Suppression of p53 response by targeting p53-Mediator binding with a stapled peptide

Transcription factors represents one of the most high-impact therapeutic targets in biomedicine. The authors developed a new strategy for blocking transcription factor function that could have broad applications to several diseases, including cancer. More than 1,500 transcription factors exist in the human body, each responsible for binding to specific sequences in DNA and transcribing or decoding the body’s genetic blueprint to instruct a cell what to do. Different transcription factors act in different kinds of cells regulating everything from inflammation to cholesterol metabolism to wound healing to controlled cell death, which is key to inhibiting cancer.

When a transcription factor is mutated, those instructions can go awry, turning a health promoting protein into a pathological protein. For instance, mutations in the p53 transcription factor, can change its function from a tumor suppressor to a tumor promoter. For years, scientists have strived to develop methods to inhibit mutated transcription factors. Because they are all molecularly similar in the regions that bind to DNA, targeting one can indirectly target others, disrupting normal cell functions. Transcription factors also contain a section, called an activation domain, that is structurally disordered, making it hard to develop a molecule that will block it. They set out to selectively inhibit p53, which is present in every kind of cell and plays a critical role in human development and in the body’s stress response. University of Colorado Boulder researchers have discovered a new way to inhibit the most commonly mutated gene underlying human tumor growth, opening the door to new therapeutic strategies for cancer and a host of other diseases.

The authors sought to test whether the same outcome could be achieved by targeting Mediator instead. They chose p53 as a test case because it is well studied and biomedically important and contains a well-characterized AD. An apparent obstacle was that Mediator is large (1.4 MDa, 26 subunits) and its p53 interaction site is not precisely defined. However, we reasoned that the p53 AD (residues 13–60) evolved to selectively interact with Mediator with high affinity; supporting this concept, the p53AD alone can selectively purify Mediator from human cell extracts, and mass spectrometry analysis of p53AD-bound factors revealed Mediator as a top hit (Tables S1 and S2). Consequently, they used the native p53AD structure and sequence as a starting point, rather than screen thousands of drug-like compounds. To directly assess p53-Mediator function, the researchers used a defined in vitro transcription system that recapitulated p53- and Mediator-dependent transcription. Biochemical results were tested further in human cells, using genome-wide approaches. Collectively, the experiments conducted establish that p53 activity can be selectively controlled by targeting its interaction with Mediator. The discovery, published in the journal Cell Reports, marks an important step forward in the decades-long quest to target transcription factors (TFs), a notoriously hard-to-block class of proteins which, when mutated or dysregulated, can disrupt cell function and drive illness.

To do so, instead of targeting p53 itself, they targeted a 26-subunit complex aptly named Mediator. Mediator attaches to p53 and other transcription factors, serving as a bridge between them and the enzyme that decodes sections of the body’s genetic blueprint. In essence, the transcription factor must click into Mediator, like a key in a lock, which then activates the decoding process.

In laboratory studies of human cancer cells, the researchers found that when they applied a novel peptide, which they designed based upon the p53 activation domain, they could prevent p53 from working. The team showed that the peptide worked by blocking p53 from clicking in to Mediator, much like jamming up the lock before the real key (p53 itself) could be inserted. According to the authors, the unique method they used using a transcription factor activation domain as a starting point rather than screening thousands of compounds could also lead to faster, cheaper ways to develop new leads for therapeutics.