK1 levels, which may display more sufficient therapeutic promise. This prompted us to develop a RIPK1 degrader that targets both kinase-dependent and kinase-independent RIPK1 functions (Figure 1).

Figure 1.

Figure 1

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Schematic model of the necroptosis regulation pathway(s). RIPK1 modification occurs during the formation of transient signaling complex I triggered by TNFR engagement. TNF-α stimulation recruits complex I components, such as RIPK1, TRADD, TRAF2, cIAP1/2, and LUBAC. cIAP and LUBAC promote K63-linked and M1-linked RIPK1 polyubiquitination, respectively, to provide a scaffold for the TAK1 and IKK complexes, thereby inducing NF-κB nuclear translocation to activate inflammatory gene transcription. In contrast, the deubiquitination of RIPK1 results in the dissociation of RIPK1 from complex I, leading to the activation of RIPK1 and the formation of complex IIa, ultimately initiating apoptosis. When caspase-8 activity is curtailed, RIPK1 assembles into a complex IIb necrosome, resulting in the activation of RIPK3 and MLKL. Activated MLKL translocates to the plasma membrane and oligomerizes to induce membrane rupture, thereby promoting necroptotic cell death. While RIPK1 inhibitors block kinase activity-mediated cell death pathways, RIPK1-PROTAC may modulate both necroptosis and NF-κB-mediated gene transcription.

Results and Discussion

Evaluation of ZB-R-55 as a RIPK1 Ligand

To develop the RIPK1 degrader, we utilized ZB-R-55 as the RIPK1 ligand, due to its unique characteristic of binding to RIPK1 via dual interacting sites, an allosteric site and an ATP binding domain,27 and the cocrystal structure of it in complex with RIPK1 was reported (PDB ID: 7FCZ) (Figure 2A).27 To validate the stable complex formation between ZB-R-55 and RIPK1, we first synthesized the biotinylated version of ZB-R-55, Biotin-ZB-R-55 (Figures 2A and S1), and conducted an in vitro pulldown analysis. To this end, Biotin-ZB-R-55 was incubated with HT-29 cell lysates for 3 h, followed by streptavidin bead pulldown. The captured proteins were eluted using SDS sample buffer. The results of the Western blot analysis demonstrated that Biotin-ZB-R-55 forms a complex with RIPK1 at concentrations above 1 μM, while no detectable binding was observed with RIPK3 or MLKL under this experimental condition (Figure 2B). To determine if the observed complex formation occurred through specific interaction, we further carried out a competition experiment. Biotin-ZB-R-55 was mixed with ZB-R-55 in HT-29 cell lysates and incubated for 3 h. The Biotin-ZB-R-55 was then pulled down with streptavidin beads to isolate the ZB-R-55/RIPK1 complex. The results confirmed that the addition of increased amounts of ZB-R-55 decreased the interaction between Biotin-ZB-R-55 and RIPK1, indicating that ZB-R-55 competed with RIPK1 for Biotin-ZB-R-55 in the lysates (Figure 2C). The findings suggest that ZB-R-55 binds to the RIPK1 protein in a targeted manner, rather than through nonspecific interactions.

Figure 2.

Figure 2

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Evaluation of ZB-R-55 as a RIPK1 ligand. (A) Left, chemical structure of RIPK1 ligand ZB-R-55 and its biotinylated compound Biotin-ZB-R-55; Right, cocrystal structure of ZB-R-55 in complex with RIPK1 (PDB ID: 7FCZ). Red dashed circle indicates the solvent exposure part. (B) ZB-R-55 binds to RIPK1 in a concentration-dependent manner. Immunoblot (IB) analysis of streptavidin pulldown and HT-29 whole-cell lysates (WCL). Biotin-ZB-R-55 at the indicated concentrations was incubated with HT-29 cell lysates for 3 h and then with streptavidin beads for an additional 3 h. The bead-bound proteins were eluted with SDS sample buffer and blotted with the indicated antibodies. (C) An increased amount of free-form ZB-R-55 competes with RIPK1 with Biotin-ZB-R-55. IB analysis of streptavidin pulldown and HT-29 WCL. Biotin-ZB-R-55 (1 μM) was incubated with HT-29 cell lysates with the indicated concentration of free ZB-R-55 for 3 h, and streptavidin pulldown was conducted. The bead-bound proteins were eluted and blotted with the RIPK1 antibody.

