Degradation Of Extracellular Proteins Research Articles

Engineered platelets as targeted protein degraders and application to breast cancer models.

Clinical application of chimeric molecules for targeted protein degradation has been limited by unfavorable drug-like properties and biosafety concerns arising from nonspecific biodistribution after systemic administration. Here we develop a method to engineer platelets for degradation of either intracellular or extracellular proteins of interest (POIs) in vivo by covalently labeling heat shock protein 90 (HSP90) in platelets with a POI ligand. The degrader platelets (DePLTs) target wound areas and undergo activation. Depending on the tethered POI ligand and transport mechanism of the prelabeled HSP90, activated DePLTs can mediate targeted protein degradation in the target cell through the ubiquitin-proteasome machinery or the lysosome. HSP90 packaged into platelet-derived microparticles uses the ubiquitin-proteasome system to degrade intracellular POIs, whereas released free HSP90 redirects extracellular POIs to lysosomal degradation. In postsurgical breast cancer mouse models, DePLTs engineered with corresponding POI ligands effectively degrade intracellular bromodomain-containing protein 4 or extracellular programmed cell death ligand 1, thereby suppressing cancer recurrence or metastasis.

Development of Integrin Targeting Chimeras (ITACs) for the Lysosomal Degradation of Extracellular Proteins.

The emerging of lysosomal targeting chimera (LYTAC) expands the field of targeted protein degradation (TPD) to include the extracellular proteins for precise depletion. However, most of the reported LYTACs either induce ubiquitous degradation of the protein of interest (POI) in a broad range of tissues or specifically target liver cells. More tissue-selective degraders are highly desirable. Herein, we describe the development of cyclic RGD (cRGD) peptide-antibody conjugates as a novel class of integrin targeting chimeras (ITACs) with potential cancer selectivity. Our results indicate that the ITACs are able to recruit integrin to induce the degradation of both soluble and membrane targets in the lysosome. We observed higher efficiency of ITACs on degrading membrane protein in cancer cells, providing a promising platform for cancer-selective TPD strategy.

Targeted protein degradation via cellular trafficking of nanoparticles.

Strategies that selectively bind proteins of interest and target them to the intracellular protein recycling machinery for targeted protein degradation have recently emerged as powerful tools for undruggable targets in biomedical research and the pharmaceutical industry. However, targeting any new protein of interest with current degradation tools requires a laborious case-by-case design for different diseases and cell types, especially for extracellular targets. Here we observe that nanoparticles can mediate specific receptor-independent internalization of a bound protein and further develop a general strategy for degradation of extracellular proteins of interest by making full use of clinically approved components. This extremely flexible strategy aids in targeted protein degradation tool development and provides knowledge for targeted drug therapies and nanomedicine design.

Development of folate receptor targeting chimeras for cancer selective degradation of extracellular proteins.

Targeted protein degradation has emerged as a novel therapeutic modality to treat human diseases by utilizing the cell's own disposal systems to remove protein target. Significant clinical benefits have been observed for degrading many intracellular proteins. Recently, the degradation of extracellular proteins in the lysosome has been developed. However, there have been limited successes in selectively degrading protein targets in disease-relevant cells or tissues, which would greatly enhance the development of precision medicine. Additionally, most degraders are not readily available due to their complexity. We report a class of easily accessible Folate Receptor TArgeting Chimeras (FRTACs) to recruit the folate receptor, primarily expressed on malignant cells, to degrade extracellular soluble and membrane cancer-related proteins in vitro and in vivo. Our results indicate that FRTAC is a general platform for developing more precise and effective chemical probes and therapeutics for the study and treatment of cancers.

Nanomotor-Driven Targeting Chimeras as Accelerators for the Degradation of Extracellular Proteins.

