Multivalent Aptamer-Based Lysosome-Targeting Chimeras (LYTACs) Platform for Mono- or Dual-Targeted Proteins Degradation on Cell Surface.

Selective protein degradation platforms have opened novel avenues in therapeutic development and biological inquiry. Antibody-based lysosome-targeting chimeras (LYTACs) have emerged as a promising technology that extends the scope of targeted protein degradation to extracellular targets. Aptamers offer an advantageous alternative owing to their potential for modification and manipulation toward a multivalent state. In this study, a chemically engineered platform of multivalent aptamer-based LYTACs (AptLYTACs) is established for the targeted degradation of either single or dual protein targets. Leveraging the biotin-streptavidin system as a molecular scaffold, this investigation reveals that trivalently mono-targeted AptLYTACs demonstrate optimum efficiency in degrading membrane proteins. The development of this multivalent AptLYTACs platform provides a principle of concept for mono-/dual-targets degradation, expanding the possibilities of targeted protein degradation.

A Nucleic Acid-Based LYTAC Plus Platform to Simultaneously Mediate Disease-Driven Protein Downregulation.

Protein degradation techniques, such as proteolysis-targeting chimeras (PROTACs) and lysosome-targeting chimeras (LYTACs), have emerged as promising therapeutic strategies for the treatment of diseases. However, the efficacy of current protein degradation methods still needs to be improved to address the complex mechanisms underlying diseases. Herein, a LYTAC Plus hydrogel engineered is proposed by nucleic acid self-assembly, which integrates a gene silencing motif into a LYTAC construct to enhance its therapeutic potential. As a proof-of-concept study, vascular endothelial growth factor receptor (VEGFR)-binding peptides and mannose-6 phosphate (M6P) moieties into a self-assembled nucleic acid hydrogel are introduced, enabling its LYTAC capability. Small interference RNAs (siRNAs) is then employed that target the angiopoietin-2 (ANG-2) gene as cross-linkers for hydrogel formation, giving the final LYTAC Plus hydrogel gene silencing ability. With dual functionalities, the LYTAC Plus hydrogel demonstrated effectiveness in simultaneously reducing the levels of VEGFR-2 and ANG-2 both in vitro and in vivo, as well as in improving therapeutic outcomes in treating neovascular age-related macular degeneration in a mouse model. As a general material platform, the LYTAC Plus hydrogel may possess great potential for the treatment of various diseases and warrant further investigation.

TAC-tics for Leveraging Proximity Biology in Drug Discovery.

Chemically induced proximity (CIP) refers to co-opting naturally occurring biological pathways using synthetic molecules to recruit neosubstrates that are not normally encountered or to enhance the affinity of naturally occurring interactions. Leveraging proximity biology through CIPs has become a rapidly evolving field and has garnered considerable interest in basic research and drug discovery. PROteolysis TArgeting Chimera (PROTAC) is a well-established CIP modality that induces the proximity between a target protein and an E3 ubiquitin ligase, causing target protein degradation via the ubiquitin-proteasome system. Inspired by PROTACs, several other induced proximity modalities have emerged to modulate both proteins and RNA over recent years. In this review, we summarize the critical advances and opportunities in the field, focusing on protein degraders, RNA degraders and non-degrader modalities such as post-translational modification (PTM) and protein-protein interaction (PPI) modulators. We envision that these emerging proximity-based drug modalities will be valuable resources for both biological research and therapeutic discovery in the future.

Diseño y evaluación computacional de LYTACS para la degradación selectiva de MMP-2 / Computational design and evaluation of LYTACS for the selective degradation of MMP-2

Los LYTACs (LYsosome TArgeting Chimeras) son una novedosa estrategia farmacológica basada en la degradación dirigida de proteínas extracelulares y transmembrana. Su mecanismo de acción se basa en la utilización de un receptor de membrana para internalizar a una proteína diana y promover su degradación lisosomal. Hasta la fecha, su desarrollo se ha basado en el uso de anticuerpos para la unión a la proteína diana, lo cual presenta ciertas desventajas desde el punto de vista farmacocinético y sintético. El objetivo de este trabajo es diseñar un LYTAC capaz de inducir la degradación selectiva de MMP-2 (LYTAC-MMP2), una metaloproteasa de la matriz que se encuentra sobreexpresada en diversos tipos de cáncer. LYTAC-MMP2 está formado por un ligando del receptor de manosa-6-fosfato independiente de cationes (CI- MPR) y un inhibidor selectivo de MMP2 previamente descrito. Se han empleado métodos computacionales de modelado por homología, docking y dinámica molecular para estudiar el receptor CI-MPR y su mecanismo de internalización, así como para la comparación del comportamiento dinámico libre en agua de un ligando de CI-MPR descrito en la bibliografía y el LYTAC-MMP2.(AU)