Synthesis of RIPK1-PROTAC Compounds

As we confirmed the formation of a stable complex between ZB-R-55 and RIPK1, we subsequently developed bivalent compounds that connect ZB-R-55 with the ligand of E3 ligase, thereby facilitating the recruitment of the protein degradation machinery to the complex. To achieve specific degradation of RIPK1, we opted to utilize the VHL and CRBN ligases, which are widely employed choices for PROTAC-based approaches.28−31 We designed and synthesized 26 compounds, incorporating 26 different linkers of varying lengths and polarities with the aim of optimizing stable ternary complexes (Figure 3A and Figure S2). To identify a functional compound that can facilitate the degradation of RIPK1 in cells, we treated HT-29 cells with the compounds at a concentration of 3 μM for a period of 24 h and subsequently evaluated the level of RIPK1 by Western blot analysis. Our data revealed a marked decrease in the level of RIPK1 protein in cells that were treated with compound 7, a VHL-based RIPK1 PROTAC (Figure 3B,C). To identify the optimal drug concentration for effectively targeting RIPK1 degradation through the use of compound 7, we treated HT-29 cells with the compound at various concentrations. Our data demonstrated that the reduction of RIPK1 protein abundance was achieved at concentrations ranging from 1 to 10 μM (Figure 3D). To increase the efficiency of RIPK1 degradation, we sought to develop a compound with chemical modifications that would enhance the effectiveness of the VHL binder. The compound MS2031 (Figure 3C), with VHL-1-Me32 instead of VHL-1 as the VHL binder (Figure S2), demonstrated effectiveness in inducing targeted degradation of RIPK1 in HT-29 and T47D cells at a concentration ranging from 0.03 to 0.3 μM (Figure 3E,F). This concentration is approximately 10 times lower than that of compound 7 (Figure 3E,F). Moreover, at higher concentration, we observed a reduction in the degradation efficacy of MS2031, possibly due to the hook effect that is frequently observed in PROTAC-mediated target degradation.33 Additionally, we verified that unlike the RIPK1 degrader MS2031, the RIPK1 ligand ZB-R-55 had a minimal impact on RIPK1 protein levels within this concentration range (Figure 3G). To further determine the optimal treatment conditions, we conducted an analysis of the drug treatment time that produced the greatest efficacy in degrading RIPK1. Following the administration of PROTAC, cells were collected at the specified time points for Western blot analysis. Our observations revealed that the highest degradation level was reached around 24 h (Figure 3H,I). Hence, we decided to set the treatment time at 24 h for all subsequent experiments.

Figure 3.

Figure 3

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Synthesis of RIPK1 PROTAC compounds for targeted degradation of RIPK1 in cells. (A) Chemical structures of RIPK1 PROTACs. (B) Screening of RIPK1 PROTACs. IB analysis of WCL derived from HT-29 cells treated with the indicated 26 RIPK1 degraders. (C) Chemical structures of compound 7 and MS2031. (D) Evaluation of the efficiency of compound 7 in inducing RIPK1 downregulation. IB analysis of WCL derived from HT-29 cells treated with compound 7. (E, F) Evaluation of the efficiency of MS2031 in inducing RIPK1 downregulation. IB analysis of WCL derived from HT-29 (E) and T47D (F) cells treated with MS2031. (G) Comparison of changes in RIPK1 protein levels following treatment with the RIPK1 inhibitor ZB-R-55 RIPK1 versus the RIPK1 degrader MS2031. IB analysis of WCL derived from HT-29 cells treated with ZB-R-55 and MS2031. (H, I) Time course of RIPK1 downregulation following treatment with MS2031. IB analysis of WCL derived from HT-29 cells treated with MS2031. Cells were harvested at the indicated time points for IB analysis (H). Quantification of RIPK1 band intensity (I). Mean ± standard deviation (SD), n = 3, *p < 0.05, **p < 0.01, ***p < 0.001.

Evaluation of the Efficacy of MS2031 in Inducing RIPK1 Proteasomal Degradation

Next, to prove that the observed RIPK1 downregulation was mediated by ternary complex formation and subsequent RIPK1 proteasomal degradation, we undertook a series of experiments using pharmacological and genetic approaches. To verify our hypothesis that the degradation of RIPK1 is mediated by the ubiquitin-proteasomal system (UPS), we carried out a cotreatment experiment using HT-29 cells and MS2031, in conjunction with two different UPS inhibitors, MG132 and MLN4924. MG132 is a 26S proteasome inhibitor,34 while MLN4924 selectively impedes the activity of CUL-RING-type E3 ligases, including CUL2VHL through inhibition of the NEDD8-activating enzyme35 (Figure 4A). Our observations revealed a partial inhibition of RIPK1 degradation upon treatment with MG132 or MLN4924 in cells (Figure 4B), indicating that the VHL-mediated RIPK1 degradation process is dependent on the proteasome. To provide additional confirmation of the specificity of RIPK1 proteasomal degradation, we synthesized a negative control compound MS2031N, by incorporating a diastereomer of VHL-1-Me to block the VHL engagement while keeping the same RIPK1 binder and linker (Figure 4C and Figure S2). Notably, MS2031N did not show an ability to degrade RIPK1, which confirms the effect of PROTAC in inducing RIPK1 degradation through CRL2VHL activity in a manner that is dependent on the proteasome (Figure 4D). In addition, we aimed to validate our findings through a genetic approach by creating VHL-knockout cells to evaluate whether VHL depletion results in resistance to MeVHL-induced RIPK1 degradation. RIPK1 degradation mediated by PROTAC was largely abolished by the VHL knockout (Figure 4F,G), thereby providing additional confirmation of the targeted proteasomal degradation of RIPK1 upon treatment with RIPK1-PROTAC.

