This episode breaks down a PNAS study that identifies a druggable pocket in the ERG transcription factor PNT domain and describes PBITE-1, a small-molecule probe that binds this pocket to inhibit ERG-driven prostate cancer models.
0:00Welcome to Base by Base, the paper cast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Thanks for having me back. So for decades now, drug hunters have looked at a very specific class of proteins, and honestly, just thrown their hands up in absolute frustration.
0:19Yeah, total frustration. Right. We are talking about transcription factors. These proteins are basically the ultimate bosses of the cell. They really are. They sit deep inside the nucleus, scanning your DNA, and they flip genetic switches on or off to dictate exactly what a cell is supposed to do.
0:37Which is a huge amount of power for one protein to have. Exactly. So you would think that when one of these boss proteins goes rogue, you know, and starts driving a massive cancer, you would just design a drug to shut it down.
0:49would think so, yeah. But the pharmaceutical industry has a long labeled these transcription factors as completely hopelessly unruggable. Undruggable. like the worst word in oncology. It is. I mean, imagine a rock climber trying to scale a perfectly smooth, solid glass skyscraper.
1:06Good luck with that. Right. When you look at the structure of a transcription factor, there are typically no natural pockets. There are no obvious crevices or uh, enzymatic active sites for a small molecule drug to wedge into.
1:21Just a flat, slippery surface. So what really happens when the most common genetic driver in a major cancer is one of these supposedly untouchable bosses. That is the $1000000 question for this deep dive.
1:34And how could finding a single hidden microscopic pocket completely change the future of cancer treatment? Well, I mean that biological paradox is what makes this so fascinating. Scientific community had largely just accepted this structural limitation.
1:47They just gave up. Essentially yes. Finding a way to drug a transcription factor wasn't just a hurdle. It was considered biochemically impossible by a lot of experts. Wow. But, you know, when a problem affects 100s of 1000s of people, accepting that limitation isn't really an option.
2:01You have to look closer at the glass to find a crack. And somebody finally did. Today, we celebrate the work of RLM Chanan and the research team at the University of Michigan, who have advanced our understanding of targeting transcription factors in cancer.
2:13It's phenomenal. It really is. They published this in July 2026 in PNAS, and it outlines a massive paradigm shift. A total landmark approach. But to understand the gravity of the science, you 1st have to look at the clinical scope of the enemy they're fighting here.
2:30Right, which is prostate cancer. Exactly. And the numbers are staggering. This disease causes roughly 313,780 new cases and about 35,770 deaths annually in the United States alone. 300,000 new cases. That is, wow, that is a massive footprint.
2:46It's huge. And behind a huge percentage of those cases is one specific genetic anomaly. Right. The researchers set their sights on this genomic villain known as the TMPRS2 dot ERG gene fusion. Which is a bit of a mouthful.
2:59I know. Yeah, TMPRSS dot ERG. But a gene fusion, just to clarify for everyone. That happens when chromosomes literally break apart and then stitch themselves back together incorrectly, right? Yes, exactly.
3:10So you end up with a hybrid gene that just never should have existed in nature. That is the exact mechanism. Two entirely separate genes are suddenly fused together. And uh, this isn't some rare one in a 1000000 anomaly either.
3:23No. No, this specific fusion occurs in approximately 50% of prostate cancers in patients of European ancestry. Wait, 50%? Half of them. It is one of the most frequent structural genomic alterations across all human solid tumors.
3:37That is insane for a single genetic event. So what does this hybrid gene actually do to the cell's internal wiring when it forms? It basically sets up a catastrophic signaling loop. So normally the TMPRS is 2 gene is under the strict control of the androgen receptor, or AR, and the androgen receptor responds to male hormones, androgens, and acts as a standard, totally normal, regulated growth signal for prostate tissue.
4:03Makes sense. But the ERG gene, on the other hand, contains the blueprint for building the ERG transcription factor. Which is one of those boss proteins we talked about earlier. Exactly. So when these 2 chromosomes abnormally fuse, the ERG protein is violently ripped from its normal regulatory environment. Oh, I see.
