Most drugs work by attaching to a specific site on a target protein to block or modulate the function of the protein. However, the activity of many proteins cannot be altered. The emerging class of drugs brings proteins closer to other molecules that unconventionally alter protein function.1–3. One such method uses drug molecules called protein breakers, which promote the labeling of proteins with ubiquitin, another small protein. The proteins labeled with the labeled elements are then broken down into small peptide molecules by the proteasome mechanism of the cell. Because of the ubiquitin-induced degradation pathway within cells, the protein degraders developed to date attack mainly intercellular targets. Writing nature, Banik et al.4 now reports a different mechanism that opens intercellular and membrane proteins for targeted cleavage.
The authors report protein degraders, which they call lysosome-directed chimeras (LYTAC), which are bifunctional (they have two binding sites; Fig. 1). At one end of the cell surface is a group of oligoglycopeptides that bind to transmembrane receptors (cation-independent mannose-6-phosphate receptor; CI-M6PR). At the other end is either an antibody or a small molecule that binds to proteins that are destroyed. These two regions are connected by a chemical switch.
The formation of the trimeric CI-M6PR – LYTAC – target complex in the plasma membrane directs the complex to the destruction of protease enzymes in the membranes in closed organs called lysosomes. LYTACs are conceptually related but complementary to proteolysis-targeting chimeras5 (PROTAC) is another bifunctional class of protein degradation that focuses on intercellular proteins by employing them in E3 ligases (enzymes that label proteins ubiquitin).
Banik et al. started the production of LYTACs of various sizes and linker compositions, the protein-binding component of which used a small molecule called biotin – biotin binds with a particularly high affinity for avidin proteins. The authors observed that these LYTACs rapidly transferred the intercellular fluorescent avidin protein to intracellular lysosomes in a manner that required binding to CI-M6PR. When the authors replaced biotin with an antibody that recognizes apolipoprotein E4 (a protein associated with neurodegenerative diseases), this protein was also internalized and degraded by lysosomes. Therefore, LYTAC may re-modify antibodies from their normal immune function to target intercellular proteins for lysosomal degradation.
Next, Banik et al. investigated whether LYTAC could induce the breakdown of membrane proteins that are targets of drug discovery. In some cancer cell lines, LYTAC has induced the internalization of the epidermal growth factor receptor (EGFR), a membrane protein that promotes cell proliferation by activating the signaling pathway, and lysosomal degradation. Decreased EGFR levels of LYTAC in cancer cell lines reduced signal activation downstream of EGFR compared to the amount observed when EGFR was blocked by antibodies alone. This result confirms the previously reported information5 an advantage when target cleavage is used in therapy rather than target blockade.
Similar results were observed with LYTAC using other single transmembrane proteins (proteins that cover the cell membrane only once), including a programmed death 1 ligand (PD-L1) that helps cancer cells bypass the immune system. The next step will be to see if LYTAC can also stimulate multiple proteins that multiple membrane-spanning, such as ubiquitous G-protein-coupled receptors and proteins that transport substances across membranes (ion channels and solvent carrier). proteins, e.g.). If so, it will be interesting to compare the performance of LYTAC, which binds to the intercellular domains of such proteins, with PROTAC, which can bind to the intercellular domains of these proteins (as recently shown).6 soluble carrier proteins).
As with any new drug, there is room for improvement. For example, Banik and colleagues were the first to target PD-L1 in LYTAC, causing only partial protein cleavage, which the authors attributed to low CI-M6PR expression in cell lines used. When the authors developed the second type of LYTAC, which includes a more potent PD-L1 antibody, degradation was increased, although in cells that expressed higher levels of CI-M6PR than did the original cell lines. This suggests that the low abundance of lysosomal transport receptors (in this case CI-M6PR) captured by LYTAC (in this case CI-M6PR) may reduce the efficiency of these degraders. Similarly, the loss of key components of E3 ligases is a common mechanism by which cells become resistant to PROTAC.7. LYTAC may use alternative lysosomal transport receptors other than CI-M6PR if resistance develops. Degraders targeting cell type-specific receptors may also have a better safety profile compared to conventional small molecule drugs, which are not always selective for cell types.
What sets PROTAC and LYTAC apart from conventional medicines is the way they work. For example, after PROTAC destroys the target protein, PROTAC is released and may induce further cycles of ubiquitin labeling and degradation, thus acting as a catalyst at low concentrations.1,5. Mechanistic studies are now needed to assess whether LYTAC acts catalytically.
Another aspect of the way PROTAC and LYTAC work is that they combine the two proteins to form a trimeric complex. A common feature of such processes is the hook effect, where at high drug concentrations the formation of a grinder and the associated biological activity are reduced. This is because dimeric complexes are usually formed at high drug concentrations, an undesirable effect that can be reduced by ensuring that all three components interact in such a way that trimer formation is more favorable than dimer formation.1.
Kinetics are also important for protein degraders. For example, stable and long-lasting trimeric complexes containing PROTAC accelerate target degradation, improving drug potency and selectivity.8. It is very important to understand how the complexes formed by LYTAC can be optimized to improve the degradation activity.
PROTAC and LYTAC are larger molecules than conventional drugs. Due to its size, PROTAC often crosses biological membranes, which may make them less potent than the biologically active groups they contain. The size of LYTAC should not cause less problems as they do not need to cross the cell membrane, although they will still need to cross biological barriers to fight diseases of the central nervous system. The development of lysosomal fissures, which are smaller and less polar than LYTAC, will be eagerly awaited and may therefore pass more easily through membranes. Small “glue” molecules that bind to E3 diseases can already do the same job as PROTAC9.
Targeted protein degradation is a promising therapeutic strategy, and the first PROTACs are in clinical trials10. LYTAC will have to do the catch-up, but they have earned their place as a tool to expand the range of proteins that can be broken down. Their development as therapies requires an understanding of their behavior in humans – their pharmacokinetics, toxicity and, for example, how they are metabolised, distributed and eliminated. It may be difficult to optimize the biological behavior of molecules with large groups, such as antibodies and oligoglycopeptides, during discovery, but this problem can be solved by further improving the structures of these groups.11. So the new approach to degradation by Banik and colleagues justifies a hands-on approach.
Researchers working on medicines are eagerly awaiting the development of LYTAC and the emergence of other methods of drug-induced protein breakdown.12. Is there no protein available to the breakers?