Author Correction: Extracellular vesicle-based targeted protein degradation platform for multiple extracellular proteins

A heterobifunctional molecule recruits cereblon to an RNA scaffold and activates its PROTAC function

Targeted protein degradation (TPD) is an emerging therapeutic approach that enables the degradation of undruggable targets via intracellular degradation systems. Extracellular vesicles (EVs) have shown potential to act as next-generation TPD platforms. However, the molecular mechanism underlying their degradation remains unknown, which restricts their application in TPD. In this study, we found that the autophagy-mediated lysosomal pathway was the major route by which EVs were degraded. MAP1LC3B recognized the LIR motifs of SQSTM1 and induced the degradation of EVs in the autophagy pathway. Based on the EV degradation mode, we developed an EV-based targeted protein degradation platform (EVTPD) using EVs loaded with the LIR motif of SQSTM1 as a degradation signal. Additionally, target protein-binding domains were integrated into the EVTPD to capture target proteins. EVTPD selectively degraded extracellular proteins without requiring receptors on target cells. Furthermore, dual-targeting EVTPD effectively degraded both TNF-α and IL-1β and exhibited potent anti-inflammatory effects in rat and goat models of intervertebral disc degeneration. This study has established a modular EV-based TPD strategy with multi-targeting potential.

BioPROTACs are heterobifunctional proteins designed for targeted protein degradation (TPD). They are useful not only for probing protein functions but also offer a therapeutic avenue for modulating disease-related proteins. To extend the use of TPD beyond just protein attenuation, we introduce a synthetic framework for logic-gated, switchable TPD to achieve conditional control of protein content. By exploiting both the cleavage and ligation functionalities of Sortase A (SrtA), we present a new strategy utilizing SrtA as the control input to direct bioPROTAC activity for switchable TPD. Furthermore, by layering the SrtA input with protease gating, conditional degradation phenotypes can be readily adapted with minimal modifications to the design. This Logic-gated AdPROM deploying SrtA-mediated Element Recombination (LASER) platform allows us to expand the possible protein degradation outcomes in mammalian cells using Boolean logic operations depending on the input combinations. The flexibility to modulate the level of multiple native intracellular proteins can potentially lead to applications from therapy to diagnostics and biotechnology.

Bruton’s tyrosine kinase (BTK) is a target for multiple generations of covalent (irreversible) and non-covalent (reversible) inhibitors due to its critical role in the proliferation and survival of B-cell malignancies. Similar to drug resistance mechanisms in other cancers, resistance to covalent BTK inhibitors (BTKi) in chronic lymphocytic leukemia (CLL), such as ibrutinib, arise through on-target BTK mutations at the BTK C481 residue (the binding site of ibrutinib), which allow escape from BTK inhibition. Non-covalent BTKi represent a new avenue to overcome resistance to the clinically approved covalent BTKi; however, we have recently discovered mechanisms of resistance to non-covalent BTK inhibition in patients with CLL (Wang, Xi, Thompson, Montoya et al NEJM 2022). We used bulk and single cell genomic analyses that identified a series of acquired BTK mutations in a cohort of CLL patients that relapsed on the phase I/II clinical trial of pirtobrutinib. We discovered mutations (BTK V416L, A428D, M437R, T474I, L528W) that occur at critical residues within the catalytic kinase domain of BTK and conferred resistance to both non-covalent and covalent BTKis. Based on structural models of BTK, we observed that these mutations physically impede drug binding, and disrupt the normal kinase activity of BTK but can, upon B-cell receptor stimulation, sustain AKT, ERK, and NF𝜅B signaling as well intracellular Ca2+ release in the presence of pirtobrutinib. In addition, CITE-seq analyses of 53,722 cells from this cohort identified pre-existing leukemic and immune cell states associated with development of resistance in patients. These findings identify novel mechanisms to engender clinical resistance to non-covalent BTKis, including contribution of the immune microenvironment to response to BTK inhibition. Together our data suggest that mutations at the BTK kinase domain may alter conformation of BTK’s non-kinase protein interaction domains thus allowing BTK to be used as a scaffold for other signaling molecules to phosphorylate phospholipase C gamma 2 (PLC𝛾2), the direct downstream target of BTK. We have performed mass spectrometry phosphoproteomics to evaluate potential signaling molecules that are active and could bypass BTK in catalytically inactive BTK mutant cells. Given the above, we decided to target BTK’s non-kinase function by investigating targeted protein degraders of BTK using BTK-degrading protein targeting chimeras (PROTACS). The immediate advantages of using protein degraders are (1) Binding may occur at any site of the target protein, and (2) PROTACS can act catalytically to bind to and degrade multiple target proteins. Our studies have shown that several of the BTK mutants that engendered clinical resistance to covalent and non-covalent BTKi are sensitive to these degraders, elucidating an exciting alternative therapeutic approach for patients who become resistant to BTKi therapies. We are continuing to test the efficacy of BTK PROTAC degraders in overcoming acquired resistance mechanisms in CLL as well as other B-cell lymphomas. Citation Format: Skye Montoya, Eric Wang, Jessie Bourcier, Sana Chaudhry, Tulasigeri Totiger, Alejandro Pardo, Gabriel Pardo, Maurizio Affer, Jacob Jahn, Anthony Mato, Omar Abdel-Wahab, Justin Taylor. Targeted protein degradation as a strategy to overcome non-covalent BTK inhibitor resistance in lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A04.

