Targeting a single protein and making it disappear from the cell is quite the magic trick, and there are various molecular tools available for this task. You can use RNA interference, which prevents a protein from being made, inhibitors that bind the protein, rendering it unavailable for use or even gene editing tools like CRISPR that can remove it from the genome. But did you know that you can target an existing protein for destruction, using the cell’s own garbage disposal system to degrade the protein? All you need is a molecule that can connect your protein to one with a role in cellular protein degradation and your protein can be destroyed.
Why is this important? There are many cellular proteins that play a role in diseases like cancer, but researchers are unable to find compounds that might block the protein activity. These proteins, transcription factors such as MYC, are called “undruggable” because they have no binding site for small molecules to act on the protein and inhibit cancer growth. But if you could specifically target the troublesome protein and essentially delete it from the cell, there is no need to continue to search for a chemical compound or two that might inhibit the protein.
PROTACs Bring Destruction to Targeted Proteins
What are these amazing protein-degrading tools? Into the ring steps the PROTAC or the proteolysis-targeting chimera. A PROTAC consists of three elements: A ligand that binds to a ubiquitin E3 ligase, a second ligand that binds the protein of interest and a linker that brings the two ligands together. The E3 ligase is part of a protein complex that tags proteins with ubiquitin, a molecular signal to clear away damaged proteins in the cell. By collecting the protein you are interested in and hitching it to the E3 ligase, the bifunctional PROTAC introduces the target protein to the protein complex that adds ubiquitin, which forces the target protein on the path to destruction. Ubiquitinated proteins are directed to the proteasome where they are broken down into their component molecules, ready to be recycled by the cell.
While PROTACs have been around for a while, in recent years, researchers have really been digging into the possibilities that targeted protein degradation may offer. If you have a method that can neutralize noncatalytic difficult-to-drug proteins that play a role in disease, that is a tool worth exploring. Even more exciting is news that the first PROTAC is entering clinical trials, bringing a higher profile to this molecular tool that can target proteins for degradation. For a great background on the history of PROTACs, check out this Nature feature article.
However, there are limitations on what PROTACs can do. They are larger molecules, which can pose a challenge getting them inside cells. The multistep process requires that the PROTACs bind both the target protein and the E3 ligase, join a complex where the target protein is ubiquitinated, direct the target protein to the proteasome and then end up degraded. At any point, there is a possibility of failure. Plus this process focuses on intracellular proteins with cytosolic domains, excluding secreted and most membrane proteins as possible targets.
PHOTACs: Making PROTACs Responsive to Light
Researchers are creative people. They like to tinker with tools others developed to see if they can improve on what’s available. PROTACs are bifunctional molecules but a recent paper posted to the preprint ChemRxiv describes a trifunctional molecule based on PROTACs. By adding a photoswitch to the ligand for E3 ligase and the ligand for the protein target, Reynders et al. generated what they called photochemically targeted chimeras (PHOTACs) that are not active until exposed to light at 380-440nm. They tested their newly developed PHOTACs with bromodomain-containing proteins (BRD2-4) and FK506 binding protein 12 (FKPB12), proteins that have been successfully degraded by existing PROTACs. They observed degradation when the cells were exposed to light while those that remained in the dark maintained their protein levels. Because photoswitches are reversible, the cells dosed with the BRD PHOTAC were exposed first to the activating light wavelength and then the deactivating wavelength. The amount of BRD2 did decrease initially but recovered more quickly than cells that were exposed to the activating light and left in the dark.
Being able to activate the degradation of a particular protein at a particular time has advantages, including minimizing off-target effects (i.e., altering the degradation of a protein other than the protein of interest) and fine tuning control of when degradation occurs. PHOTAC reversibility means that you can also control how long the protein is targeted for degradation. The technique is new, but the authors expand on its possible applications.
LYTACs: A New Tool for Protein Degradation
Because PROTACs act on proteins with domains exposed to the cytosol, there are membrane proteins and secreted proteins that elude this strategy. But what if you could target proteins to the lysosome, rather than the proteasome? That particular degradation method was described in an article posted to the ChemRxiv as a way to target some of those proteins not served by the PROTACs. By exploiting a known lysosome targeting receptor [cation-independent mannose-6-phosphate receptor (CI-M6PR)] that directs proteins to the lysosome and an antibody specific to the protein, Banik et al. describe how they generated lysozyme targeting chimeras (LYTACs). To test their concept, they used a polypeptide with multiple ligands for CI-M6PR attached to biotin and examined if NeurAvidin was taken up by the cells. Avidin and biotin have a high affinity for each other. In the presence of the biotin LYTAC, the NeurAvidin was colocalized to endosomes and lysosomes.
Could they use an antibody to target a protein to the lysosome? After showing that they could conjugate glycoprotein ligands to an antibody, the LYTAC was incubated with a fluorescently labeled mouse IgG and the fluorescence was 40-fold higher in the lysosome relative to controls. This demonstrated an extracellular protein could be sent to the lysosome using the engineered LYTAC. If the protein was bound to its antigen, would the LYTAC still work? When using a fluorescent reporter mCherry bound to mouse anti-mCherry antibody or a protein implicated in neurodegenerative disease, apoliporotein E4 (ApoE4), bound to mouse anti-ApoE4 antibody with the LYTAC, there was at least an order of magnitude increase in lysosomal uptake of the protein compared to untreated cells.
Would LYTACs also work for membrane proteins? Like with PROTACs, the membrane protein and CI-M6PR would have to bind the LYTAC simultaneously. To target epidermal growth factor receptor (EGFR), which plays a role in cancer proliferation, cetuximab, an EGFR-blocking antibody, was conjugated to a glycopeptide with multiple CI-M6PR ligands. When cells were treated with the EGFR LYTAC for 24 hours, the amount of EGFR was substantially decreased compared to control treatment, indicating the targeted protein had been degraded. When tested with other membrane proteins like programmed death-ligand 1 (PD-L1), a receptor overexpressed on cancer cells, the amount of PD-L1 on the cell surface decreased in two different cell lines: 33% decrease in a cell line that expressed CI-M6PR at low levels and 50% decrease in a Hodgkin’s lymphoma cell line with higher levels of CI-M6PR. Taking all the data together, the proof-of-concept by Banik et al. shows that LYTACs are an approach to target extracellular proteins for degradation in the lysosome.
Targeting proteins for degradation is a strategy to circumvent the difficult-to-drug proteins that have known roles in diseases like cancer. PROTACs are one approach researchers are taking to mitigate the effect a particular protein has in a cellular pathway. This method has brought at least one drug to clinical trials and based on the investment in PROTAC research by pharmabiotech companies, there may be more in the pipeline. With modified PROTACs like PHOTACs and complementary protein degradation schemes like LYTACs, there may be even greater potential for targeted protein degradation as a method for blocking a protein that plays a role in cellular dysfunction.
Want to learn more?
Learn more about targeted protein degradation and PROTACS here.