A Fragment Screen Identifies Acrylamide Covalent Inhibitors of the TEAD/YAP Protein-Protein Interaction

This study identifies acrylamide fragments that covalently bind to a conserved cysteine in the TEAD palmitate pocket, thereby inhibiting the TEAD-YAP protein-protein interaction and providing a foundation for developing allosteric covalent inhibitors of the Hippo pathway.

The Gist

The Big Picture: Stopping a Cancer "Super-Team"

Imagine your body is a bustling city. Inside the cells, there is a security system called the Hippo Pathway. Its job is to keep cell growth in check, like a traffic light preventing too many cars from entering an intersection.

In healthy cells, this traffic light works perfectly. But in cancer, the light gets stuck on "green." This allows two key proteins, YAP and TEAD, to team up. Think of them as a Master Key (YAP) and a Lock (TEAD). When they click together, they unlock a door that tells the cell to grow, divide, and spread uncontrollably. This leads to tumors.

Scientists have tried to break this team up before, but the place where they hold hands (the "interface") is smooth and slippery—like trying to pry apart two smooth glass plates with a tiny tool. It's considered "undruggable."

The Clever Hack: The Hidden Back Door

However, the TEAD protein (the Lock) has a secret feature. It has a deep, hidden palm-sized pocket (called the palmitate pocket) on its side, far away from where it holds hands with YAP. Usually, the cell fills this pocket with a fatty oil called palmitate to keep the lock stable.

The researchers realized: If we can jam a tool into this hidden pocket, we might be able to twist the lock just enough so it can't hold the Master Key anymore. This is called allosteric inhibition—changing the shape of the lock from the inside out to break the connection on the outside.

The Search: Fishing for the Right "Screw"

The team needed a tool small enough to fit in the pocket but strong enough to stick. They used a fragment screen, which is like throwing a net full of thousands of tiny, different-shaped Lego bricks (fragments) into a bucket of TEAD proteins to see which ones stick.

They were looking for a specific type of brick: an acrylamide. Think of this as a "sticky screw." It doesn't just sit in the pocket; it chemically bonds (screws itself) into a specific spot inside the pocket.

The Results:

• They found a few "hits." One specific fragment, named Fragment 1, was the winner.
• It successfully screwed itself into the TEAD protein.
• Crucially: When they tested it, the TEAD protein could no longer hold hands with YAP. The "Master Key" fell off.

The Upgrade: Tuning the Screw

The first fragment worked, but it was a bit slow and weak. So, the scientists acted like master mechanics. They took the basic design of Fragment 1 and built four new, improved versions (Derivatives 14–17).

They tweaked the shape slightly:

• They made the "handle" of the screw stiffer.
• They moved a chemical group (a trifluoromethyl group) to different positions, like moving a weight on a seesaw to find the perfect balance.

The Best Result:
One new version, Compound 14, was a superstar. It screwed into the TEAD protein much faster and tighter than the original. It was like upgrading from a rusty screwdriver to a high-speed power drill.

The Surprise: Different Locks, Different Keys

Here is where it gets fascinating. The human body has four versions of this "Lock" (TEAD1, TEAD2, TEAD3, and TEAD4). They look almost identical, like four slightly different models of the same car.

The researchers expected their new "power drill" (Compound 14) to work equally well on all four. It didn't.

• TEAD1 and TEAD3: The drill worked incredibly fast.
• TEAD2 and TEAD4: The drill was much slower.

Why?
When they looked under the microscope (using X-ray crystallography), they saw the drill was sitting in the pocket differently depending on which model of the lock it was in.

• In TEAD3, the drill sat deep and straight, mimicking the natural oil perfectly.
• In TEAD2, the drill had to twist and turn to fit because a tiny bump in the pocket blocked the straight path.

This taught them that even tiny differences in the "lock's" internal shape change how well the "key" fits. This explains why the drug works better on some cancer types than others.

The "Allosteric" Magic: How a Side Pocket Stops a Handshake

You might wonder: If the screw is in a pocket on the side, how does it stop the handshake in the middle?

Imagine a door with a heavy spring. The door handle (the handshake) is on the front. The researchers put a wedge (the drug) into a hidden latch on the side of the door frame.