LYTACs (LYsosome TArgeting Chimeras) are a novel pharmacological strategy based on the targeted protein degradation of extracellular and transmembrane proteins. Their mechanism of action is based on the use of a membrane receptor to internalize a target protein and mediate its lysosomal degradation. To date, its development has been focused on the use of antibodies for target binding, which has certain disadvantages from the pharmacokinetic and synthetic point of view. The aim of this work is to design a LYTAC capable of inducing the selective degradation of MMP-2 (LYTAC-MMP2), a matrix metalloprotease that is overexpressed in many types of cancer. LYTAC-MMP2 consists of a cation-independent mannose-6-phosphate receptor (CI-MPR) ligand and a selective MMP-2 inhibitor developed by our research group. Computational methods of homology modelling, docking and molecular dynamics have been used to study the CI-MPR receptor and its internalization mechanism, as well as for the comparison of the dynamic behaviour in water of a CI-MPR ligand described in the literature and LYTAC-MMP2.(AU)

Nano-LYTACs for Degradation of Membrane Proteins and Inhibition of CD24/Siglec-10 Signaling Pathway.

Lysosome-targeting chimeras (LYTACs) are an emerging therapeutic modality that effectively degrade cancer cell membranes and extracellular target proteins. In this study, a nanosphere-based LYTAC degradation system is developed. The amphiphilic peptide-modified N-acetylgalactosamine (GalNAc) can self-assemble into nanospheres with a strong affinity for asialoglycoprotein receptor targets. They can degrade different membranes and extracellular proteins by linking with the relevant antibodies. CD24, a heavily glycosylated glycosylphosphatidylinositol-anchored surface protein, interacts with Siglec-10 to modulate the tumor immune response. The novel Nanosphere-AntiCD24, synthesized by linking nanospheres with CD24 antibody, accurately regulates the degradation of CD24 protein and partially restores the phagocytic function of macrophages toward tumor cells by blocking the CD24/Siglec-10 signaling pathway. When Nanosphere-AntiCD24 is combined with glucose oxidase, an enzyme promoting the oxidative decomposition of glucose, the combination not only effectively restores the function of macrophages in vitro but also suppresses tumor growth in xenograft mouse models without detectable toxicity to normal tissues. The results indicate that GalNAc-modified nanospheres, as a part of LYTACs, can be successfully internalized and are an effective drug-loading platform and a modular degradation strategy for the lysosomal degradation of cell membrane and extracellular proteins, which can be broadly applied in the fields of biochemistry and tumor therapeutics.

Aptamer-LYTACs for Targeted Degradation of Extracellular and Membrane Proteins.

Recently, lysosome targeting chimeras (LYTACs) have emerged as a promising technology that expands the scope of targeted protein degradation to extracellular targets. However, the preparation of chimeras by conjugation of the antibody and trivalent N-acetylgalactosamine (tri-GalNAc) is a complex and time-consuming process. The large uncertainty in number and position and the large molecular weights of the chimeras result in low internalization efficiency. To circumvent these problems, we developed the first aptamer-based LYTAC (Apt-LYTAC) to realize liver-cell-specific degradation of extracellular and membrane proteins by conjugating aptamers to tri-GalNAc. Taking advantage of the facile synthesis and low molecular weight of the aptamer, the Apt-LYTACs can efficiently and quickly degrade the extracellular protein PDGF and the membrane protein PTK7 through a lysosomal degradation pathway. We anticipate that the novel Apt-LYTACs will expand the usage of aptamers and provide a new dimension for targeted protein degradation.

Protac: redirigiendo los sistemas de degradación de proteínas a nuevos sustratos / Protac: redirecting protein degradation systems to new substrates