4:20And it gets placed directly under the control of the antigen receptor. So the cell's normal hormone signals get completely hijacked. Whenever the androgen receptor just says grow for basic maintenance, it doesn't just trigger normal stuff.
4:32It pumps out massive, toxic amounts of this ERG transcription factor. Massive aberrant overexpression. And once ERG is over expressed to that degree, It takes over as a trunkle oncogenic driver. It fundamentally reprograms the cell's DNA landscape.
4:50But wait, how does a transcription factor actually reprogram the landscape? Like, it doesn't mutate the DNA itself, does it? No, no, it alters accessibility. What do you mean? Well, your DNA is normally wound up very tightly around these spool-like proteins called histones.
5:03Right, to keep it organized. Yeah, exactly. But ERG physically hijacks massive molecular machines in the cell, specifically chromatin remodeling complexes like this YSNF complex. Yeah, Suisway SNF. It normally slides those histones around just to expose necessary genes when you need them.
5:21But the overexpressed ERG forces the squeezes NF complex to persistently unspool the specific regions of DNA that promote massive tumor cell proliferation and invasion. Oh, wow. Yeah, it basically forces the cell to read the cancer playbook on an endless loop.
5:38That makes you think of like a hot wired car. The engine, which in this case is ERG, is constantly redlining, because the ignition, the TMPRS2 promoter driven by androgens is just jammed in the on position.
5:51That is a perfect visualization. The ignition is completely jammed, and that brings up a really crucial clinical problem, which is that standard therapies eventually fail. they're targeting the wrong thing.
6:01Well, the frontline medical approach for advanced prostate cancer has historically been androgen deprivation therapy, or next generation AR inhibitors, like the drug and zleudomide. Okay, so trying to shut off the hormones.
6:12Right. The logic is totally sound. If the ignition is jammed, try to cut the fuel supply. But the tumor is incredibly adaptable, right? If you block the androgens, the cancer eventually figures out a workaround.
6:23Tumors are under immense evolutionary pressure. Over time, the cancer cells inevitably mutate the androgen receptor, or they amplify it, or they just find bypass pathways to restore that signaling. And the moment that IR signaling is restored.
6:38The TMPRSS 2.ERG fusion kicks right back in, and that oncogenic ERG engine is running at full speed all over again. Which means cutting the fuel just isn't enough. If you want to stop a treatment resistant tumor, you have to destroy the engine itself.
6:51Exactly. You have to target ERG directly. But going back to our earlier point, the entire field believed ERG was that perfectly smooth glass skyscraper with literally nowhere for a drug to bind. And that is exactly where this research team decided to challenge the dogma.
7:06But before you spend years hunting for a microscopic pocket on an undrugable protein, you have to be absolutely certain that destroying the protein will actually kill the cancer. Right. You have to prove the tumor is biologically addicted to ERG first.
7:19Precisely. So how do you definitively prove that addiction in a lab without just, you know, dumping toxins on a cell and hoping for the best? They turned to computational biology and genetic engineering.
7:28The researchers 1st query the cancer dependency map or debt map. Yeah, it's this exhaustive public database where scientists use CRISPR cast 9 to systematically knock out or delete individual genes across over 1770 different cancer cell lines.
7:45Oh, so they computationally sifted through 1000s of cell lines just to see what happens when the ERG gene is deleted. Yes, exactly. And the debt map data definitively showed that knocking out Erie was highly lethal to a specific metastatic prostate cancer soline called VCAP.
8:00Let me guess, VCAP harbors that exact TMPRS2.erg fusion. You guessed it. Interestingly, it was also essential for survival in certain leukemia cell lines, like REH and ALM 16. Okay, so the computational data flags ERG as a vital dependency.
8:15But a database is just a starting point. How did they validate that inside a living tumor model? The engineered VCAP cells with an inducible short hairpin RNA or SHRNA system? Right. Think of HHRNA as a customized molecular assassin that specifically hunts down and destroys the Messenger RNA instructions for ERG before the protein can even be built.