1. The wedge jams the latch.
2. This causes the whole door frame to warp slightly.
3. Even though the wedge isn't touching the handle, the warping makes the handle too stiff to turn.

The drug doesn't block the handshake directly; it changes the shape of the TEAD protein so that the handshake becomes weak and falls apart.

The Bottom Line

This paper is a blueprint for a new type of cancer medicine.

1. The Problem: Cancer proteins hold hands too tightly, and we couldn't pull them apart.
2. The Solution: We found a hidden pocket on one of the proteins and jammed a "sticky screw" into it.
3. The Result: This screw twists the protein's shape, causing the cancer-promoting handshake to fall apart.
4. The Future: By understanding exactly how these screws fit into different versions of the protein, scientists can now design even better, more precise drugs to stop cancer growth without hurting healthy cells.

It's a classic case of finding a back door when the front door is locked tight.

Technical Summary

1. Problem Statement

The Hippo pathway is a critical regulator of cell growth and organ size. Dysregulation of this pathway, specifically the hyperactivation of the TEAD•YAP/TAZ protein-protein interaction (PPI), drives tumorigenesis and metastasis in various cancers.

• The Challenge: The TEAD•YAP interface is a large, flat surface (>1000 Ų) lacking a deep binding pocket, rendering it traditionally "undruggable" by small molecules.
• The Opportunity: TEAD proteins possess a unique, deep palmitate-binding pocket located outside the PPI interface. This pocket contains a highly conserved cysteine residue (Cys367 in TEAD4) that forms a thioester bond with the native palmitate lipid.
• The Goal: To identify small-molecule covalent inhibitors that target this palmitate pocket, react with the conserved cysteine, and allosterically disrupt the TEAD•YAP interaction. While previous covalent inhibitors (e.g., TED-347) and non-covalent binders exist, there is a need for new chemical scaffolds, particularly acrylamide-based fragments, to explore structure-activity relationships (SAR) and improve potency/selectivity.

2. Methodology

The authors employed a multi-disciplinary approach combining fragment-based drug discovery (FBDD), biophysics, structural biology, and kinetics:

• Fragment Screen: A library of 372 acrylamide electrophile fragments was screened against TEAD4 using a Fluorescence Polarization (FP) assay. The assay utilized a fluorescently labeled YAP peptide (FAM-YAP60–99) to detect inhibition of TEAD4•YAP binding.
• Hit Validation:
  • Mutant Analysis: Hits were tested against the TEAD4C367S mutant to confirm covalent mechanism (loss of inhibition in the mutant implies reaction at Cys367).
  • Mass Spectrometry (MS): Whole-protein MS was used to confirm covalent adduct formation and determine adduct mass.
  • Dialysis Studies: To verify the irreversibility of the inhibition.
• SAR and Derivative Synthesis: The lead fragment (Compound 1, ACR-021) was used as a scaffold to synthesize four derivatives (Compounds 14–17) with modifications to the warhead rigidity and trifluoromethyl positioning.
• Kinetic Profiling: Time- and concentration-dependent studies were conducted for all four TEAD paralogs (TEAD1–4) to determine:
  • $k_{obs}$: Pseudo-first-order rate constants.
  • $k_{inact}$: Maximum rate of inactivation.
  • $K_I$: Binding constant.
  • $k_{inact}/K_I$: Second-order rate constant (measure of overall potency).
  • $t_{1/2}^{\infty}$: Reaction half-life.
• Structural Biology: Co-crystal structures of TEAD2 and TEAD3 in complex with the lead derivatives were solved to visualize binding modes and explain selectivity.
• Allosteric Mechanism Testing: Site-directed mutagenesis (F393A, F415A in TEAD4) was performed to test the hypothesis that engagement of specific pocket residues drives allosteric inhibition.

3. Key Contributions

• Identification of Novel Acrylamide Fragments: The study successfully identified acrylamide fragments that covalently modify the palmitate pocket cysteine and inhibit TEAD•YAP binding, expanding the chemical space for TEAD inhibitors.
• Discovery of Selectivity via Binding Mode: The paper reveals that subtle differences in the amino acid composition of the palmitate pocket (specifically TEAD2 vs. TEAD3) lead to drastically different binding modes and kinetic profiles for the same compound.
• Mechanistic Insight into Allostery: The authors provide structural evidence that the inhibition mechanism involves local conformational changes induced by the compound's engagement with specific pocket residues, rather than global protein unfolding.
• Kinetic Characterization: Comprehensive kinetic data ($k_{inact}$, $K_I$, $k_{inact}/K_I$) for four TEAD paralogs across multiple compounds, highlighting the importance of reaction rates over simple binding affinity for covalent allosteric inhibitors.

