Dual protein analysis approach offers potential way to slow cancer growth

Effectively fighting cancer often requires stopping cancer cells from multiplying, which requires understanding the proteins that cells rely on to survive. Protein profiling plays a critical role in this process by helping researchers identify which proteins (and specific parts of them) future drugs should target. But when used alone, past approaches aren’t detailed enough to highlight all potential protein targets, leading to some being missed.

By combining two methods of protein analysis, a team of chemists at Scripps Research has mapped more than 300 small-molecule-responsive cancer proteins and their small-molecule binding sites. Revealing key protein targets that, when disrupted by certain chemicals (or small molecules), stop cancer cell growth could eventually lead to the development of more effective and precise cancer treatments. The findings are published in Chemistry of nature.

“One method gave us a big picture of which proteins were interacting with which chemicals, and the second method showed exactly where those interactions were happening,” says co-senior author Benjamin Cravatt, Ph.D., Norton B. Gilula Professor of Biology and Chemistry at Scripps Research.

Both methods are forms of activity-based protein profiling (ABPP), a technique Cravatt pioneered to capture protein activity on a global scale. The research team used their dual approach to identify both proteins and protein sites that interacted with a library of stereoprobes, chemical compounds designed to selectively bind to proteins. Stereoprobes are used to study protein functions and identify potential drug targets.

“We made a conscious effort to design our stereoprobes with chemical features that tend to be underrepresented in compounds typically used in drug discovery,” says co-senior author Bruno Melillo, Ph.D., a research scientist in the Department of Chemistry at Scripps Research. “This strategy increases our chances of making discoveries that can advance biology and ultimately translate into improvements in human health.”

The research team’s stereo probes were electrophilic, meaning they were designed to irreversibly bind to proteins, particularly cysteine. This amino acid is ubiquitous in proteins, including those found in cancer cells, and it helps form important structural bonds. When chemicals react with cysteine, they can disrupt these bonds and cause the proteins to malfunction, interfering with cell growth. Many cancer drugs bind irreversibly to cysteines in proteins.

“We also focused on cysteine ​​because it is the most nucleophilic amino acid,” says first author Evert Njomen, Ph.D., HHMI Hanna H. Gray Fellow at Scripps Research and a postdoctoral research associate in Cravatt’s lab.

To find out which specific proteins would bind to the stereoprobes, the team turned to a method called protein-directed ABPP. Using this approach, the researchers discovered more than 300 individual proteins that reacted with the stereoprobe compounds. But they still wanted to dig deeper and identify the precise locations of the reactions.

The second method, called cysteine-directed ABPP, pinpointed exactly where the stereoprobes bind to proteins. This allowed the team to “zoom in” on a specific protein pocket and examine whether the cysteine ​​it contains reacts with the stereoprobes, similar to focusing on a single point on a puzzle board to see if a particular piece fits there.

Each stereoprobe molecule has two main components: the binding part and the electrophilic part. Once the binding part recognizes the protein pocket of the cancer cell, the stereoprobe molecule can enter it, like a key must fit into a lock. When a stereoprobe remains in a pocket that is essential to the functioning of the cancer cell, it prevents the protein from binding to other proteins, which ultimately prevents cell division.

“By targeting these very specific steps in the cell cycle, it’s possible to slow the growth of cancer cells,” Njomen says. “A cancer cell would remain in a state that almost looks like two cells, and your body’s immune system would detect it as defective and tell it to die.”

Identifying specific protein regions essential to cancer cell survival could help researchers develop more targeted treatments to stop the cells from multiplying.

Among the team’s other key findings was confirmation that their two-pronged approach provided a more accurate picture of protein and stereo probe reactivity than a single method.

“We always knew that both methods had their drawbacks, but we didn’t know exactly how much information was lost by using just one technique,” ​​Njomen says. “We were surprised to find that a significant number of protein targets were missed when we used one platform over the other.”

The team hopes their findings will one day lead to the development of new cancer therapies that target cell division. In the meantime, Njomen wants to design new libraries of stereoscopic probes to uncover pockets of proteins involved in diseases other than cancer, including inflammatory disorders.

“There are many proteins that are involved in diseases, but we don’t have stereoscopic probes to study them,” she said. “In the future, I would like to find more proteins that we can study for drug discovery purposes.”