Although PROTAC technology is very promising, its dependence on specific E3 ligase expression and proteasome pathways may limit its potential applications in specific cell types or proteasome-resistant proteins.

Recently, new technologies about lysosomal degradation pathways have emerged: LYTAC, AUTAC, ATTEC technologies, which are a major degradation pathway independent of the proteasome. Lysosomal degradation pathways include endosomal/lysosomal pathways and autophagy pathways. The endosomal/lysosomal pathway involves sequential processing of several membrane-bound intracellular compartments: endocytosed material is bound and undergoes subsequent hydrolysis through early endosomes, endosomal carrier vesicles, late endosomes, and lysosomes. The autophagy pathway begins with an isolated membrane structure called a phagocytic carrier, which is derived from the lipid bilayer and lipidated LC3 protein. This phagosome will swell and phagocytize the cargo (autophagy substrate) in the cell, including proteins and other biomolecules, and even organelles, thereby isolating them in a double-layered vesicle called autophagosome. The loaded autophagosome will mature as the protein degrades. Therefore, the endosomal/lysosomal pathway and the autophagy pathway can degrade target substances.

LYTAC (lysosomal targeted chimera) technology

LYTAC (lysosomal targeted chimera) technology is a promising strategy to degrade POI using the endosomal/lysosomal pathway. LYTAC molecules can act on extracellular proteins and membrane-bound proteins, such as EGFR. These types of proteins are usually resistant to PROTAC, which mainly targets proteins in cells. The LYTAC molecule is composed of an antibody against a specific POI, and 20- or 90-mer mannose-6-phosphate (M6P) is covalently linked to the antibody. M6P is a sugar chain that targets proteins for degradation by binding to the M6P receptor (CI-M6PR) that is not related to cations. Therefore, the M6P-bound antibody, the LYTAC molecule, is recognized by this endogenous system for the lysosomal degradation of glycosylated proteins, and the polysaccharide and POI are taken to the lysosome for degradation.

The advantage of LYTAC technology is that it can degrade extracellular and membrane-related POI, and utilize the ubiquitously expressed endogenous degradation pathway. The main limitation is that the LYTAC molecule is relatively large, so it loses the desired properties of small molecule drugs.

Autophagy Targeted Chimera (AUTAC) Technology

LYTAC technology is aimed at POI outside the cell or on the membrane, pointing to the endosomal/lysosome pathway, and not applicable to cytoplasmic proteins. However, these can become targets for autophagy, and autophagy targeted degradation technology is ideal for cytoplasmic POI, especially those POIs that are resistant to PROTAC molecules. Two degradant technologies using the autophagy pathway have recently been reported.

The design of autophagy targeting chimera (AUTAC) is similar to PROTAC technology. Both AUTAC and PROTAC molecules function through ubiquitination. However, the AUTAC molecule does not tether the POI to the E3 ligase subunit and triggers K48 polyubiquitination, but triggers K63 polyubiquitination. K63 polyubiquitination is recognized by the selective autophagy pathway, resulting in the target POI degradation. The AUTAC molecule contains a degradation tag (guanine derivative) and a POI ligand to provide target specificity. The degradation tag mimics S-guanylic acid, which is a post-translational modification of Cys-cGMP formed by treatment with 8-nitro-cGMP or similar compounds. Studies have shown that AUTAC molecules can degrade POI and organelles, such as damaged mitochondria. For AUTAC, its mechanism of action is unclear, especially how S-guanylic acid induces K63 polyubiquitination. This can be solved by identifying the interactome of this modified protein or by biochemical screening of S-guanylic acid-induced K63 polyubiquitination modifiers.

Autophagy-Tethered Complex (ATTEC)

Autophagy-tethered complex (ATTEC) technology is a more direct strategy to use autophagy to degrade POI. Unlike PROTAC and AUTAC, ATTEC molecules do not rely on ubiquitination. ATTEC molecules bind POI and autophagosomes by directly binding POI and the key autophagosome protein LC3. A proof-of-concept study established a high-throughput screening strategy to identify compounds against mutant HTT protein, a protein that causes Huntington’s disease (HD), with expanded polyglutamine (PolyQ). Studies have shown that these compounds can degrade mHTT in cells and animal models, and can rescue HDR-related phenotypes. ATTEC molecules can also degrade other diseases that cause PolyQ proteins, such as the mutant ATXN3 that causes spinocerebellar ataxia type III. Some ATTEC molecules can pass through the blood-brain barrier and work at a concentration of about 100 nm, which provides a unique entry point for drug discovery.

ATTEC molecules directly interact with the autophagy protein LC3, bypassing the ubiquitination process, and have great potential to degrade different types of targets, including non-protein cargo recognized by autophagy, such as DNA/RNA molecules, damaged organelles, etc. However, whether AUTAC affects systemic autophagy remains to be elucidated to avoid non-specific degradation of functional proteins and organelles.

Summary
In addition to the recent hot PROTAC technology and its further development, the newly emerging LYTAC, AUTAC, and ATTEC lysosomal degradation technologies have recently been developed. While each approach has its advantages and limitations, and emerging degradant technologies are also in their infancy and need to address some key issues, they greatly expand the potential applications of degradant technologies and may open up new therapies in the field of targeted degradation.