How Scientists Used Molecular Matchmakers to Confirm a Cancer Target
Imagine if you couldn't fix a broken machine part by simply pressing pause—you needed to remove the faulty component entirely. This is the fundamental difference between traditional drugs and an exciting new technology called PROteolysis TArgeting Chimeras (PROTACs). While conventional medications work by temporarily inhibiting problematic proteins, PROTACs act as cellular demolition crews, completely removing disease-causing proteins from our cells 1 6 .
Temporarily inhibit proteins by binding to active sites, requiring continuous presence to maintain effect.
Completely remove target proteins by marking them for degradation, offering permanent solution until new proteins are synthesized.
This revolutionary approach is particularly valuable for tackling the "undruggable" proteins that have long frustrated scientists. These proteins lack the clear pockets or binding sites that traditional small-molecule drugs need to work effectively. By eliminating rather than merely inhibiting these proteins, PROTACs open up new therapeutic possibilities for conditions ranging from cancer to neurodegenerative diseases 5 7 .
Over 40 PROTAC-based therapies are currently undergoing clinical trials, targeting proteins involved in cancer, autoimmune disorders, and other conditions .
PROTAC molecule binds to the target protein using its target-binding ligand.
PROTAC simultaneously recruits an E3 ubiquitin ligase using its E3-binding ligand.
The E3 ligase attaches ubiquitin chains to the target protein, marking it for destruction.
The ubiquitinated protein is recognized and degraded by the proteasome.
The PROTAC molecule is released and can catalyze another round of degradation.
Our cells have their own sophisticated protein-recycling systems, primarily the ubiquitin-proteasome system 1 6 . This natural process works like a cellular garbage disposal: proteins are first tagged with a molecule called ubiquitin, then recognized and broken down into their component parts by a structure called the proteasome 9 .
PROTACs cleverly hijack this system by creating an artificial connection between a specific target protein and an E3 ubiquitin ligase—the enzyme responsible for attaching the ubiquitin tags 2 6 .
Perhaps the most remarkable feature of PROTACs is their catalytic nature. Unlike traditional drugs that remain bound to their targets, a single PROTAC molecule can detach after facilitating ubiquitination and move on to destroy multiple copies of the target protein 2 7 .
This represents a fundamental shift from what scientists call "occupancy-driven pharmacology" (simply blocking a protein's function) to "event-driven pharmacology" (triggering its complete removal) 1 .
Instead of temporarily blocking a problematic protein, PROTACs offer a more permanent solution by eliminating it entirely, forcing the cell to make new copies if the function needs to be restored 5 .
Our story focuses on a particular protein called pirin, a mysterious member of the cupin superfamily that binds iron and may regulate transcription factors 8 . Scientists had discovered that pirin appears to play a role in cancer progression through the HSF1 stress pathway, making it a potentially valuable therapeutic target 8 .
Researchers had developed a chemical probe called CCT251236 that bound strongly to pirin in test tubes, but they faced a formidable challenge: demonstrating that this binding also occurred inside living cells 8 . This problem—known as target engagement—is common in drug discovery, especially for proteins like pirin that lack enzymatic activity or known biomarkers to easily confirm when they're being targeted 8 .
The research team devised a clever strategy: they would convert their pirin-binding probe into a PROTAC molecule. If their original probe was indeed binding to pirin inside cells, the PROTAC version would degrade pirin, and this degradation could be measured. If not, no degradation would occur 8 .
This approach allowed researchers to "demonstrate chemical probe 1 binding to pirin within living cells by developing a pirin-targeting protein degradation probe" 8 .
This method served as both validation of their original probe and a potential therapeutic approach in its own right. The team focused on using a CRBN-targeting thalidomide derivative as their E3 ligase recruiter, chosen for its low molecular weight and well-characterized properties 8 .
The researchers began by examining the crystal structure of their original probe bound to pirin, identifying a solvent-exposed region where they could attach a linker without disrupting the binding interaction 8 . Their initial design—PDP 3—connected the pirin-binding moiety to the CRBN-recruiting ligand through a 15-atom linker containing two amide groups 8 .
This first-generation PROTAC showed excellent binding to pirin in biochemical tests and moderate affinity for the CRBN-DDB1 complex. However, when tested in human cancer cell lines, it failed to degrade pirin even at high concentrations over extended periods 8 .
Rather than abandoning the approach, the researchers systematically analyzed what might have gone wrong. They identified several potential issues:
Focusing on the latter possibility, they noted that although PDP 3 had acceptable lipophilicity, the linker had introduced two new hydrogen bond donors that could impair cell membrane permeability 8 .
