Scientists Identify Drug-Binding Site on FOXA1

Forkhead box protein 1 (FOXA1) is a difficult-to-target protein that is frequently involved in hormone-driven malignancies, such as those of the breast and prostate. Mutations in FOXA1 may facilitate the growth and multiplication of these malignancies. Currently, medications are notoriously tough to block FOXA1, but this could soon change.

Researchers at Scripps Research have discovered a critical binding location on FOXA1 that may open the door to new cancer therapies. The team's research also mapped out the interactions between the protein and small molecules, which are chemical compounds that resemble drugs. The findings were published in the journal Molecular Cell.

While studying large-scale protein interactions, researchers in the Norton B. Gilula Chair in Biology and Chemistry lab of Co-Corresponding Author Benjamin Cravatt, PhD, found that small molecules might interact with FOXA1.

FOXA1 had historically been considered undruggable. It is thought to lack the types of surfaces that small molecule drugs can bind to, which is likely why it is been so difficult to target the protein.”

Benjamin Cravatt, Co-Corresponding Author, Scripps Research

To better understand how those chemicals might impact the functions of FOXA1, Cravatt's group collaborated with the lab of Michael Erb, PhD, when it was discovered.

Activity-based protein profiling (ABPP), a method developed by Cravatt's group to measure protein activity globally, was employed by both Cravatt and Erb in two different ways. Thanks to the dual method, they were able to identify the precise binding site and whether a tiny chemical could attach to FOAX1 at all.

Erb and his team are especially interested in how proteins known as transcription factors “turn on” and “off” specific genes and how this results in cell states that contribute to cancer. Certain DNA sequences are bound by transcription factors such as FOXA1, which regulate whether a gene is repressed (turned “off”) or activated (turned “on”).

The way cells work and react to changes, like in hormone-driven malignancies, which frequently rely on FOXA1 for growth, depends on this control.

FOXA1 is a master regulator of gene control, or what we call a lineage-defining factor. We found a specific site on FOXA1 that can bind to small molecules, which is a tremendously important discovery since transcription factors like FOXA1 are not only attractive targets for cancer but also many other diseases.”

Michael Erb, Study Co-Corresponding Author and Associate Professor, Department of Chemistry, Scripps Research

The finding was surprising because small molecule binding sites on transcription factors are quite uncommon.

A common analogy is that drugs bind to proteins like keys inside a lock, but the prevailing attitude is that most transcription factors do not have binding sites to unlock. The binding site on FOXA1 is like a hidden lock; without the ABPP technology as it exists today, it is hard to imagine how we would have discovered it.”

Michael Erb, Study Co-Corresponding Author and Associate Professor, Department of Chemistry, Scripps Research

Another unexpected discovery is that FOXA1 ordinarily attaches to a specific sequence of DNA bases to regulate gene expression. However, when FOXA1 was bound to tiny molecules, the sequences it preferred were altered, enabling the protein to target different genes than it would normally.

Future studies may benefit from this discovery by better understanding how these chemicals impact cancer gene regulation. Tiny chemicals that change the DNA preferences of FOXA1 could impact the progression of cancer by affecting which genes are activated or inactive.

“We found small molecules could impact FOXA1’s ability to interpret the information written into the genome,” said Erb.

The group also discovered that specific mutations in FOXA1 impacted regions around the protein's small molecule attachment sites. Similar to the tiny molecules, these alterations altered how FOXA1 interacted with DNA.

“This suggests that a hotspot for cancer-associated mutations is also a hotspot for small molecule binding events,” said Erb.

The researchers discovered that tiny compounds couldn't just bind to FOXA1 by itself, which was different from what they had initially believed. The efficiency of small molecules as cancer treatments most likely depends on FOXA1's interactions with DNA, as they were only able to connect to the protein when it was already bound to DNA sequences.

Erb and Cravatt also intend to employ ABPP to look for small-molecule binding sites on transcription factors other than FOXA1 that are currently thought to be undruggable and investigate the optimization of FOXA1 ligands into antagonists of its function and cancer progression.

“Now that we have created chemical probes to study FOXA1, we hope our research inspires the development of drugs that can target the protein,” said Cravatt.