Targeted protein degradation (TPD) is emerging as a therapeutic paradigm and a serviceable research tool in chemical biology and disease treatment. However, without driving sources, most targeting chimeras (TACs) lack the capability of self-diffusion and active searching in biological environments, which significantly impedes degradation efficiency. Herein, nanomotor-driven targeting chimeras (MotorTACs) are ingeniously designed to achieve effective internalization and degradation of extracellular platelet-derived growth factor (PDGF), a driver to cancer invasion and metastasis. Catalyzed by endogenous H2O2, MotorTACs diffused rapidly and searched actively in living cells, as visualized at the single-particle level under the dark-field mode. Hydrolysis efficiency is significantly enhanced as target protein degradation is complete in only 4h. Furthermore, MotorTACs-mediated degradation of PDGF is found to be via the lysosome and ubiquitin-proteasome dual-degradation pathways. Taking advantage of the properties, it is anticipated that MotorTACs provide a unique strategy against extracellular undruggable proteins, thus advancing the development of therapeutic interventions in chemical biology and disease treatment.

Programming DNA Nanoassemblies into Polyvalent Lysosomal Degraders for Potent Degradation of Pathogenic Membrane Proteins.

Lysosome-targeting chimera (LYTAC) shows great promise for protein-based therapeutics by targeted degradation of disease-associated membrane or extracellular proteins, yet its efficiency is constrained by the limited binding affinity between LYTAC reagents and designated proteins. Here, we established a programmable and multivalent LYTAC system by tandem assembly of DNA into a high-affinity protein degrader, a heterodimer aptamer nanostructure targeting both pathogenic membrane protein and lysosome-targeting receptor (insulin-like growth factor 2 receptor, IGF2R) with adjustable spatial distribution or organization pattern. The DNA-based multivalent LYTACs showed enhanced efficacy in removing immune-checkpoint protein programmable death-ligand 1 (PD-L1) and vascular endothelial growth factor receptor 2 (VEGFR2) in tumor cell membrane that respectively motivated a significant increase in T cell activity and a potent effect on cancer cell growth inhibition. With high programmability and versatility, this multivalent LYTAC system holds considerable promise for realizing protein therapeutics with enhanced activity.

A plug-and-play monofunctional platform for targeted degradation of extracellular proteins and vesicles

Existing strategies use bifunctional chimaeras to mediate extracellular protein degradation. However, these strategies rely on specific lysosome-trafficking receptors to facilitate lysosomal delivery, which may raise resistance concerns due to intrinsic cell-to-cell variation in receptor expression and mutations or downregulation of the receptors. Another challenge is establishing a universal platform applicable in multiple scenarios. Here, we develop MONOTAB (MOdified NanOparticle with TArgeting Binders), a plug-and-play monofunctional degradation platform that can drag extracellular targets into lysosomes for degradation. MONOTAB harnesses the inherent lysosome-targeting ability of certain nanoparticles to obviate specific receptor dependency and the hook effect. To achieve high modularity and programmable target specificity, we utilize the streptavidin-biotin interaction to immobilize antibodies or other targeting molecules on nanoparticles, through an antibody mounting approach or by direct binding. Our study reveals that MONOTAB can induce efficient degradation of diverse therapeutic targets, including membrane proteins, secreted proteins, and even extracellular vesicles.

Targeted degradation of membrane and extracellular proteins with LYTACs.

Targeted protein degradation technology has gained substantial momentum over the past two decades as a revolutionary strategy for eliminating pathogenic proteins that are otherwise refractory to treatment. Among the various approaches developed to harness the body's innate protein homeostasis mechanisms for this purpose, lysosome targeting chimeras (LYTACs) that exploit the lysosomal degradation pathway by coupling the target proteins with lysosome-trafficking receptors represent the latest innovation. These chimeras are uniquely tailored to degrade proteins that are membrane-bound and extracellular, encompassing approximately 40% of all proteome. Several novel LYTAC formulas have been developed recently, providing valuable insights for the design and development of therapeutic degraders. This review delineates the recent progresses of LYTAC technology, its practical applications, and the factors that dictate target degradation efficiency. The potential and emerging trends of this technology are discussed as well. LYTAC technology offers a promising avenue for targeted protein degradation, potentially revolutionizing the therapeutic landscape for numerous diseases.