8:38Well, that's clever. But they made it inducible, meaning it is controlled by a chemical switch. So they can let the cancer cells grow normally and then flip a switch to suddenly rip the ERG protein away.
8:50Exactly. The switch in this case is the antibiotic doxycycline. Okay. When they added doxycycline to the V capsules, the HHRNA activated, and the ERG protein levels just plummeted. The results were immediate.
9:02What happened? The cancer cells stopped dividing and underwent massive apoptosis. They basically triggered their own cellular self-destruct sequence. Okay, target validated. If you take out ERG, the cancer crashes.
9:13But knowing the target is essential, still doesn't give you a drug. No, it doesn't. This is where the structural biology comes in, right? They had to look at the ERG protein and find a vulnerability somewhere in that glass building.
9:25And that's hard because transcription factors are notoriously floppy and unstructured in many regions, which makes them terrible drug targets. Just totally chaotic. Exactly. But ERG has a specific, highly structured region called the N terminal pointed domain or PNT moment.
9:41PNT domain. Yeah, it is a bundle of 4 alpha helices. Because it has a stable, rigid 3D shape. It presented really the only logical place to hunt for a hidden pocket. So to find a chemical key that might fit into this hypothetical pocket.
9:56The team utilize a technique called differential scanning fluorimetry or DSF. And they screened 1655 different chemical compounds. Walk us through how you screen compounds by melting them. Well, it all comes down to the thermodynamics of protein folding.
10:12Proteins naturally fold into specific energy efficient 3D shapes and they usually hide their water repelling hydrophobic amino acids in the deep inner core. Okay. Keeping the water hating parts inside.
10:23Right. Now if you slowly heat a protein up, you increase its kinetic energy until it eventually denatures or unfolds, exposing that hidden core. Got it. In a DSF assay, you add a sparilized fluorescent dye that only lights up when it binds to those exposed hydrophobic regions.
10:39So you put the protein in a tube. Slowly crank up the heat, and the moment the protein melts and unfolds, the dye binds and the whole sample lights up. You can pinpoint the exact melting temperature. Precisely.
10:50Now, if you add a small molecule drug to that tube and it physically wedges into a pocket on the protein, it lowers the free energy of the folded state. It acts like a structural anchor. Exactly, because the protein is physically stabilized by the drug.
11:05It takes significantly more thermal energy, more heat to force it to unfold. Wait, so they essentially boiled the proteins. Is that like adding a heavy steel beam to a wobbly wooden bridge to see if it survives a windstorm?
11:17That is exactly it. That is an excellent way to conceptualize the thermodynamics at play. You apply kinetic stress. The windstorm or the heat, and you measure if the compound provides structural integrity.
11:27Wow, okay. And out of the 1655 chemical keys they tested, they found a single hit. Just one. Just one. A compound named F0341. When they added F0341 to the PNT domain at the concentration of 400 micromolar, the melting temperature shifted by a massive 7.2 degrees Celsius.
11:47That is a huge stabilization. So they knew as 0341 was physically grabbing onto this supposedly undruggable protein. Yes. And they didn't just trust the test tube, right? They used a method called the cellular thermal shift essay, or seiza to verify this inside living cells.
12:04is critical because a cell is a chaotic, crowded environment. Totally unlike purified water in a test tube. Right. Right. There's a lot of other stuff there. Exactly. Seats approved that F0341 could penetrate the cell membrane, navigate the cellular machinery and still successfully bind and thermally stabilize the ERG protein in Vivo.
12:23But F0341 was just a starting point. It proof the glass skyscraper had a microscopic handhold, but it wasn't a potent drug yet. No, it was a very weak diner. To turn a weak chemical hit into a viable molecular probe.
12:36You have to enter the grueling phase of medicinal chemistry known as structure activity relationship, or SAR optimization. Which is basically the trial and error phase. The chemists take the F0341 molecule and just start modifying it atom by atom.
12:49Exactly. They systematically alter the electron density and the steric bulk of the molecule. They actually synthesize over 100 structural analogs. Over 100. Yeah, they would tweak a piper dine ring or, you know, shift in a man mean group, add a methyl group, and then constantly retest the new molecule to see if it bound tighter or possessed better solubility.
