Cells must integrate information from their environments to make decisions. To do this, they have developed complex signaling networks that govern proliferation, survival, and migration in response to various cues. Cell signaling is frequently dysregulated in cancers, leading to uncontrolled tumor growth or spread to distant sites in the body.
Kinases are a family of proteins that function as critical integrators of cell signaling. Mechanistically, they transfer phosphate groups from a donor ATP molecule to various acceptor proteins. Phosphorylation induces a conformational change that typically “activates” the acceptor protein so that it can propagate a signal through the appropriate cascade. Different kinases target different acceptor proteins, but the regions that bind to ATP are highly conserved across kinases. This makes it extremely challenging for researchers to pharmacologically target specific kinases because drugs that interfere with ATP binding have dramatic off-target effects.
Focal adhesion kinase (FAK) can be activated by several external signals and has important roles in coordinating cell migration, proliferation, and survival. FAK expression is elevated in certain types of breast and ovarian cancers, making it a potential clinical drug target, but inhibiting the protein with small molecule, ATP-competitive drugs to treat these cancers has been challenging because of the off-target effects.
Dr. Magnus Bauer in the Baker lab at the University of Washington and Dr. Behnam Nabet in the Human Biology Division wanted to solve this problem using protein design.
To start, Bauer used RFdiffusion, an AI tool developed by the Baker lab, to design proteins that bind the FAK kinase domain. Using existing structures of FAK in its active and inactive states as guides, the model generated candidate binders designed to interact with different conformations of the protein. They recombinantly expressed 96 of these candidate binders and tested their ability to inhibit or activate FAK. Of these, they found that three designs enhanced FAK activity, while 30 other designs inhibited FAK.
From there, the team chose the two most potent FAK inhibitors and the two most potent activators to validate biochemically. They tested FAK activity in the presence of all four binders by measuring the incorporation of a radiolabeled phosphate group into peptide substrates. Unsurprisingly, they saw that the activators increased phosphate incorporation, while the inhibitors decreased incorporation in a dose-dependent manner. All binders had low-nanomolar affinities, indicating that they bind strongly to FAK.
Next, Bauer and his team determined the crystal structure of FAK bound to one activating binder, which closely matched the computational design. Comparisons with AlphaFold3-predicted FAK-binder complexes suggested that activating binders stabilize a compact, active-like ATP-binding pocket, while inhibitory binders favor a more open, displaced pocket. Together, these findings support the group’s approach to designing proteins that modulate kinase activity.
They then tested whether their new inhibitors were truly specific for FAK by treating other related kinases with the designed proteins. They found that the inhibitory effect on the other kinases ranged from substantially reduced to negligible, indicating that these effects were indeed specific to FAK. Taken together, the structural data and specificity of the inhibition indicate that this type of AI protein design is a powerful approach to modulate the activity of specific kinases.
After comprehensively validating the new binders, Dr. Saurav Kumar and Mia Donald-Paladino in the Nabet lab set out to test how well they performed inside a cell.
Their first challenge was establishing model systems to express the binders and then confirming that they were actually engaging FAK inside the crowded environment of a cell. To test this, the researchers took advantage of a proteolysis-targeting chimera (PROTAC) developed by the Nabet group. The PROTAC was designed to bind FAK and recruit the cell’s protein-disposal machinery to degrade it.
The team hypothesized that if the binders were truly engaging FAK and competing for binding, the PROTAC would be unable to engage the FAK active site to promote degradation. Excitingly, they saw that each new FAK binder prevented PROTAC-mediated degradation, indicating that the binders were engaging FAK instead. They next measured FAK phosphorylation, as an output for its activated state, in the cells expressing each binder. In the cells expressing the inhibitory binders, they observed less FAK phosphorylation, while cells expressing the activating binders had more FAK phosphorylation, indicating that the binders behave as expected in the cells. The team then set out to study whether the changes in FAK altered cell behaviors. They evaluated the cell’s ability to spread and adhere to coated surfaces, which are critical responses as a cell moves, as well as their growth in 3D-settings. The expression of the binders inhibited or promoted changes in cell state, highlighting their potential as tools to control cell signaling outcomes.
To Bauer, this project is a powerful example of using protein design to address a problem that has been plaguing the field for a long time. “The cool part is if you can [design an inhibitor and genetically encode it] for one kinase, because they are so similar, you can probably also adapt it to all other kinases fairly easily,” he explained. In the future, the pair hopes that expressing the new binders in cells will become more feasible. “There’s so much we can continue to learn for how these can become rapidly designed and usable tools for people in the community, and I think we’ve learned a lot in the past couple of years in getting it to work and come to fruition,” says Nabet.
This work was supported by funding from Worldwide Cancer Research, a Generation of Knowledge Grant from MICIU and FEDER/UE, the National Institutes of Health, and the Robert L Fine Cancer Research Foundation.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Member Drs. Behnam Nabet and David Baker contributed to this research.
Bauer MS, Kumar S, Donald-Paladino M, Li D, Klupt KA, Glögl M, Fernández Escamilla AM, Schlichthaerle T, Muratspahić E, Wang X, Schmiderer L, Kenny S, Coventry B, Faezov B, Chen W, Shida AF, Lee GR, Hsia Y, Kibler RD, Elowitz MB, Lietha D, Nabet B, Baker D. 2026. De novo design of selective kinase modulators. bioRxiv. 2026 Jul 13:2026.07.10.737808. doi: 10.64898/2026.07.10.737808.
Kelsey Woodruff is a PhD candidate in the Termini Lab at Fred Hutch Cancer Center. She studies how acute myeloid leukemia cells remodel the sugars on their membranes to reprogram cancer cell signaling. Originally from Indiana, she holds a bachelor's degree in Biochemistry from Ball State University. Outside of lab, you can find her crocheting and enjoying the Seattle summers.