TANK-binding kinase 1 (TBK1) is essential for interferon beta (IFN-β) production and innate antiviral immunity. However, other, additional functions of TBK1 have remained elusive. Here, we showed that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. Further evidence showed that TBK1 could also be self-ubiquitylated in vivo Importantly, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Mechanistically, TBK1 phosphorylated multiple picornavirus VP3 proteins at serine residues and ubiquitinated them via K63-linked ubiquitination at lysine residues. In addition, the C426 and C605 residues of TBK1 were not essential for TBK1 innate immunity activity; however, these residues were required for degradation of multiple picornavirus VP3 proteins and for its E3 ubiquitin ligase activity. Hence, our findings identified a novel role of TBK1 in regulating the virus life cycle and provided new insights into the molecular mechanisms of TBK1-mediated antiviral response.IMPORTANCE TBK1 is an important adaptor protein required for innate immune response to viruses, but its other functions were unknown. In this study, we found that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. In addition, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Our report provides evidence that TBK1 plays a role in viral protein degradation.

Targeted protein degradation has generated excitement in chemical biology and drug discovery throughout academia and industry. By hijacking the machinery responsible for protein degradation via the ubiquitin proteasome system (UPS), various cellular targets have been selectively degraded. However, since the tools used, often termed PROteolysis TArgeting Chimeras (PROTACs), hijack the intracellular quality control machinery, this technology can only access targets within the cell. Extracellular targets such as growth factors, cytokines, and chemokines bind to cell surface receptors, often initiating aberrant signaling in multiple diseases such as cancer and inflammation. However, efforts to develop small molecule inhibitors for these extracellular target proteins have been challenging. Herein, we developed a proof-of-concept approach to evaluate if extracellular proteins can be internalized and degraded via the receptor-mediated endolysosomal pathway. Using a heterodimeric molecule, termed “ENDosome TArgeting Chimera” (ENDTAC), internalization and degradation of an extracellular recombinant eGFP-HT7 fusion protein was achieved by hijacking the decoy GPCR receptor, CXCR7. This proof-of-concept study suggests that using ENDTACs to co-opt the endosomal–lysosomal degradation pathway, in contrast to PROTACs using the UPS, may provide an avenue for degrading extracellular targets such as cytokines. Overall, the technology described herein provides a novel expansion to the field of targeted protein degradation.

The ATP-binding cassette (ABC) transporter ABCB6 is a mitochondrial porphyrin transporter that activates porphyrin biosynthesis. ABCB6 lacks a canonical mitochondrial targeting sequence but reportedly traffics to other cellular compartments such as the plasma membrane. How ABCB6 reaches these destinations is unknown. In this study, we show that endogenous ABCB6 is glycosylated in multiple cell types, indicating trafficking through the endoplasmic reticulum (ER), and has only one atypical site for glycosylation (NXC) in its amino terminus. ABCB6 remained glycosylated when the highly conserved cysteine (Cys-8) was substituted with serine to make a consensus site, NXS. However, this substitution blocked ER exit and produced ABCB6 degradation, which was mostly reversed by the proteasomal inhibitor MG132. The amino terminus of ABCB6 has an additional highly conserved ER luminal cysteine (Cys-26). When Cys-26 was mutated alone or in combination with Cys-8, it also resulted in instability and ER retention. Further analysis revealed that these two cysteines form a disulfide bond. We discovered that other ABC transporters with an amino terminus in the ER had similarly configured conserved cysteines. This analysis led to the discovery of a disease-causing mutation in the sulfonylurea receptor 1 (SUR1)/ABCC8 from a patient with hyperinsulinemic hypoglycemia. The mutant allele only contains a mutation in a conserved amino-terminal cysteine, producing SUR1 that fails to reach the cell surface. These results suggest that for ABC transporters the propensity to form a disulfide bond in the ER defines a unique checkpoint that determines whether a protein is ER-retained.

