Most drugs act by binding to a specific site in a target protein to block or modulate the function of the protein. However, the activity of many proteins cannot be altered in this way. Instead, the emerging class of drugs will bring proteins closer to other molecules, which then change the function of the protein in an unconventional way.1–3One such approach uses drug molecules called protein degraders that promote the labeling of proteins with ubiquitin, another small protein. The labeled proteins are then divided into small peptide molecules by the cell’s protease apparatus. But because the ubiquitin-mediated degradation pathway occurs intracellularly, the protein degraders developed to date attack mainly intracellular targets. Writing in nature, Coal miner et al.4 now disclose another mechanism that opens up extracellular and membrane-bound proteins for targeted degradation.
The authors report protein degraders called lysosome-targeted chimeras (LYTAC), which are bifunctional (have two binding regions; Fig. 1). One end carries an oligoglycopeptide moiety that binds to the transmembrane receptor (cation-independent mannose-6-phosphate receptor; CI-M6PR) on the cell surface. The other end carries either an antibody or a small molecule that binds to a protein targeted for destruction. These two regions are connected by a chemical linker.
The formation of the CI-M6PR – LYTAC trimeric complex on the plasma membrane directs the complex to be destroyed by protease enzymes in organelles called lysosomes enclosed on the membrane. LYTACs are conceptually linked, but complementary, to proteolysis-targeted chimeras5 (PROTACs) – another bifunctional class of protein degrader that focuses mainly on intracellular proteins by obtaining them into E3 ligases (enzymes that label proteins with ubiquitin).
coal miner et al. began with the production of LYTACs of various sizes and linker compositions, and which used a small molecule called biotin as a protein binding component – biotin binds with an extremely high affinity for avidin proteins. The authors observed that these LYTACs rapidly sealed the extracellular fluorescent avidin protein 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 implicated in neurodegenerative diseases), this protein was also internalized and degraded by lysosomes. LYTACs can therefore reuse antibodies from their normal immune function to target extracellular proteins to lysosomal degradation.
Next, Banik et al. investigated whether LYTAC could induce degradation of membrane proteins that are targets of drug discovery. Indeed, in several cancer cell lines, LYTACs induced internalization and lysosomal degradation of the epidermal growth factor receptor (EGFR), a membrane protein that controls cell proliferation by activating the signaling pathway. Depletion of EGFR levels by LYTAC in cancer cell lines reduced EGFR signal activation compared to the amount observed when EGFR was blocked by antibodies alone. This result confirms previously reported data5 The advantage of using target degradation in therapeutic applications, rather than blocking the target.
Similar results have been observed with LYTAC for other single-pass transmembrane proteins (proteins that span the cell membrane only once), including the programmed death ligand 1 (PD-L1), which helps cancer cells avoid the immune system. The next step will be to see if LYTAC can also induce the degradation of multi-pass proteins that span the membrane several times, such as ubiquitous G-protein coupled receptors and proteins that transport materials across membranes (ion channels and solute carriers) proteins, for example). If so, it will be interesting to compare the performance of LYTACs that would bind to the extracellular domains of such proteins with the effects of PROTACs that can bind to the intracellular domains of such proteins (as recently demonstrated).6 for proteins dissolved in a carrier).
As with any new drug modality, there is room for improvement. For example, Banik and colleagues the first LYTACs targeting PD-L1 produced only partial degradation of the protein, which the authors attributed to the low expression of CI-M6PR in the cell lines used. When the authors made a second type of LYTAC, which contains a stronger PD-L1 antibody, the degradation increased, although in cells that expressed higher levels of CI-M6PR than the original cell lines. This indicates that the low abundance of the lysosome receptor that is abducted by LYTAC (in this case CI-M6PR) may reduce the efficacy of these degraders. Similarly, the loss of nuclear components of E3 ligases is a common mechanism by which cells become resistant to PROTAC.7If resistance occurs, LYTACs could use LYTACs as alternatives to lysosome-blocking receptors other than CI-M6PR. Degraders that target cell-type receptors may also have better safety profiles compared to conventional small molecule drugs, which are not always cell-type selective.
What distinguishes PROTAC and LYTAC from conventional drugs is their mode of action. For example, after PROTAC causes destruction of the target protein, PROTAC is released and can induce further cycles of ubiquitin labeling and degradation, thereby acting as a catalyst at low concentrations.1.5Mechanistic studies are now warranted to determine whether LYTAC also functions catalytically.
Another aspect of the way PROTAC and LYTAC work is that they combine two proteins to form a trimeric complex. A common feature of such processes is the hook effect, in which trimer formation and associated biological activity is reduced at high drug concentrations. This is because dimeric complexes are generally formed preferentially at high drug concentrations – an adverse effect that can be alleviated by ensuring that all three components interact in such a way that trimer formation is more favorable than dimer formation.1.
The kinetics also depend on the degradation of proteins. For example, stable and long-lived trimeric complexes that include PROTAC accelerate target degradation, improve drug potency, and selectivity.8It will be important to understand how LYTAC complexes 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, making them less effective drugs than the biologically active groups they contain. Size should not be a problem for LYTAC, as they do not have to cross the cell membrane, although they would still need to cross biological barriers to fight central nervous system diseases. The development of lysosomal degraders that are smaller and less polar than LYTAC and are therefore able to cross membranes is eagerly expected. Small adhesive 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 currently in clinical trials.10, LYTAC will have to catch up, but have gained their place as a tool to expand the range of proteins that can be degraded. Their development in therapies will require an understanding of their behavior in the human body – for example, their pharmacokinetics, toxicity, and the way they are metabolized, distributed, and excreted. Optimizing the biological behavior of molecules that contain large groups, such as antibodies and oligoglycopeptides, during drug discovery can be challenging, but this problem can be overcome by further engineering the structures of these groups.11, The new approach of Banika and colleagues to degradation therefore requires access on board.
Researchers working on drug research will eagerly await the development of LYTAC and the discovery of other methods for drug-induced protein degradation.12Is there no protein out of reach of degraders?