For their second attempt, the team made strategic modifications to reduce hydrogen bond donors and improve membrane permeability while maintaining the same linker length and CRBN-targeting ligand 8 . The resulting molecule—PDP 16—represented a dramatic improvement.
| Property | First-Generation (PDP 3) | Second-Generation (PDP 16) |
|---|---|---|
| Molecular Weight | Higher | Reduced |
| Hydrogen Bond Donors | 7 | 5 |
| Calculated tPSA (Ų) | 199 | 159 |
| Permeability | Poor | Improved |
| Pirin Degradation | None | Significant |
Most importantly, PDP 16 achieved what its predecessor could not: significant degradation of pirin in SK-OV-3 ovarian carcinoma cells, with maximum degradation observed within 8 hours of treatment 8 . This success not only confirmed that their original probe was indeed engaging pirin inside cells but also demonstrated that rationally designed PROTACs could degrade challenging targets like pirin.
Despite good biochemical binding, this first-generation PROTAC failed to degrade pirin in cells due to poor cellular permeability caused by excessive hydrogen bond donors.
Strategic modifications reduced hydrogen bond donors and improved membrane permeability, resulting in significant pirin degradation within 8 hours of treatment.
| Reagent/Tool | Function in PROTAC Development |
|---|---|
| E3 Ligase Ligands | Recruit the ubiquitin-tagging machinery; common examples include thalidomide derivatives for CRBN and VHL-targeting compounds |
| Target Protein Binders | Recognize and bind to the protein of interest; these are often derived from known inhibitors or chemical probes |
| Chemical Linkers | Connect E3 ligands to target binders; length and composition critically affect PROTAC activity |
| Cell Lines with E3 Expression | Provide cellular context for testing; must express relevant E3 ligases (e.g., SK-OV-3 for CRBN) |
| Affinity Assays | Measure binding strength to target proteins and E3 ligases; includes Surface Plasmon Resonance (SPR) |
| Proteomic Analysis | Evaluate degradation efficiency and selectivity; measures changes in target protein levels over time |
The pirin case study highlights the critical role of linker optimization in PROTAC development. The linker does more than simply connect two binding moieties—it plays an active role in facilitating the formation of what scientists call the ternary complex (the productive interaction between the target protein, PROTAC, and E3 ligase) 8 .
Researchers can vary linker length, composition, and flexibility to optimize this three-way interaction. As the pirin project demonstrated, even with excellent target-binding and E3-recruiting components, an imperfect linker can result in complete PROTAC failure 8 .
Developing effective PROTACs requires sophisticated analytical methods to confirm both binding and degradation:
These tools help researchers iterate through multiple PROTAC designs to identify molecules with optimal degradation activity.
The successful demonstration of pirin degradation represents more than just a technical achievement—it validates PROTACs as both research tools and potential therapeutics. This dual application makes the technology particularly valuable: the same molecules that help scientists understand protein function in the lab might eventually become medicines 9 .
The clinical potential of PROTACs is already being realized. As of 2025, multiple PROTAC-based therapies have advanced to late-stage clinical trials:
| PROTAC Name | Target | Indication | Phase |
|---|---|---|---|
| Vepdegestrant (ARV-471) | Estrogen Receptor | Breast Cancer | Phase 3 |
| BMS-986365 | Androgen Receptor | Prostate Cancer | Phase 3 |
| BGB-16673 | BTK | B-cell Malignancies | Phase 3 |
| ARV-110 | Androgen Receptor | Prostate Cancer | Phase 2 |
| KT-474 | IRAK4 | Inflammatory Diseases | Phase 2 |
PROTACs offer several potential benefits compared to traditional small-molecule drugs:
Despite their promise, PROTACs face several hurdles. Their relatively large size can limit cellular permeability and oral bioavailability 1 7 . The "hook effect"—where high PROTAC concentrations actually reduce degradation efficiency—requires careful dosing considerations 2 . Additionally, cells may develop resistance by downregulating E3 ligases or mutating binding sites 7 .
Researchers are addressing these challenges through innovative approaches like molecular glues (smaller molecules that induce protein-protein interactions without a linker), dual-action PROTACs, and advanced delivery systems such as nanoparticles 7 .
The story of pirin degradation exemplifies how PROTAC technology is revolutionizing both basic research and therapeutic development. What began as a method to confirm target engagement has blossomed into a powerful strategy for exploring cellular function and developing precision medicines.
PROTACs represent a shift from "occupancy-driven" to "event-driven" pharmacology 1 . Instead of merely blocking protein function, we can now eliminate problematic proteins entirely—a capability that could transform treatment for countless conditions.
The journey from understanding a protein's role in cancer cells to designing molecular machines that remove it represents the cutting edge of biomedical science. As PROTACs continue to advance through clinical trials, we move closer to a future where precisely removing disease-causing proteins becomes a standard therapeutic approach, offering new hope for patients with conditions once considered untreatable.