Advances and opportunities in targeted protein degradation

Recently, targeted protein degradation (TPD) strategies have emerged as an effective tool for addressing undruggable targets in both biomedical research and the pharmaceutical industry, selectively binding proteins of interest and targeting them to the intracellular degradation machinery for degradation. However, the targeting of extracellular proteins with current degradation tools requires a tedious, case-specific selection and design process based on lysosomal trafficking of cell surface receptors. Here, we introduce Macropinocytosis-Targeting Chimeras (MapTACs), a TPD platform that exploits macropinocytosis, a receptor-independent endocytic process, to deliver extracellular proteins to lysosomes for degradation. Using dextran as a versatile scaffold conjugated to protein-binding aptamers or antibodies, we demonstrate that MapTACs efficiently degrade monocyte chemotactic protein-1 (MCP-1) in a time-, dose-, and macropinocytosis-dependent manner. Importantly, without the need for receptor-specific modifications, MapTACs exhibit broad applicability to a variety of cell types and extracellular protein targets (MCP-1, tumor necrosis factor-α, and interferon-γ). In vivo, TNF-α-targeting MapTACs effectively and specifically reduce the levels of TNF-α in an LPS-induced acute inflammation model, attenuating lung injury, with a half-life of approximately 0.72 h and predominant accumulation in the liver and lung, where F4/80-positive monocytes/macrophages serve as the primary uptake cells. By overcoming the limitations of the receptor-based TPD strategy, MapTAC provides a universal, cost-effective, and scalable platform for extracellular protein degradation, facilitating the development of targeted protein degradation tools and opening opportunities for therapeutic intervention in cancer, inflammation, and other diseases.

The budding yeast, Saccharomyces cerevisiae, has three cullin proteins, which act as platforms for Cullin-based E3 ubiquitin ligases. Genetic evidence indicates that Cul8, together with Mms1, Mms22, and Esc4, is involved in the repair of DNA damage that can occur during DNA replication. Cul8 is thought to form a complex with these proteins, but the composition and the function of Cul8-based E3 ubiquitin ligases remain largely uncharacterized. Herein, we report a comprehensive biochemical analysis of Cul8 complexes. Cul8 was found to form a Cul8-Mms1-Mms22-Esc4 complex under physiological conditions, with Mms1 bridging Cul8 and Mms22 and Mms22 bridging Mms1 and Esc4. Domain analysis demonstrated that the N-terminal region of Mms1 and the C-terminal region of Mms22 are required for the Mms1-Mms22 interaction, whereas the N-terminal region of Mms22 is required for the Mms22-Esc4 interaction. We also found other Cul8-Mms1-binding proteins Ctf4, Esc2, and Orc5 using yeast two-hybrid screening. Esc4 and Ctf4 bound to Mms22 directly and bound to Cul8-Mms1 in the presence of Mms22, whereas Esc2 and Orc5 interacted with both Cul8 and Mms1, independently. We found that Cul8, Mms1, and Mms22 participated in the regulation of transcriptional silencing of yeast telomeres. These results suggest that Cul8-Mms1, as part of various protein complexes, is involved in the regulation of chromatin metabolism.

