In an intricate dance, missteps or errant gestures between two partners can disturb the entire ensemble. Such is the molecular dance that is life. Here, the partners that need to hit their cues, execute the right moves, and follow the choreography are proteins. And when the partners slip or stumble—when protein-protein interactions (PPIs) fail to progress as they should—the consequences can be dire. Cellular processes such as signal transduction, enzyme regulation, and gene activation can go awry, giving rise to disease.

Because PPIs are fundamental to cellular function and dysfunction, they are of intense interest to researchers and drug developers. However, PPIs often defy analysis. They can be subtle and fleeting, and they can involve low-abundance proteins. Moreover, PPIs can be challenging to preserve outside of the dynamic environment of living cells. Finally, PPIs can involve so-called undruggable proteins, which lack well-defined binding sites for small-molecule drugs. Such proteins complicate the search for new drugs.

The difficulties posed by PPIs leave pioneering companies, several of which are featured here, undaunted. They describe how they are improving their PPI analysis techniques and enabling the development of molecular entities that promise to treat cancer and other diseases.

Monitoring PPIs in live cells

Marie Schwinn, PhD, senior research scientist at Promega, asserts that the company’s technology is ideal for studying specific PPIs in living cells. “Promega’s techniques,” she says, “can detect close proximity—on the order of 10 nm—between two proteins with high specificity and sensitivity while preserving all signaling pathways.”

Promega’s platform is centered around bioluminescent signals that are generated when two proteins interact. “Bioluminescence involves the emission of light by living organisms,” she notes. “People are fascinated to learn that our bioluminescent enzymes were engineered from the juice of a deep-sea shrimp.”

Promega uses two main techniques to measure PPIs in live cells: NanoBiT and NanoBRET. NanoBiT is based on splitting a bioluminescent enzyme into two subunits. When two tagged proteins interact, the two subunits recombine, producing a bright luminescent signal.

Promega’s NanoBRET proximity assays diagram
In Promega’s NanoBRET proximity assays, energy from a blue-emitting donor is transferred to a red-emitting acceptor, allowing superior spectral separation, increased signal, and lower background. The energy transfer is depicted here. The donor is a NanoLuc-Protein A fusion, and the acceptor is a fluorescently labeled HaloTag-Protein B fusion. Upon interaction of Protein A and Protein B, the fluorescent signal can be detected in real time. HL: HaloTag-NanoBRET 618 ligand; HT: HaloTag protein.

NanoBRET, on the other hand, is based on a technique called bioluminescence resonance energy transfer (BRET). One protein is tagged with the bioluminescent enzyme, while the other is tagged with an enzyme that binds a fluorescent ligand. When the two proteins interact, a fluorescent signal is generated.

Schwinn stresses that bioluminescence is particularly useful for studying interactions at the molecular level. She adds, “Compared with many fluorescence techniques, bioluminescence produces a clear signal with low background noise and excellent sensitivity.”

Targeting undruggable proteins

At Belharra Therapeutics, chief business officer Rachel Lane, PhD, and vice president of proteomics Sherry Niessen, PhD, agree that finding a way to modulate PPIs with conventional drug discovery techniques is difficult. These techniques are best suited to identifying small molecules that bind to specific sites on the surface of proteins.

“Up to 90% of human proteins are undruggable,” Lane points out. “Novel approaches are needed to complement traditional drug discovery techniques.” She adds that a promising way to access undruggable protein targets is to use chemical probes. Although they are not drugs, chemical probes can be utilized as drug-like starting points for medicinal chemistry. They can also help to determine specific binding sites on a protein.

“We often compare chemical probes to climbing,” Lane says. “When you are climbing, you are always searching for a handhold or toehold. Probes are doing that on the face of the protein, trying to find a structure that they can fit within.”

protein-protein interaction
Belharra Therapeutics uses a chemo-proteomics approach and a library of drug-like molecular probes to discover novel druggable pockets across a range of mechanisms including active sites, allosteric sites, and protein-protein interactions. In this image, the mechanism is a protein-protein interaction. Yellow: region where a chemical probe crosslinks. Gray: cyclin-dependent kinase 1. White: cyclin B1.

To date, Belharra has profiled over 10,000 chemical probes and mapped novel druggable pockets for more than 4,000 proteins. “The pockets that we have been identifying include everything from active sites to allosteric sites to pockets at the interface of key PPIs,” Niessen reports.

Niessen emphasizes that Belharra’s library of chemical probes is designed with chemical diversity in mind to cover as much proteome pocket space as possible. “About 85% of our library consists of enantiomers,” she notes. “These are molecules that are paired, mirror images of each other.” The preferential binding of one of the enantiomers gives Belharra confidence that an actual pocket has been identified.

