Most drugs act by binding to a specific site in the target protein to block or modulate protein function. However, the activity of many proteins cannot be altered in this way. The new class of drugs instead brings proteins next to other molecules, which then change the function of the protein in unconventional ways1–3. One such approach uses drug molecules called protein degraders, which promote the labeling of proteins with ubiquitin, another small protein. These proteins are then cleaved into small peptide molecules by the cell proteasome mechanism. But because the ubiquitin-mediated degradation pathway occurs within the cell, the developed protein degradators have so far attacked predominantly intracellular targets. Letter to Nature, Banik etc.4 we now report another mechanism that opens up extracellular and membrane-bound proteins for targeted degradation.
The authors report protein degradators, which they call lysosome-oriented chimeras (LYTACs), which are bifunctional (they have two binding regions; Fig. 1). One end contains an oligoglycopeptide group that binds to the transmembrane receptor (cation-independent mannose-6-phosphate receptor; CI-M6PR) on the cell surface. The other end contains either an antibody or a small molecule that binds to a protein to be destroyed. These two areas are connected by a chemical linker.
The formation of a three-dimensional complex CI-M6PR – LYTAC – targets on the plasma membrane directs the complex for the destruction of protease enzymes in the organelles enclosed by the membrane, called lysosomes. LYTACs are conceptually related, but complementary, chimeras focused on proteolysis5 (PROTAC) is another bifunctional class of protein degradator that mainly targets intracellular proteins by attracting them to E3 ligases (enzymes that label proteins with ubiquitin).
Banik etc. began with the manufacture of LYTAC of various sizes and linker composition, which used a small molecule called biotin as a protein-binding component – biotin binds with extremely high affinity for avidin proteins. The authors noted that these LYTACs rapidly migrated the extracellular fluorescent protein avidin to intracellular lysosomes in a manner that required interaction with CI-M6PR. When the authors replaced biotin with an antibody that recognizes apolipoprotein E4 (a protein involved in neurodegenerative diseases), this protein was also internalized and degraded by lysosomes. Thus, LYTAC can replace antibodies from its normal immune function to direct extracellular proteins to lysosomal degradation.
Next, Banick etc. investigated whether LYTAC can induce degradation of membrane proteins that are targets for drug detection. Several LYTAC cancer cell lines have indeed induced internalization and lysosomal degradation of the epidermal growth factor receptor (EGFR), a membrane protein that drives cell proliferation by activating a signaling pathway. Depletion of EGFR LYTAC levels in cancer cell lines reduces signal activation downstream of EGFR compared to when EGFR was blocked by antibodies. This result confirms the previously reported5 The advantage of using target degradation in therapeutic applications rather than target blocking.
Similar results have been observed with LYTAC for other single-pass transmembrane proteins (proteins that span the cell membrane only once), including programmed death ligand 1 (PD-L1), which helps cancer cells evade the immune system. The next step will be to determine whether LYTAC can also induce the degradation of multi-pass proteins that span the membrane several times, such as ubiquitous receptors associated with G-proteins and proteins that transport materials across membranes (ion channels and solvent-carrying proteins). , example). If so, it will be interesting to compare the efficacy of LYTAC, which would bind to the extracellular domains of such proteins, with the indicators of PROTAC, which may bind to the intracellular domains of these proteins (as recently demonstrated).6 for protein solvents).
Like any new drug modality, there is room for improvement. For example, the first LYTACs, targeted at Banik and colleagues, caused only partial protein degradation, which the authors attributed to low CI-M6PR expression in the cell lines used. When the authors developed a second type of LYTAC, which included a more potent PD-L1 antibody, degradation increased, although in cells that expressed higher levels of CI-M6PR than in the original cell lines. This suggests that the low number of lysosomal receptors stolen by LYTAC (in this case CI-M6PR) may reduce the effectiveness of these degradants. Similarly, the loss of major components of E3 ligases is a common mechanism by which cells become resistant to PROTAC7. LYTAC can be used as alternative lysosome receptors other than CI-M6PR as an alternative. Degradators targeting cell-type-specific receptors may also improve safety profiles compared to conventional small molecule therapies, which are not always cell-selective.
What distinguishes PROTAC and LYTAC from conventional drugs is their mode of action. For example, after PROTAC has destroyed the target protein, PROTAC is released and can induce further cycles of labeling and degradation of ubiquitin, thereby acting as a catalyst at low concentrations.1,,5. Mechanical studies are now guaranteed to determine if LYTAC is also catalytic.
Another aspect of the mode of action of both PROTAC and LYTAC is that they combine the two proteins together to form a trimeric complex. A common feature of such processes is the hook effect, due to which the formation of high trimers and, accordingly, biological activity decreases at high drug concentrations. This is due to the fact that dimer complexes are usually formed mainly at high concentrations of the drug – an undesirable effect that can be facilitated by ensuring the interaction of all three components so that the formation of the trimer is more favorable than dimeric1.
Kinetics are also important for protein degraders. For example, stable long-term trimeric complexes, including PROTAC, accelerate target degradation, improving drug potency and selectivity.8. It is important to understand how the complexes formed by LYTAC can be optimized to improve degradation activity.
PROTAC and LYTAC are larger molecules than conventional drugs. Due to their size, PROTACs often do not penetrate well through biological membranes, which can make them less potent drugs than the biologically active groups they contain. Size should be less of a problem for LYTAC, as they do not need to cross the cell membrane, although they still need to cross biological barriers to fight central nervous system disease. Unexpectedly, the development of lysosomal degradators is expected, smaller and less polar than LYTAC, and therefore more able to pass through membranes. Small ‘glue’ molecules that bind to E3 ligases can already do the same job as PROTAC9.
Targeted protein degradation is a promising therapeutic strategy, and the first PROTACs are now in clinical trials.10. LYTAC will have to play on the rise, but they have earned their place as a tool ready to expand the range of proteins that can be degraded. Their development as a therapy will require an understanding of their behavior in the human body – for example, their pharmacokinetics, toxicity and methods of metabolism, distribution and excretion, for example. Optimizing the biological behavior of molecules that include large groups, such as antibodies and oligoglycopeptides, during drug detection can be difficult, but this problem can be overcome by further engineering the structures of these groups.11. The new approach of Banik and colleagues to degradation, therefore, requires a comprehensive approach.
Drug researchers are looking forward to the development of LYTAC and the emergence of other methods of drug-induced protein degradation.12. Is there no protein beyond the reach of degradators?