Targeting extracellular and membrane proteins for degradation remains a frontier challenge in the field of targeted protein degradation (TPD), largely due to the intracellular confinement of existing proteolysis systems and reliance on bulky biologics. Here, we develop a novel TPD platform, human epidermal growth factor receptor 2 (HER2)‐targeted lysosome‐tethering chimeras (HerTACs), which co‐opts the tumor overexpressed, endocytic, and lysosomal trafficking capability of HER2. Starting from the HER2‐binding peptide LTVSPWY, we engineered the first‐generation HerTAC (LP), a conjugate of the HER2‐binding peptide and a PD‐L1 ligand, to degrade programmed death ligand 1 (PD‐L1) in HER2‐positive cells. Guided by AlphaFold modeling and alanine scanning, we developed a stapled peptide‐based HerTAC (L2,5P) with enhanced degradative efficacy (DC50 = 156 nM), stability, and pharmacokinetics. HerTAC L2,5P showed potent antitumor activity and low systemic toxicity in HER2+ breast cancer animal models. The HerTAC strategy was further extended to other clinically relevant inaccessible membrane and extracellular targets (i.e., V‐domain Ig suppressor of T cell activation [VISTA] and macrophage migration inhibitory factor [MIF]), highlighting its generality and broad applicability. This work establishes a tumor‐selective, lysosome‐directed TPD strategy that expands the druggable proteome and offers a clinically transformable approach for precision oncology.

Targeting extracellular and membrane proteins for degradation remains a frontier challenge in the field of targeted protein degradation (TPD), largely due to the intracellular confinement of existing proteolysis systems and reliance on bulky biologics. Here, we develop a novel TPD platform, human epidermal growth factor receptor 2 (HER2)-targeted lysosome-tethering chimeras (HerTACs), which co-opts the tumor overexpressed, endocytic, and lysosomal trafficking capability of HER2. Starting from the HER2-binding peptide LTVSPWY, we engineered the first-generation HerTAC (LP), a conjugate of the HER2-binding peptide and a PD-L1 ligand, to degrade programmed death ligand 1 (PD-L1) in HER2-positive cells. Guided by AlphaFold modeling and alanine scanning, we developed a stapled peptide-based HerTAC (L2,5P) with enhanced degradativeefficacy (DC50=156nM), stability, and pharmacokinetics. HerTAC L2,5P showed potent antitumor activity and low systemic toxicity in HER2+ breast cancer animal models. The HerTAC strategy was further extended to other clinically relevant inaccessible membrane and extracellular targets (i.e., V-domain Ig suppressor of T cell activation [VISTA] and macrophage migration inhibitory factor [MIF]), highlighting its generality and broad applicability. This work establishes a tumor-selective, lysosome-directed TPD strategy that expands the druggable proteome and offers a clinically transformable approach for precision oncology.

Selective elimination of proteins associated with the pathogenesis of diseases is an emerging therapeutic modality with distinct advantages over traditional inhibitor-based approaches. This strategy, called targeted protein degradation (TPD), is based on hijacking the cellular proteolytic machinery using chimeric degrader molecules that physically link the target protein of interest with the degradation effectors. The TPD era began with the development of PROteolysis TAtrgeting Chimeras (PROTACs) in 2001, with various methods and applications currently available. Classical PROTAC molecules are heterobifunctional chimeras linking target proteins with E3 ubiquitin ligases. This induced interaction leads to the ubiquitylation of the target protein, which is needed for its recognition and subsequent degradation by the cellular proteasomes. However, this technology is limited to intracellular proteins since the effectors involved (E3 ubiquitin ligases and proteasomes) are located in the cytosol. The related methods for selective destruction of proteins present in the extracellular space have only emerged recently and are collectively termed extracellular TPD (eTPD). The prototypic eTPD technology utilizes LYsosomal TArgeting Chimeras (LYTACs) that link extracellular target proteins (secreted or membrane-associated) to lysosome-targeting receptors (LTRs) on the cell surface. The resulting complex is then internalized by endocytosis and trafficked to lysosomes, where the target protein is degraded. The successful elimination of various extracellular proteins via LYTACs and related approaches has been reported, including several important targets in oncology that drive tumor growth and dissemination. This review summarizes current progress in the eTPD field and focuses primarily on the respective technological developments. It discusses the design principles and diversity of degrader molecules and the landscape of available targets and effectors that can be employed for eTPD. Finally, it emphasizes current open questions, challenges, and perspectives of this technological platform to promote the expansion of the eTPD toolkit and further development of its therapeutic applications.

The aptamer-based RNA-PROTAC

Discovery of novel potential CRBN modulators through structure-based virtual screening and bioassay