13:09It is an exhaustive process of mapping the chemical space. And through that whole chemical evolution, they created their lead compound, PDEEU. That stands for PNT binding inhibitor of the transcription factor, ERG.
13:22PBE one was the breakthrough. It pushed the thermal stabilization even higher, hitting a melting temperature shift of 8.7 degrees Celsius. More importantly, it improved the binding affinity, dropping the dissociation constant, or KD, to 264 micromolar.
13:38Okay, hold on. If you look at standard approved clinical drugs, They often have a KD in the anomaler range. Isn't 264 micromolar objectively very weak for a drug? As a Finnish clinical therapeutic, yes, it is incredibly weak.
13:51An anomola drug binds 1000s of times more tightly, but, you know, you have to view this in context. Right, the context of it being impossible. Exactly. This is a transcription factor that has divide all drug discovery efforts for decades, achieving a verifiable KD of 264 micromolar on a flat, featureless protein surface is a monumental proof of concept.
14:12It proved the pocket exists and is legandable. It's the wedge in the door. To figure out exactly where that wedge was sitting, they utilized nuclear magnetic resonance spectroscopy, or NMR, alongside computational molecular docking.
14:27NMR is vital here because the pocket they were targeting involved a flexible loop on the protein. Why does that matter? Well, x-ray crystallography, which is the standard way to take a picture of a protein, requires freezing the protein into a rigid crystal.
14:40Oh, and a flexible loop wouldn't freeze well. Exactly. But NMR allows you to look at the protein in solution at an atomic resolution while it is moving and breathing. So they mapped the exact footprint of P by U1.
14:52It wasn't just sticking to the surface randomly. Not at all. It was slotting perfectly into a highly discrete pocket, formed by that flexible loop, and 2 specific alpha helises, designated H2 and H6. The NMR data showed exactly which amino acids were interacting with the drug.
15:08PDDR1 wedges right into the hydrophobic side chains of Voline 125, Isolusine 126, and Voline 127. Achieving that level of atomic resolution on a dynamic protein domain is just a triumph of structural biology.
15:25But, you know, a chemical binding in a tube or even stabilizing a protein in a cell is only half the battle. If key by D1 is going to mean anything for prostate cancer, it has to actually stop the tumor cells from growing.
15:37Which is why the researchers tested PBD one across multiple biological models, they used the VCAP cell line, they grew 3D tumor organoids derived from human patients, and they even used mice xenographs where human prostate tumors were implanted into mice.
15:50And the biological response was pretty undeniable. When they treated these models with TBR one, it profoundly suppressed colony formations. The cancer cells lost their ability to cluster and expand, and it induced massive widespread apoctosis.
16:03And you can track that cellular death through specific biochemical markers. For instance, the researchers observed a sharp increase in a marker called cleaved PRP. What exactly does cleaved PRP tell us about the cells internal state?
16:15PRP is a protein involved in DNA repair. When a cell commits to apoptosis, It activates these specialized executioner enzymes called cast spaces that literally chop up the cell's vital proteins to ensure it dies cleanly.
16:30It chops up its own repair proteins. Right. So when we see Cleave PRP on a Western blot, we know the cast bases are active and the cell's self-destruct sequence is fully underway. They also saw an increase in P21, right?
16:42Yes, P21 is a master regulator that forcefully hits the brakes on the cell division cycle. Making sure it doesn't replicate. Right. And concurrently, they saw a significant decrease in C mic, which is one of the most potent oncogenic growth signals in human biology.
16:55So not only was the cell dying, but the researchers proved P by PO was actually stopping ERG from doing its specific job as a transcription factor in the 1st place. It silenced the downstream genes that ERG normally forces open genes with names like TDRD1, PAT, and PLA1A.
17:13The boss was effectively gagged. Completely silenced. But wait, if it's causing this much apoptosis, is it just a toxic sledgehammer? Does it kill healthy cells too? That is always the primary concern when evaluating a new compound, right?