Lysosome Targeting Chimaeras for Glut1-Facilitated Targeted Protein Degradation.

Targeted protein degradation technology holds great potential in biomedicine, particularly in treating tumors and other protein-related diseases. Research on intracellular protein degradation using molecular glues and PROTAC technology is leading, while research on the degradation of membrane proteins and extracellular proteins through the lysosomal pathway is still in the preclinical stage. The scarcity of useful targets is an immense limitation to technological advancement, making it essential to explore novel, potentially effective approaches for targeted lysosomal degradation. Here, we employed the glucose transporter Glut1 as an innovative lysosome-targeting receptor and devised the Glut1-Facilitated Lysosomal Degradation (GFLD) strategy. We synthesized potential Glut1 ligands via reversible addition-fragmentation chain transfer (RAFT) polymerization and acquired antibody-glycooligomer conjugates through bioorthogonal reactions as lysosome-targeting protein degradation molecules, utilized in the management of PD-L1 high-expressing triple-negative breast cancer. The glucose transporter Glut1 as a lysosome-targeting receptor exhibits potential for the advancement of a broader array of medications in the future.

Bifunctional Molecules That Induce Both Targeted Degradation and Transcytosis of Extracellular Proteins in Brain Cells

Bifunctional Molecules That Induce Both Targeted Degradation and Transcytosis of Extracellular Proteins in Brain Cells

In vitro and in vivo proof-of-concept studies of extracellular protein degradation using novel M6PR-targeting chimeras (MTACs).

Abstract The mannose-6-phosphate-receptor (M6PR) traffics proteins from extracellular and intracellular locales to late endosomes/lysosomes for degradation and is ubiquitously expressed. This natural endogenous function of the mannose-6-phosphate-receptor (M6PR) may be harnessed for degradation of soluble circulating proteins and cell specific degradation of membrane proteins. Here we describe the design and development of novel M6PR targeting chimeras (MTACs). MTACs are heterobifunctional molecules containing Avilar’s novel, potent, small molecule M6PR-binding ligands conjugated to ligands that bind a target protein of interest for degradation. For proof-of-concept studies, MTACs were designed to target the extracellular protein IgG, the second most abundant circulating protein with a long plasma half-life. Reduction of pathogenic IgG autoantibodies is a clinically validated strategy for treating multiple debilitating autoimmune diseases. In vitro characterization of tridentate and monodentate MTACs revealed potent biochemical binding to M6PR and IgG, and MTAC-mediated cellular uptake and degradation of IgG via the endolysosomal pathway in K562 and LNCaP cells. In vivo MTAC-mediated depletion of human IgG was demonstrated using a heterologous rat PK/PD model.

Directed evolution of genetically encoded LYTACs for cell-mediated delivery

Lysosome-targeting chimeras (LYTACs) are a promising therapeutic modality to drive the degradation of extracellular proteins. However, early versions of LYTAC contain synthetic glycopeptides that cannot be genetically encoded. Here, we present our designs for a fully genetically encodable LYTAC (GELYTAC), making our tool compatible with integration into therapeutic cells for targeted delivery at diseased sites. To achieve this, we replaced the glycopeptide portion of LYTACs with the protein insulin-like growth factor 2 (IGF2). After showing initial efficacy with wild-type IGF2, we increased the potency of GELYTAC using directed evolution. Subsequently, we demonstrated that our engineered GELYTAC construct not only secretes from HEK293T cells but also from human primary T-cells to drive the uptake of various targets into receiver cells. Immune cells engineered to secrete GELYTAC thus represent a promising avenue for spatially selective targeted protein degradation.

Targeted Extracellular Protein Degradation by Dendronized DNA Chimeras.