Another major advantage of Belharra’s platform is that it enables screening within live cells and thereby preserves the native conformations of proteins. Niessen highlights how the company has identified several novel pockets that are present only in live cells.

Once chemical probe experiments have been performed, cells are lysed, and relevant compounds are identified through quantitative mass spectrometry. Ultimately, the final output is the identification of which proteins have been engaged by a given probe.

Investigating very slow binding events

Thomas Garner, PhD, principal scientist at Genentech, works in a small-molecule biophysics team. His research involves developing novel
drugs to repair aberrant PPIs.

Garner uses a label-free optical technique called surface plasmon resonance (SPR) to visualize binding events for drug discovery. “The great thing about SPR,” he says, “is that it provides a wealth of data, including the speed at which binding and unbinding occurs, the strength of binding, and even the mechanism of binding.”

He explains that SPR is based on refraction, the same phenomenon that makes a spoon standing in a glass of water appear to bend. Proteins have a higher refractive index than water, allowing them to be detected using a refractometer.

“By measuring the strength of the interaction between two proteins, you determine how strongly a small-molecule compound needs to bind to disrupt the PPI and qualify as a ‘hit,’” Garner continues. “That is the starting point for your new medicine.” Such binding measurements can also determine whether early starting points for medicines are altering PPIs.

Garner is particularly interested in very strong and stable PPIs. “In the past, it was not possible to measure reactions occurring over days to weeks with SPR,” he recalls. “You couldn’t record data continuously for more than a few hours.” But now, Garner and his colleagues are developing an SPR method that allows for the accurate monitoring of slow processes. This method, which is called the chaser method, involves measuring the binding of a competitive binder.

“When the PPI begins to fall apart, the competitor may take its place,” Garner explains. “We can measure this interaction very accurately to determine whether the protein-protein complex is coming apart.”

Developing novel drugs to inhibit PPIs

Chris Smith, PhD, executive director of medicinal chemistry and drug discovery at Mirati Therapeutics, explains that his goal is to create targeted therapies for people living with cancer. “By focusing on PPIs,” he says, “we are able to specifically direct our cancer-killing molecules to the tumor and avoid healthy tissue.”

Smith is particularly interested in the deletion of a gene called MTAP, which occurs in up to 15% of tumors. This deletion ultimately leads to the formation of a complex called PRMT5/MTA. According to Smith, this complex is important because it “exists only in tumor cells and is not present in healthy cells.”

Mirati is currently working on a new investigational compound called MRTX1719 to treat MTAP-deleted cancers. Smith explains that the drug selectively binds the PRMT5/MTA complex, inhibiting PRMT5 function.

To develop this drug, Mirati used a technique called fragment-based lead discovery. Specifically, Mirati used SPR to screen a commercially available library of 1,000 low-molecular-weight fragments (150 to 300 Da). Mirati scientists (Smith et al. J. Med. Chem. 2022; 65: 1749–1766.) that they prioritized SPR due to “several anticipated advantages: high sensitivity, ability to detect weak binding, and in combination with a clean screen, the ability to detect nonspecific binding and reduce false positives.”

X-ray cocrystallographic data, show MRTX1719  bound to PRMT5/MTA.
Focusing on protein-protein interactions enabled Mirati Therapeutics to develop MRTX1719, a drug candidate that selectively binds to PRMT5/MTA, a protein complex that occurs in MTAP-deleted cancers but not in healthy, nontumor cells. These images, which are based on X-ray cocrystallographic data, show MRTX1719 (magenta) bound to PRMT5/MTA.

Smith notes that MRTX1719 is currently in Phase I/II trials in patients with advanced solid tumors harboring the MTAP deletion. He adds that early signs of clinical activity were observed along with objective responses in patients with a wide variety of MTAP-deleted cancers—including melanoma, gallbladder adenocarcinoma, mesothelioma, non-small cell lung cancer, and metastatic peripheral nerve sheath tumors.

According to Smith, MRTX1719 combines positive clinical data with a favorable safety profile. He asserts that these findings “demonstrate the development of a possible therapeutic approach for a significant population of patients with MTAP-deleted cancers in need of new treatment options.”

Looking to the future

Promega’s Schwinn notes that most technologies provide unique and complementary information. “I don’t think any of these technologies are mutually exclusive,” she continues. “They can be used together to provide a comprehensive picture of what is going on.” Although various techniques are improving in sensitivity, Schwinn stresses that certain interactions are still hard to detect—either because they are short lived or not very common. “We do a great job,” she says, “but there are still some very, very low-abundance interactions out there.”

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