4. Key Results

Fragment Screening and Lead Identification

• Hits: From 372 fragments, 13 showed >40% inhibition. Only Fragment 1 (ACR-021) and Fragment 2 (ACR-362) showed covalent inhibition dependent on Cys367 (no inhibition of TEAD4C367S).
• Lead Compound 1 (ACR-021):
  • IC50 (TEAD4): 4.6 µM (24h incubation).
  • Mechanism: Irreversible covalent adduct formation (+284 Da mass shift) confirmed by MS.
  • Selectivity: Showed high reactivity toward TEAD1 and TEAD3, but lower reactivity toward TEAD2 and TEAD4.

Derivative Optimization (Compounds 14–17)

• Compound 14 (ACR-374): A rigidified derivative of Fragment 1.
  • Potency: Significantly improved kinetics. For TEAD3, $k_{inact}/K_I$ reached 128 M⁻¹s⁻¹ (compared to 41.4 for the parent).
  • Selectivity: Highly selective for TEAD1 and TEAD3. It showed minimal inhibition of TEAD2 and TEAD4 binding to YAP at 100 µM, despite covalent binding.
  • Kinetics: $t_{1/2}^{\infty}$ for TEAD3 was reduced from ~9 hours (parent) to <1 minute.

Structural Insights (Crystallography)

• TEAD2 (Monomer A & B): Compound 14 binds covalently to Cys380 (the conserved cysteine). In Monomer A, the trifluoromethylphenyl group occupies the "lower" sub-pocket. In Monomer B, the compound binds to a non-conserved Cys343 (closer to the YAP interface) while maintaining the same orientation of the aromatic ring.
• TEAD3: Compound 14 binds covalently to Cys368. Crucially, the binding mode mimics native palmitate, with the trifluoromethylphenyl group deeply occupying the "lower" sub-pocket.
• Selectivity Explanation: The authors propose that the palmitate-mimicking mode in TEAD3 stabilizes the protein, potentially enhancing YAP binding or failing to induce the necessary allosteric disruption. Conversely, the binding mode in TEAD2/TEAD4 (occupying the middle/upper pockets) induces conformational changes that disrupt the PPI. This explains why TEAD3 is highly reactive (fast covalent bond) but poorly inhibited functionally.

Allosteric Validation

• Mutating Phe393 and Phe415 in TEAD4 (residues contacting the compound in the crystal structure) to Alanine resulted in enhanced inhibition by Compound 14 and a previous inhibitor (TED-642).
• This supports the hypothesis that steric clash or specific engagement of these phenylalanine residues is required to trigger the allosteric conformational change that disrupts YAP binding.

5. Significance

• New Chemical Scaffolds: This work introduces acrylamide fragments as a viable starting point for developing covalent TEAD inhibitors, distinct from previous vinyl sulfone or cyanamide warheads.
• Paralog Selectivity: The study highlights the extreme difficulty in achieving pan-TEAD inhibition due to subtle structural differences in the palmitate pocket. It suggests that "reactivity" (covalent bond formation) does not always equate to "functional inhibition" (PPI disruption).
• Allosteric Drug Design: The findings reinforce that for allosteric covalent inhibitors, the reaction rate ($k_{inact}$) and the specific conformational change induced are more critical than high binding affinity ($K_I$). High affinity might even stabilize the active conformation, counteracting inhibition.
• Future Directions: The authors suggest that future optimization should focus on the central and upper sub-pockets of the palmitate site to maximize the allosteric disruption of the TEAD•YAP interface, rather than simply deepening the lower pocket (which mimics palmitate).

In summary, this paper provides a rigorous structural and kinetic dissection of how acrylamide fragments inhibit TEAD proteins, revealing that successful inhibition requires not just covalent binding, but a specific binding geometry that triggers allosteric destabilization of the PPI interface.