Extracellular soluble proteins are key agents in the development of various diseases. However, strategies to remove therapeutically relevant extracellular targets are still scarce. Here, we establish dendronized DNA chimera (DENTAC) as an efficient approach for targeted degradation of the extracellular protein of interest (ePOI). DENTAC consists of a DNA dendron against cell-surface scavenger receptors (SRs), a protein ligand, and a connecting linker, which harnesses SRs as a lysosome-trafficking receptor to mediate the lysosomal degradation of the ePOI. We interrogate and optimize structure-activity relationships of DENTAC. Using neutravidin as a model ePOI, we show that both branch number and DNA length in the DNA dendron are important determinants for efficient lysosomal delivery and degradation of the protein. We demonstrate three branches and 10 nucleotide-length polythymidine as the optimal DNA dendron components to construct DENTAC. We further exemplify the anticancer application of DENTAC by targeting matrix metalloproteinase-9 (MMP-9), where we find linker property as another factor important for DENTAC performance. We reveal that MMP-9-targeting DENTAC effectively restrain cancer cell proliferation, migration, and invasion. This study thus provides a potent strategy to delete extracellular proteins that are commonly difficult to target.

Anti-tumor immunotherapy using engineered bacterial outer membrane vesicles fused to lysosome-targeting chimeras mediated by transferrin receptor

The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not invivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth invivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for invivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.

Discovery of Repurposable Drugs in the Combination Therapy of Breast Cancer: A Virtual Drug Screening Study

Cathepsin D (Cat D) is a lysosomal aspartic acid protease encoded by CTSD gene and has significant biological roles such as degradation of extracellular and intracellular proteins, regulation of apoptosis, hormone processing, antigen processing etc. Furthermore, it is overexpressed by breast cancer cells and it acts a role in many processes affecting the cancer prognosis such as metastasis, angiogenesis, invasion, and drug resistance through regulation of the metabolic pathways and digesting the extracellular matrix (ECM) proteins. Due to that there is no drug targeting Cat D in clinical trial phases, a virtual drug screening in order to reveal possible drugs with high Cat D inhibitory activity from a library composed of 12,111 ligands is carried out with this study. Results have demonstrated that ZINC000003922429 (Adozelesin), ZINC000012358610 (Phthalocyanine), ZINC000051951669 (Bemcentinib), ZINC000003786250 (YM022), and ZINC000150338819 (Ledipasvir) have high binding affinity to Cat D. Among these chemical ligands, YM022 from Drugs in Clinical Trials dataset has been evaluated as most promising one that might be repurposed in the treatment of breast cancer due to its high affinity, convenient ADME and Toxicity properties, and highest bioactivity profiles. However, the possible activity of YM022 should be analyzed with further molecular dynamics (MD) simulations, in vitro and in vivo studies.

Click chemistry in the development of PROTACs.

Proteolysis-targeting chimeras or PROTACs are hetero-bifunctional molecules designed to mediate the disposal of a target protein via recruitment of the ubiquitination-proteasome degradation machinery. Because of the chimeric nature of such molecules, their synthesis requires a key step of "assembling" whether in the lab or in situ. Furthermore, targeted PROTACs often are hetero-trifunctional and require a second "assembling" step. Click chemistry has the unique advantages of tethering two or more molecular entities of choice under near physiological conditions and therefore has been applied to the development of PROTACs in various ways. This review provides a succinct summary of this field with a critical analysis of various factors that need to be considered for optimal results. Specifically, we examine issues including applications of click chemistry in in situ assembly for improved delivery, conjugation with a targeting group for selectivity, rapid synthesis for linker optimization, and lysosomal degradation of extracellular and membrane-associated proteins. We also examine reaction kinetics issues whenever possible or warranted.

Hepatocyte targeting via the asialoglycoprotein receptor.

This review highlights the potential of asialoglycoprotein receptor (ASGPR)-mediated targeting in advancing liver-specific treatments and underscores the ongoing progress in the field. First, we provide a comprehensive examination of the nature of ASGPR ligands, both natural and synthetic. Next, we explore various drug delivery strategies leveraging ASGPR, with a particular emphasis on the delivery of therapeutic nucleic acids such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). An in-depth analysis of the current status of RNA interference (RNAi) and ASO-based therapeutics is included, detailing approved therapies and those in various stages of clinical development (phases 1 to 3). Afterwards, we give an overview of other ASGPR-targeted conjugates, such as those with peptide nucleic acids or aptamers. Finally, targeted protein degradation of extracellular proteins through ASGPR is briefly discussed.