17:27A drug that kills cancer by killing everything isn't a drug. It's a poison. Exactly. But this is where P byte one demonstrates its most crucial characteristic. Selectivity. Oh, really? Yeah. When the researchers applied P byte one to models that did not have the TMPRSS2.erG fusion cells, there were ERG negative, the compound was largely ignored.
17:48just passed right by them. It spared the healthy models entirely. Yeah. It only induced apoptosis in the specific prostate cancer cells that were entirely addicted to the ERG oncagene. The selectivity is the real victory here.
18:01So stepping back from the assays and the NMR data, what does the structural breakthrough actually mean for the future of medicine? It completely overturns a massive restrictive paradigm in cancer biology.
18:12The ERG protein belongs to a larger family of related proteins called the ETS family of transcription factors. Okay, the ETS. And for years, the entire ETS family was deemed undruggable. This research proves that the PNT domain is officially a tractable, legandable target.
18:28The glass skyscraper actually has a structural flaw we can exploit. Now, the authors are very careful in the paper. They explicitly describe P byte one as a molecular probe, rather than a final pharmacy ready therapeutic.
18:42It's really a tool for discovery. Absolutely. Pea byte one, with its weak binding affinity is not going into human clinical trials tomorrow. Right. But it provides the exact chemical scaffold. The exact atomic blueprint needed for the next era of therapeutics.
18:57And what makes this so incredibly exciting is how it opens the door for a new technology called Protax. Wait, a Protax Protex stands for proteolysis targeting Cameras, right? How does a protac differ from a traditional inhibitor, like P byte one?
19:10So traditional drugs bind to a target and try to inhibit its function. They try to block the gears of the machine. Like jamming a wrench in it. Exactly. Protax are entirely different. There are essentially 2 headed molecules.
19:21One head binds to your specific target protein. The other head recruits an enzyme from the cell's own natural garbage disposal system, an enzyme called an E3 ubiquitan lygus. So the Protac grabs the rogue ERG protein with one hand, grabs the cell's trash disposal unit with the other, and pulls them together.
19:41That physical proximity allows the E3 Legus to slap a biochemical trash sticker, a chain of ubiquitous molecules onto the ERG protein. Right, and then what? Once ERG is tagged with ubiquitan, the cells produce sum, which is basically the cellular shredder, recognizes the tag, reels the protein in, and completely dismantles it into harmless amino acids.
20:03You don't just inhibit the target, you erase it from existence. And the brilliant strategic advantage of a protect is that the binding head doesn't need to block a functional site perfectly. Oh because it's just grabbing it.
20:15Right. It doesn't need an animal or binding affinity to hold the gears in place. It just needs to grab onto the protein anywhere. for a fraction of a second, just long enough to deliver the trash tag. And because P byte one binds so specifically to the ERGPNT domain, it can serve as the perfect targeting head for a future ERG destroying protac.
20:34So P Bit one is like planting a homing beacon on the glass building, so a wrecking ball knows exactly where to strike. That analogy hits the nail on the head. We now have the coordinates for the homing beacon, and that changes everything for patients with advanced treatment resistant prostate cancer driven by this fusion.
20:48It really is a phenomenal journey of discovery. So let's distill everything you've heard today down to the core takeaway. For decades, the ERG transcription factor was an untouchable trunkal driver of prostate cancer, leaving patients with resistant disease basically out of targeted options.
21:06But by meticulously discovering a hidden microscopic pocket in the ERGPNT domain, and engineering a bespoke molecule to fit perfectly inside it, researchers have finally rendered this elusive oncogene druggable.
21:20They have shattered the dogma and mapped an entirely new route to treat resistant cancers. It serves as a really powerful reminder that in biochemistry, the label undruggable is rarely permanent. It usually just means we haven't analyzed the structure from the right angle or, you know, under the right conditions yet.
21:38It really makes you wonder, what does this mean for the 100s of other undruggable proteins hiding in the human genome? just waiting for their own molecular keys to be discovered? It's brand new frontier.
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