Lytacs: Novel Approaches for Lysosomal Degradation

Targeted protein degradation has emerged as a promising strategy for eliminating disease-causing proteins. While proteasome-based approaches like PROTACs have gained attention, they are limited to cytosolic and nuclear proteins. Lysosome-targeting chimeras (LYTACs) offer an alternative by directing extracellular and membrane-bound proteins to lysosomes for degradation, expanding the range of druggable targets.

Researchers are investigating LYTACs for treating cancer, neurodegenerative diseases, and immune disorders. By leveraging endogenous cellular pathways, these molecules provide a potentially efficient and selective approach to degrading harmful proteins.

Key Components

LYTACs rely on a modular structure designed to selectively degrade extracellular and membrane-associated proteins. These bifunctional molecules consist of a ligand that binds to a lysosomal targeting receptor and a moiety that engages the target protein. This dual specificity enables LYTACs to harness endocytic pathways, shuttling unwanted proteins into lysosomes. The choice of receptor-binding ligand and target-binding domain significantly impacts efficacy and selectivity.

A common receptor-binding ligand in LYTACs is a glycoprotein-based motif that interacts with lysosomal targeting receptors like the cation-independent mannose-6-phosphate receptor (CI-M6PR) or the asialoglycoprotein receptor (ASGPR). These receptors naturally mediate glycoprotein trafficking to lysosomes, providing an efficient degradation mechanism. Synthetic glycopolymers or engineered glycopeptides enhance receptor affinity, improving internalization and lysosomal delivery. The specificity of these ligands is critical, as off-target interactions could degrade unintended proteins, necessitating precise molecular engineering.

The target-binding domain determines which proteins can be degraded. Typically an antibody or peptide, this domain binds with high affinity to the extracellular region of the target protein. Monoclonal antibodies are widely used due to their specificity and well-characterized properties. Advances in protein engineering have also led to smaller, high-affinity binding domains such as nanobodies and designed ankyrin repeat proteins (DARPins), offering better tissue penetration and reduced immunogenicity. The modular nature of LYTACs allows for interchangeable binding domains, broadening their potential applications.

Asialoglycoprotein Receptor Binding

The asialoglycoprotein receptor (ASGPR) plays a key role in LYTAC function by mediating the internalization of glycoproteins lacking terminal sialic acid residues. Predominantly expressed in hepatocytes, ASGPR recognizes glycoproteins with exposed galactose or N-acetylgalactosamine (GalNAc) residues, facilitating endocytosis and lysosomal trafficking. Its strong affinity for glycoproteins and rapid recycling make it an attractive target for liver-specific protein degradation.

LYTAC-ASGPR binding relies on multivalent carbohydrate-protein interactions. ASGPR, a hetero-oligomeric complex of major (ASGR1) and minor (ASGR2) subunits, forms a binding pocket with high selectivity for galactosylated ligands. To maximize receptor engagement, LYTACs are engineered with synthetic glycopolymers or glycopeptides presenting multiple GalNAc or galactose residues in an optimized spatial arrangement. Studies show that increasing ligand valency enhances receptor avidity, improving internalization and lysosomal trafficking.

Receptor recycling dynamics significantly impact LYTAC performance. ASGPR undergoes constitutive endocytosis, returning to the plasma membrane within minutes, allowing repeated ligand binding and internalization. This rapid turnover supports continuous degradation of target proteins without receptor saturation. However, excessive engagement can lead to receptor downregulation, diminishing LYTAC efficacy. Researchers are addressing this by tuning glycopolymer densities and modifying ligands to balance receptor binding with sustained ASGPR availability.

Trafficking Through Lysosomes

After a LYTAC binds its receptor, the complex undergoes endocytosis and enters early endosomes, where sorting decisions occur. The acidic environment destabilizes weaker interactions, ensuring only tightly bound complexes proceed. Proteins destined for degradation remain in maturing endosomes, which acidify and fuse with late endosomes, forming a pre-lysosomal compartment.

Molecular machinery such as Rab GTPases and ESCRT complexes guide LYTAC-bound proteins toward late endosomes, where lysosomal degradation is finalized. As acidity increases, receptors like ASGPR or CI-M6PR dissociate from their ligands and recycle to the plasma membrane, maintaining receptor availability for further internalization. This recycling process sustains protein degradation without depleting the internalization machinery.

Upon lysosomal fusion, cargo encounters hydrolases, including proteases, lipases, and glycosidases, which break down proteins into amino acids. The highly acidic lysosomal pH (around 4.5 to 5.0) enhances enzymatic efficiency. Fluorescence-based degradation assays confirm that degradation rates vary depending on protein stability, with glycosylated and highly structured proteins requiring longer processing times. Cellular conditions, such as nutrient availability and autophagic flux, also influence lysosomal activity and LYTAC efficacy.

Molecular Engineering Approaches

Optimizing LYTACs requires precise molecular engineering to enhance specificity, improve intracellular trafficking, and maximize degradation efficiency. Modifications to the receptor-binding domain and target-binding moiety significantly impact performance. Fine-tuning ligand valency plays a crucial role—higher glycan motif density strengthens receptor engagement but can also lead to receptor saturation and downregulation, reducing long-term efficacy. Researchers are developing tunable glycopolymers to balance binding strength with receptor recycling.

Stability and half-life are also key considerations. Peptide-based linkers connecting the receptor-binding and target-binding domains must resist enzymatic degradation while maintaining flexibility for target engagement. PEGylation or non-natural amino acids in these linkers extend systemic stability without compromising function. Additionally, altering glycan composition on receptor-binding domains can modulate serum half-life, as certain glycoforms evade clearance pathways more effectively.

Analytical Methods

Evaluating LYTAC efficacy and specificity requires biochemical, cellular, and imaging-based techniques. These methods assess target protein degradation, receptor engagement, and trafficking efficiency, providing critical insights for optimization.

Western blotting and enzyme-linked immunosorbent assays (ELISA) are standard approaches for measuring protein abundance before and after LYTAC treatment. Mass spectrometry-based proteomics identifies degradation products and confirms specificity. Pulse-chase experiments with radiolabeled proteins track degradation kinetics over time. Fluorescence-based reporters, such as GFP-tagged proteins or Förster resonance energy transfer (FRET) biosensors, allow real-time visualization of LYTAC activity.

Tracking intracellular trafficking is essential for understanding LYTAC performance. Confocal and super-resolution microscopy visualize LYTAC-receptor complexes as they move through the endosomal-lysosomal pathway. Colocalization studies using lysosome-specific dyes or immunostaining for lysosomal markers like LAMP1 confirm successful delivery. Flow cytometry-based internalization assays quantify receptor-mediated uptake, distinguishing surface-bound from internalized LYTACs. Live-cell imaging with pH-sensitive fluorescent probes provides insights into endosomal acidification and receptor recycling, refining the understanding of LYTAC trafficking dynamics.