Fighting cancer effectively often involves halting the multiplication of cancer cells, which requires a deep understanding of the proteins that support these cells’ survival. Protein profiling is crucial in this effort, as it helps researchers identify specific proteins and their components that future drugs should aim to target. However, previous methods have often lacked the precision necessary to identify all possible protein targets, resulting in some being overlooked. Recently, a team of chemists has successfully mapped over 300 cancer proteins that interact with small molecules and pinpointed their binding sites by combining two analytical techniques.
Effectively combatting cancer often necessitates hindering the replication of cancer cells, which relies on a solid grasp of the proteins these cells depend on for survival. Protein profiling is vital in this context, as it assists researchers in identifying the proteins—and their specific segments—that future medications should aim for. Nonetheless, previous techniques used independently have not been detailed enough to uncover all possible protein targets, resulting in some being missed.
A collaborative effort at Scripps Research has led to the mapping of over 300 small molecule-reactive cancer proteins and their respective binding sites. This discovery highlights essential protein targets that, when disrupted by specific chemical compounds (or small molecules), can inhibit cancer cell growth, paving the way for more effective and targeted cancer treatments. These results were published in Nature Chemistry on August 13, 2024.
“One method provided us with a broad overview of the proteins interacting with the chemicals, while the other method illustrated the precise locations of these interactions,” stated co-senior author Benjamin Cravatt, PhD, the Norton B. Gilula Chair in Biology and Chemistry at Scripps Research.
Both techniques are forms of activity-based protein profiling (ABPP), a method that Cravatt developed to observe protein activity comprehensively. The research team employed this dual approach to identify both the proteins and specific sites on those proteins that bonded with a library of stereoprobes—chemical compounds designed to selectively and permanently attach to proteins. Stereoprobes are valuable for examining protein functions and identifying potential drug targets.
“We intentionally designed our stereoprobes to include chemical features that are often underutilized in typical drug discovery scenarios,” remarked co-senior author Bruno Melillo, PhD, an institute investigator in the Department of Chemistry at Scripps Research. “This approach boosts our chances of uncovering discoveries that can enhance biological understanding and, eventually, improve human health.”
The stereoprobes developed by the research team were electrophilic, meaning they were engineered to form irreversible bonds with proteins—specifically targeting cysteine. Cysteine is a common amino acid found in numerous proteins, including those in cancer cells, where it plays a role in forming key structural links. When chemicals interact with cysteine, they can disrupt these vital bonds, causing proteins to malfunction and thereby hindering cell growth. Numerous cancer therapies work by irreversibly binding to protein cysteines.
“We focused on cysteine since it’s the most nucleophilic amino acid,” explained first author Evert Njomen, PhD, an HHMI Hanna H. Gray Fellow and postdoctoral research associate in Cravatt’s lab.
To identify which proteins specifically bonded with the stereoprobes, the team utilized a method known as protein-directed ABPP. This technique helped them discover over 300 individual proteins that reacted with the stereoprobe compounds. Yet, they aimed to delve even deeper to pinpoint the exact locations of these reactions.
The second method, called cysteine-directed ABPP, accurately located where the stereoprobes bonded on the proteins, allowing the team to focus closely on individual protein pockets to assess whether the cysteine reacted with the stereoprobes—similar to examining a specific piece of a puzzle to determine how well it fits.
Every stereoprobe molecule consists of two essential elements: the binding portion and the electrophilic segment. Once the binding part interacts with the cancer cell’s protein pocket, the stereoprobe can ideally penetrate—akin to a key fitting into a lock. When a stereoprobe stays in a pocket critical for the cancer cell’s operation, it prevents the protein from binding with others, consequently halting cell division.
“By targeting these very precise stages in the cell cycle, we can potentially slow the growth of cancer cells,” stated Njomen. “A cancer cell might remain in an almost-divided state, prompting the body’s immune system to recognize it as defective and signal for its death.”
Identifying exact protein regions essential for cancer cell survival could lead researchers toward more targeted treatments that prevent cell proliferation.
Additionally, the research team confirmed that their combined approach produced a more comprehensive understanding of protein-stereoprobe interactions than either method alone.
“We’ve always acknowledged that both methods have limitations, yet we were unaware of how much information was omitted when only applying one technique,” Njomen reflected. “It was surprising to discover that a significant number of protein targets were overlooked when relying solely on one platform.”
The team aspires that their findings will significantly contribute to the development of new cancer therapies aimed at cell division. Meanwhile, Njomen is eager to create new stereoprobe libraries to explore protein pockets associated with various diseases beyond cancer, including inflammatory disorders.
“Many proteins have been linked to various diseases, yet we lack stereoprobes to investigate them,” she mentioned. “Going forward, I hope to discover further protein pockets that we can examine for drug discovery initiatives.”
Alongside Cravatt, Njomen, and co-senior author Bruno Melillo, the authors of the study, “Multi-Tiered Chemical Proteomic Maps of Tryptoline Acrylamide-Protein Interactions in Cancer Cells” include Rachel E. Hayward, Kristen E. DeMeester, Daisuke Ogasawara, and Melissa M. Dix from Scripps Research; Tracey Nguyen, Paige Ashby, and Gabriel M. Simon from Vividion Therapeutics; as well as Stuart L. Schreiber from the Broad Institute.
This research and the participating investigators were supported financially by the National Institutes of Health (U19 AI142784 and R35 CA231991); Cancer Research UK (CGCATF-2021/100012 and CGCATF-2021/100021) the National Cancer Institute (OT2CA278688 and OT2CA278692); the Howard Hughes Medical Institute Hanna H. Gray Fellowship (NGT15176), the Jane Coffin Childs Memorial Fellowship, and Vividion Therapeutics.