Tradicionalmente el diseño de fármacos se ha basado en el diseño de moléculas pequeñas hasta la aparición de terapias basadas en ácidos nucleicos, ya sea por la modificación de ciertos genes o por impedir que las proteínas se transcriban de forma efectiva. Empleando estas nuevas aproximaciones se han podido modificar dianas que hasta el momento se consideraban inmodificables o al menos no lo podían ser por moléculas pequeñas.Sin embargo, estas nuevas aproximaciones no están carentes de limitaciones como la baja biodisponibilidad debido a su limitada estabilidad y dificultad para poder atravesar las barreras celulares. Además, en muchas ocasiones las modificaciones que generan son irreversibles con el consiguiente riesgo de padecer efectos adversos de forma crónica.Como alternativa, han surgido con fuerza una serie de compuestos quiméricos heterobifuncionales denominados PROTACs (Protein Targeting Chimeras). Estos PROTACs son capaces de mantener en la proximidad de la ligasa E3 a una proteína de interés, marcándola con ubiquitina y finalmente, promoviendo su degradación mediada por el proteasoma. Esta aproximación permite la generación de diferentes estructuras de PROTAC por diseño racional o basado en la estructura y, además, permite las modificaciones estructurales necesarias para mejorar su perfil de estabilidad y farmacocinético manteniendo su actividad.Esta revisión pretende dar una visión general de qué son los PROTACs, qué ligasas E3 se emplean por el momento, factores relevantes a la hora de desarrollar un PROTAC y otras aproximaciones similares que no emplean el proteasoma como ruta de degradación. (AU)

The small molecules development has dominated the design of new drugs until the rise of nucleic acid-based therapies, either by modifying a gene or by preventing it from being effectively transcribed. Taking advantage of this new approaches, the pharmacological intervention in therapeutic targets that are considered unmodifiable up to now with small molecules were allowed.However, these new approaches are not devoid of defects such as low bioavailability due to their stability and pharmacokinetic problems, in addition to being irreversible DNA modifications in many cases, with the subsequent risk of suffering chronic adverse effects.Alternatively, a series of chimeric heterobifunctional compounds, called PROTACs (Protein Targeting Chimeras), have emerged with force in recent years. These PROTACs are able to bring E3 ligases closer with proteins of interest in space to label them with ubiquitin. Finally, it was degraded by the proteasome. This approach enables the generation of different PROTACs structures by rational design and, also, allows the chemical structure modification to improve their stability and pharmacokinetic profile keeping their activity.This review aims to give a comprehensive approach of what PROTACs are, what E3 ligases recruit, relevant factors in PROTAC development, and other approaches similar to this but that use non-proteasomal degradation pathways. (AU)

Recent advances in the development of EGFR degraders: PROTACs and LYTACs.

Epidermal Growth Factor Receptor (EGFR), a transmembrane tyrosine kinase receptor, belongs to the ErbB receptor family, also known as HER1 or ErbB1. Its abnormal expression and activation contribute to tumor development, especially in non-small cell lung cancer (NCSCL). The first-to fourth-generation inhibitors of EGFR were developed to solve mutations at different sites, but the problem of resistance has not been fundamentally addressed. Targeted protein degradation (TPD) technologies, including PROteolysis Targeting Chimeras (PROTACs) and LYsosome Targeting Chimeras (LYTACs), take advantages of protein destruction mechanism in cells, which make up for shortcomings of traditional small molecular occupancy-driven inhibitors. PROTACs based heterobifunctional EGFR degraders were recently developed by making use of wild-type (WT) and mutated EGFR inhibitors. These degraders compared with EGFR inhibitors showed better efficiency in their cellular potency, inhibition and toxicity profiles. In this review, we first introduce the structural properties of EGFR, the inhibitors that have been developed against WT/mutated EGFR, and then mainly focuses on the recent advances of EGFR-targeting degraders along with its limitations and unlimited prospects.

Protac: redirigiendo los sistemas de degradación de proteínas a nuevos sustratos / Protac: redirecting protein degradation systems to new substrates

Tradicionalmente el diseño de fármacos se ha basado en el diseño de moléculas pequeñas hasta la aparición de terapias basadas en ácidos nucleicos, ya sea por la modificación de ciertos genes o por impedir que las proteínas se transcriban de forma efectiva. Empleando estas nuevas aproximaciones se han podido modificar dianas que hasta el momento se consideraban inmodificables o al menos no lo podían ser por moléculas pequeñas. Sin embargo, estas nuevas aproximaciones no están carentes de limitaciones como la baja biodisponibilidad debido a su limitada estabilidad y dificultad para poder atravesar las barreras celulares. Además, en muchas ocasiones las modificaciones que generan son irreversibles con el consiguiente riesgo de padecer efectos adversos de forma crónica. Como alternativa, han surgido con fuerza una serie de compuestos quiméricos heterobifuncionales denominados PROTACs (Protein Targeting Chimeras). Estos PROTACs son capaces de mantener en la proximidad de la ligasa E3 a una proteína de interés, marcándola con ubiquitina y finalmente, promoviendo su degradación mediada por el proteasoma. Esta aproximación permite la generación de diferentes estructuras de PROTAC por diseño racional o basado en la estructura y, además, permite las modificaciones estructurales necesarias para mejorar su perfil de estabilidad y farmacocinético manteniendo su actividad. Esta revisión pretende dar una visión general de qué son los PROTACs, qué ligasas E3 se emplean por el momento, factores relevantes a la hora de desarrollar un PROTAC y otras aproximaciones similares que no emplean el proteasoma como ruta de degradación.(AU)