Figure 4.

Figure 4

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Evaluation of the efficacy of MS2031 in inducing RIPK1 proteasomal degradation in cells. (A, B) Cullin-RING E3 ligase (CRL) and proteasomal inhibition suppress MS2031-mediated RIPK1 downregulation in cells. IB analysis of WCL derived from HT-29 cells treated with MS2031, NEDD8-activating enzyme (NAE) inhibitor MLN4924, and proteasomal inhibitor MG132 (A). p27 is a representative target of CRL and proteasome-dependent degradation. Quantification of RIPK1 band intensity (B). (C) Chemical structure of MS2031N, incapable of binding to VHL. (D, E) MS2031N fails to downregulate RIPK1 in cells. IB analysis of WCL derived from HT-29 cells treated with the indicated compounds (D). Quantification of the RIPK1 band intensity (E). (F, G) VHL knockout suppresses RIPK1 degradation mediated by MS2031. IB analysis of WCL derived from control, and VHL-knockout HeLa cells treated with MS2031 were indicated (F). Quantification of RIPK1 band intensity (G). (B, D) Mean ± SD, n = 3, one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001.

PROTAC-Mediated Inhibition of Necroptotic Signaling and Cell Death

Subsequently, we sought to determine whether treatment with PROTACs impairs the necroptotic signaling pathway. HT-29 cells were pretreated with MS2031 and subsequently stimulated with a combination of TNF-α, SM164, and zVAD-Fmk (TSZ) to activate the necroptotic signaling pathway. We found that treatment with MS2031 inhibited RIPK3 and MLKL phosphorylation (Figure 5A). Given that RIPK3 and MLKL phosphorylation serves as biomarkers of necroptosis, we sought to determine whether TSZ-induced necroptotic cell death could be inhibited by administering MS2031. The cell viability assay demonstrated that treatment with MS2031 at concentrations of 0.1 and 0.3 μM resulted in a substantial increase in cell viability (Figure 5B and Figure S3). We also evaluated the effectiveness of PROTAC treatment under glucose deprivation conditions, which we recently reported to trigger RIPK1 activation and subsequent necroptotic cell death.36 Consistent with this finding, we found that PROTAC treatment protected against cell death induced by glucose starvation in both HT-29 and T47D cell lines (Figure 5C,D and Figure S3). These findings indicate that MS2031 can modulate the necroptotic pathway.

Figure 5.

Figure 5

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MS2031-mediated inhibition of necroptotic signaling and cell death. (A) MS2031 suppresses the activation of RIPK1 downstream from necroptotic signaling. IB analysis of WCL derived from HT-29 cells. After pretreatment with MS2031 for 24 h, cells were stimulated with TNF-α (10 ng/mL), SMC164 (50 nM), or zVAD-Fmk (25 μM) (denoted as TSZ) before harvesting at the indicated time points. (B) MS2031 increases the cell viability of necroptosis-induced HT-29. Cells were pretreated with MS2031 for 24 h and then treated with TNF-α (10 ng/mL), SMC164 (50 nM), or zVAD-Fmk (25 μM) for 30 h. (C) After pretreatment with MS2031 for 24 h, HT-29 cells were deprived of glucose for the indicated time periods before harvesting. IB analysis of WCL derived from HT-29 cells with the indicated treatment. (D) MS2031 increased HT-29 cell viability under glucose-deprived conditions. Cells were pretreated with MS2031 for 24 h and deprived of glucose for the indicated time periods. Cell viability was measured using the CCK-8 assay. (B, D) Mean ± SD, n = 6 or 4, one-way ANOVA, **p < 0.01, ***p < 0.001.

