Transcription factors (TFs) are crucial for gene expression but have long been tough drug targets. Peptides, which can bind to broad and shallow protein surfaces, offer a promising way to reach these targets inside cells.
Researchers at the University of Bath, UK, developed a method that found a strong inhibitor of the transcription factor cJun, a protein linked to melanoma and other cancers. They used a large peptide library to find candidates that could bond with a specific cysteine (C269) in cJun’s DNA-binding region.
A key feature of their method was adding a “warhead” — a chemical group that reacts with the target site. In this case, the warhead formed a permanent bond with the cysteine in cJun, creating an irreversible and selective inhibitor. When designed well, such covalent inhibitors can be more powerful and longer-lasting.
To do so, the team swapped a reversible bonding cysteine in a peptide for the synthetic warhead to make the bond permanent. The final product, called HW33, was stronger, more selective, and more effective in cell tests. In melanoma cells, HW33 lowered cJun levels and reduced cell survival, opening the door to similar irreversible inhibitors for other hard-to-target proteins.
We spoke with lead researcher Jody Mason about the difficulties of using this method in live cells and how their peptide library might help find new treatments for tough-to-target proteins like transcription factors.
You used a lab test to find drugs that permanently block transcription factors, which help control gene activity. What were the challenges in changing this test to look for permanent blockers, and why might these be better than temporary blockers?
One of the biggest surprises was how difficult it was to make the special kind of peptide needed to form a permanent bond with the target. We spent a lot of time getting the chemistry just right. And in the end, that version wasn’t even the most effective one.
Another major challenge was making sure these permanent blockers didn’t result in non-specific toxicity or off-target effects, especially in a live-cell context. That’s where our screening platform really helps. Because it works inside living cells, it automatically filters out any molecules that are harmful or do not work as intended.
The key advantage of permanent blockers over temporary ones is that once they attach to the target, they stay attached. This makes them more powerful and longer lasting. That is especially helpful when dealing with targets like transcription factors, which are usually very hard to block with traditional drugs.
You found one specific spot on the cJun protein that’s best for locking it down with a drug. Did anything surprise you about how you picked that spot or how you designed the drug to fit it?
What surprised us was that the best spot to attach the drug, called C269, is not buried deep inside the cJun protein. That made it more accessible than we expected.
We also found that the way the peptides fit and interacted with the protein inside the cell had a big impact on how well the drug worked. These effects are very hard to predict just by looking at the protein structure in a computer model.
Another interesting finding was that certain peptides sequences kept placing the active part right next to C269. This showed that both the peptide sequence and the position of the reactive group were key to getting a strong and specific effect.
Overall, it shows how powerful our screening method is. Because it works in live cells, it can uncover drug binding spots and behaviours that you might miss with standard lab tests.
You tried different chemical methods to make the drug stick permanently to the protein. What helped you decide which worked best, and do you think other types of “sticky” chemicals could work in the future?
The final choice was driven by how well the peptide could react, how specific it was, how well it fit in the right position, and whether it stayed stable inside cells. The best ones were those that only formed a strong bond when the peptide was holding them in just the right place, which helped avoid unwanted sticking to other proteins.
There is definitely room to explore other types of “sticky” chemicals in the future. Some may let us finetune how reactive they are or target different parts of a protein, not just cysteine. That could open the door to designing drugs for a wider range of hard-to-treat targets.
Your drug was able to stick to cJun without accidentally sticking to CREB1, a similar protein. How did you make it so specific, and what else could you do to ensure it’s targeting the right protein?
We made the drug highly specific by carefully designing the peptide to bind to cJun and testing how it worked in living cells. The design made sure that the reactive part of the drug lined up perfectly with the part of cJun called C269.
Even though the similar protein CREB1 also has this same spot, the peptide did not stick to it. That is because the first step, where the peptide attaches to the protein, does not happen with CREB1. Without that first connection, the chemical bond never forms.
To be even more sure the drug only targets cJun, we could do future tests that look across all proteins in the cell to check for any unwanted binding. We could also change the C269 site in cJun to confirm that the drug really needs that exact location to work.
How could this approach be improved to make it safe?
There are several ways we can make this approach safe. One option is to finetune how reactive the chemical part of the drug is, so it only forms a bond when the peptide is sitting in exactly the right place on the target protein.
We already use structure-based design to help the peptide fit its target very precisely and to keep the reactive part away from other proteins. We can also use proteomics. This is a method that looks at all the proteins in a cell to check for any unwanted effects early in the process.
Another idea is to use special types of reactive groups that only become active in certain conditions, such as in the low oxygen or acidic environment of a tumor. This would help limit the effect to cancer cells and reduce the risk to healthy tissue.
Are there particular cancers or disease models where you see the greatest potential for covalent transcription factor inhibitors beyond cJun?
This approach has the potential to be used in many different diseases, not just cancer. Our screening method, called TBS, works inside living cells and does not depend on a specific target. That means we can use it to find blockers for many different transcription factors, which are proteins that help turn genes on and off.
These proteins are often overactive in cancer, so that is a clear area where this method could be very useful. But they also play a role in autoimmune diseases and possibly even in neurodegeneration. We believe this platform could help create new treatments for all these areas by finding powerful and specific blockers for the key proteins involved.
What improvements in intracellular delivery methods would be needed to enhance the therapeutic viability of these peptide inhibitors?
We have already shown that these peptides can enter cancer cells and directly reach their target, cJun. That is an important first step.
However, to turn these into medicines, we may need to improve how well they travel through the body and how much of the drug reaches the right cells. This could involve better delivery systems that protect the peptides, help them stay in the bloodstream longer, or guide them more directly into tumour cells.
Making these improvements will help us move from promising lab results to treatments that can work safely and effectively in patients.
What are the biggest challenges in turning these special protein-targeting drugs into real medicines that can work safely and effectively inside the body?
One of the biggest challenges is making sure these peptides can sufficiently enter into cells in the body at the right amount to have an effect. Like any drug, they also need to stay stable in the body for long enough to work and have the right balance of how they are absorbed, distributed, and cleared. We also need to prove that they only stick to the right target, and that they work across different types of cells without causing side effects. Finally, it is important to show that they can be made in large enough amounts and at high purity for use in real medicines. However, this is usually more straightforward with peptides than with many other drug types as they are built up from amino acid modules.
Could your approach be generalized to other difficult-to-drug targets?
Absolutely. The real strength of our approach is that it lets us test large libraries of peptides directly inside living cells, while also adding chemical groups that can form permanent bonds.
This method is very flexible and can be adapted to many tough targets, not just transcription factors, but also proteins that help hold other proteins together or those without clear binding pockets. These are the kinds of proteins that traditional drugs often cannot reach.
By carefully designing the peptide and choosing where to place the reactive chemical group, we can create strong and selective binders for each new target. This gives us a powerful, target independent way to find new treatments for diseases that were previously out of reach.
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