The small molecules development has dominated the design of new drugs until the rise of nucleic acid-based therapies, either by modifying a gene or by preventing it from being effectively transcribed. Taking advantage of this new approaches, the pharmacological intervention in therapeutic targets that are considered unmodifiable up to now with small molecules were allowed. However, these new approaches are not devoid of defects such as low bioavailability due to their stability and pharmacokinetic problems, in addition to being irreversible DNA modifications in many cases, with the subsequent risk of suffering chronic adverse effects. Alternatively, a series of chimeric heterobifunctional compounds, called PROTACs (Protein Targeting Chimeras), have emerged with force in recent years. These PROTACs are able to bring E3 ligases closer with proteins of interest in space to label them with ubiquitin. Finally, it was degraded by the proteasome. This approach enables the generation of different PROTACs structures by rational design and, also, allows the chemical structure modification to improve their stability and pharmacokinetic profile keeping their activity. This review aims to give a comprehensive approach of what PROTACs are, what E3 ligases recruit, relevant factors in PROTAC development, and other approaches similar to this but that use non-proteasomal degradation pathways.(AU)

New Frontiers in the Discovery and Development of PROTACs.

Proteolysis targeting chimeras (PROTACs) are an emerging class of targeted protein degraders that coopt the intracellular degradation machinery to selectively deplete their respective targets. PROTACs act as bifunctional degraders that comprise ubiquitin E3 ligase- and target-binding moieties connected by chemical linkers with appropriate physicochemical properties. Through this bivalent structure, PROTACs induce the degradation of their targets via proximity-based pharmacology. Compared to conventional inhibitors, PROTACs exhibit superior pharmacologic properties in terms of efficacy, potency, selectivity, the durability of response, and efficacy against undruggable proteins. Over the last few years, the scientific community has witnessed significant endeavors to advance this field and expand the armamentarium of PROTACs. In this perspective, we highlight these advances with an emphasis on emerging PROTAC variants, PROTACtability and degradability of protein targets, expression-guided PROTACs, multivalent PROTACs, preclinical resistance, candidates evaluated in clinical trials, and prospects for the use of PROTACs as a therapeutic modality.

Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs.

Of late, targeted protein degradation (TPD) has surfaced as a novel and innovative chemical tool and therapeutic modality. By co-opting protein degradation pathways, TPD facilitates complete removal of the protein molecules from within or outside the cell. While the pioneering Proteolysis-Targeting Chimera (PROTAC) technology and molecular glues hijack the ubiquitin-proteasome system, newer modalities co-opt autophagy or the endo-lysosomal pathway. Using this mechanism, TPD is posited to largely expand the druggable space far beyond small-molecule inhibitors. In this review, we discuss the major advances in TPD, highlight our current understanding, and explore outstanding questions in the field.

Degradation from the outside in: Targeting extracellular and membrane proteins for degradation through the endolysosomal pathway.

Targeted protein degradation (TPD) is a promising strategy to remove deleterious proteins for therapeutic benefit and to probe biological pathways. The past two decades have witnessed a surge in the development of technologies that rely on intracellular machinery to degrade challenging cytosolic targets. However, these TPD platforms leave the majority of extracellular and membrane proteins untouched. To enable degradation of these classes of proteins, internalizing receptors can be co-opted to traffic extracellular proteins to the lysosome. Sweeping antibodies and Seldegs use Fc receptors in conjunction with engineered antibodies to degrade soluble proteins. Recently, lysosome-targeting chimeras (LYTACs) have emerged as a strategy to degrade both secreted and membrane-anchored targets. Together with other newcomer technologies, including antibody-based proteolysis-targeting chimeras, modalities that degrade extracellular proteins have promising translational potential. This perspective will give an overview of TPD platforms that degrade proteins via outside-in approaches and focus on the recent development of LYTACs.

PROTAC Compatibilities, Degrading Cell-Surface Receptors, and the Sticky Problem of Finding a Molecular Glue.

PROteolysis TArgeting Chimeras (PROTACs) are emerging as critical tools in biomedicinal chemistry and drug design, but they have limitations. These limitations include a lack of guiding principles when selecting suitable E3 ligases to induce ubiquitinylation, and problems degrading cell-surface receptors. This Highlight outlines some recent advances that circumvent some of these limitations, and new alternative methods to induce selective protein degradation.