Conclusion

RIPK1 dysregulation has been found to contribute to the onset and progression of a range of autoimmune and inflammatory diseases,21 such as neurodegenerative diseases,8,37 autoinflammatory disease,14,15 cardiovascular disease,38,39 and chronic obstructive pulmonary disease.40 Therefore, targeting RIPK1 is considered a potential therapeutic strategy. Several RIPK1 inhibitors have progressed to Phase I clinical trials and have demonstrated efficacy.41,42 However, despite ongoing testing for active ulcerative colitis43 and psoriasis,44 none have been approved for clinical use. Although still in the developmental stage, PROTACs have the potential to supplement existing drugs. In this study, we aimed to design and synthesize a RIPK1-PROTAC. The PROTAC technology has several potential advantages over traditional inhibitors, including its ability to deliver enhanced efficacy, increased selectivity, and improved safety. (1) Enhanced efficacy: one of the key advantages of PROTAC is that it functions through a ubiquitination-mediated protein knockdown mechanism, which allows for a more comprehensive elimination of RIPK1 protein and function. This approach targets not only the kinase activity of RIPK1,but also its scaffolding action. It is anticipated to be particularly suitable for clinical conditions with higher RIPK1 expression, e.g., Alzheimer’s disease brains with increased RIPK1 signal.45 (2) Increased selectivity: the use of PROTAC-induced RIPK1 knockdown allows for a reduction in the concentration required for effective utilization of the compound, resulting in increased selectivity toward target cells or tissues and enhanced therapeutic potential for RIPK1-PROTAC. (3) Enhanced safety profile: the selectivity of RIPK1-PROTAC is expected to minimize adverse effects on normal tissues by reducing off-target effects, thereby enhancing the overall safety. Furthermore, the degradation approach itself may more clearly reveal dose-dependent effects than the inhibition approach by nature, particularly in the presence of heterogeneous enzymatic activity, like RIPK1. In this study, we developed a compound, MS2031, that induces the proteasomal degradation of RIPK1. The design of our drug incorporated two key elements to improve its knockdown efficiency. (1) RIPK1 binder: we employed ZB-R-55 due to its ability to bind RIPK1 through both an allosteric site and ATP binding domain, thereby increasing its specificity and affinity for RIPK1. (2) VHL binder: we made chemical modifications to the original VHL ligand to create VHL-1-Me,32 which enhanced the efficacy of VHL interaction. Our study aligns with the promising results obtained from the use of R1-ICR-5 and LD4172, recently reported RIPK1 degraders, which demonstrated significant potential for targeted inhibition of RIPK1 function. In both in vitro and in vivo settings, R1-ICR-5 and LD4172 effectively degraded RIPK1 by utilizing a VHL ligand.46,47 Importantly, R1-ICR-5 and LD4172 have shown considerable promise in boosting the effectiveness of the immune checkpoint blockade (ICB) against tumors in mouse models. In contrast, the RIPK1 inhibitor did not affect ICB efficacy.47 These findings highlight the potential of the PROTAC therapeutic approach for clinical applications beyond cancer treatment, particularly for addressing neurodegenerative diseases. To this end, elevated RIPK1 protein levels or activity have been observed in neurodegenerative conditions, such as Alzheimer’s disease (AD)48,49 and amyotrophic lateral sclerosis (ALS)13,50,51 in mice or humans. Moreover, RIPK1 is crucial for triggering neuronal inflammation,13 which is the primary cause of these conditions, making it a key therapeutic target. Notably, administration of the RIPK1 inhibitor Nec-1 alleviated AD and ALS symptoms in mouse models,13,49,50 although suboptimal pharmacodynamics may hinder accurate assessment of the effectiveness of the inhibitor.52 Thus, the use of degraders may address this concern, owing to their advantages, as discussed above. Although our compound requires further substantial improvement due to the mild knockdown efficiency compared to the reported degraders, our findings provide new proof-of-concept of PROTAC-mediated RIPK1 degradation, which warrants further optimization and in vivo efficacy testing. Collectively, the RIPK1-PROTAC concept exhibits the potential for therapeutic applications by targeting a variety of inflammatory conditions associated with neurodegenerative diseases and cancer.

Acknowledgments

This work was supported in part by the NIH Grant R35CA253027 (W.W.). J.J. acknowledges the support by an endowed professorship from the Icahn School of Medicine at Mount Sinai. This work utilized the NMR Spectrometer Systems at Mount Sinai acquired with funding from the NIH SIG Grants 1S10OD025132 and 1S10OD028504.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.4c00421.

  • Figures S1–S3, compound synthesis and experimental details, and NMR and LC-MS spectra (PDF)

Author Contributions

# H.I., C.Q, and Y.Q. contributed equally.

The authors declare the following competing financial interest(s): J.J. is a cofounder and equity shareholder in Cullgen, Inc., a scientific cofounder and scientific advisory board member of Onsero Therapeutics, Inc., and a consultant for Cullgen, Inc., EpiCypher, Inc., Accent Therapeutics, Inc., and Tavotek Biotherapeutics, Inc. The Jin laboratory received research funds from Celgene Corporation, Levo Therapeutics, Inc., Cullgen, Inc., and Cullinan Oncology, Inc.

Supplementary Material

pt4c00421_si_001.pdf (697.5KB, pdf)

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