IGF2 Peptide-Based LYTACs for Targeted Degradation of Extracellular and Transmembrane Proteins.

Lysosome-targeting chimeras (LYTACs) have recently been developed to facilitate the lysosomal degradation of specific extracellular and transmembrane molecular targets. However, the LYTAC particles described to date are based on glycopeptide conjugates, which are difficult to prepare and produce on a large scale. Here, we report on the development of pure protein LYTACs based on the non-glycosylated IGF2 peptides, which can be readily produced in virtually any facility capable of monoclonal antibody production. These chimeras utilize the IGF2R/CI-M6PR pathway for lysosomal shuttling and, in our illustrative example, target programmed death ligand 1 (PD-L1), eliciting physiological effects analogous to immune checkpoint blockade. Results from in vitro assays significantly exceed the effects of anti-PD-L1 antibodies alone.

Insulin-like Growth Factor 2 (IGF2)-Fused Lysosomal Targeting Chimeras for Degradation of Extracellular and Membrane Proteins.

Targeted degradation of the cell-surface and extracellular proteins via the endogenous lysosomal degradation pathways, such as lysosome-targeting chimeras (LYTACs), has recently emerged as an attractive tool to expand the scope of extracellular chemical biology. Herein, we report a series of recombinant proteins genetically fused to insulin-like growth factor 2 (IGF2), which we termed iLYTACs, that can be conveniently obtained in high yield by standard cloning and bacterial expression in a matter of days. We showed that both type-I iLYTACs, in which IGF2 was fused to a suitable affibody or nanobody capable of binding to a specific protein target, and type-II iLYTAC (or IGF2-Z), in which IGF2 was fused to the IgG-binding Z domain that served as a universal antibody-binding adaptor, could be used for effective lysosomal targeting and degradation of various extracellular and membrane-bound proteins-of-interest. These heterobifunctional iLYTACs are fully genetically encoded and can be produced on a large scale from conventional E. coli expression systems without any form of chemical modification. In the current study, we showed that iLYTACs successfully facilitated the cell uptake, lysosomal localization, and efficient lysosomal degradation of various disease-relevant protein targets from different mammalian cell lines, including EGFR, PD-L1, CD20, and α-synuclein. The antitumor properties of iLYTACs were further validated in a mouse xenograft model. Overall, iLYTACs represent a general and modular strategy for convenient and selective targeted protein degradation, thus expanding the potential applications of current LYTACs and related techniques.

Conjugation with glucagon like peptide-1 enables targeted protein degradation

Lysosome-targeting chimeras (LYTACs) have emerged as a promising technique to extend the scope of targeted protein degradation to extracellular proteins, e.g., secreted proteins and membrane-anchored proteins. However, up to now, only a small number of lysosomal targeting receptors (LTRs), such as cation-independent mannose 6-phosphate receptor (CI-M6PR) and asialoglycoprotein receptor (ASGPR), were reported to build LYTACs for degradation of extracellular proteins. Therefore, it is important to explore more functionalized ligands for the relevant LTRs to expand the LYTAC framework. Herein, we demonstrate a new LTR ligand-glucagon like peptide 1 (GLP-1) based targeted degradation platform, termed GLP-1 receptor-targeting chimeras (GLP-1-LYTAC). GLP-1-LYTACs are formed by conjugating GLP-1 with targeted binder (such as antibody) through Click Chemistry, showing efficiently lysosomal degradation of both extracellular proteins (GFP and Neutravidin) as well as cell membrane proteins (EGFR and PD-L1). We believe that this novel GLP-1-LYTAC will open up a new dimension for targeted protein breakdown.