Cereblon already sits at the center of thalidomide-derived therapies for some hematologic malignancies and many targeted protein degradation strategies. Yet drug discovery around this E3 ubiquitin ligase substrate adapter has focused overwhelmingly on one place: its orthosteric thalidomide-binding site.

That made the first clue in this study particularly intriguing. A screen intended to find compounds that displaced an orthosteric probe instead produced several compounds that increased its signal. Further work narrowed the result to SB-405483. Biochemical experiments indicated cooperative binding, while X-ray crystallography revealed a previously unrecognized, evolutionarily conserved allosteric pocket on cereblon.

The 2026 Nature study “Identification of an allosteric site on the E3 ligase adapter cereblon” reports the discovery and its functional consequences.

The paper’s first author, Dr. Vanessa Dippon, is an incoming postdoctoral fellow at the Broad Institute of MIT and Harvard and a former graduate student in Harvard’s Chemistry and Chemical Biology Department advised under Professor Christina Woo. Speaking with Pelago Bioscience, she described how that unexpected result became a five-year research project.

“This was found completely serendipitously. It was not what they were planning on looking for. They were not screening for this effect.”

A pharmaceutical generated the initial screening, biochemical, and crystallographic evidence. The Woo Laboratory at Harvard then took the project on and investigated what the finding meant in cells and for cereblon function. Scripps Research later contributed structural work that helped explain the functional results.

The result that allowed the project to continue

Dippon was only a few months into graduate school when the compound arrived at The Woo Laboratory. The in vitro results and crystal structure made a strong case for the new pocket, but her first task was more practical: could the allosteric ligand engage cereblon inside a cell?

This distinction mattered to her. “At least for me, I always like seeing the cellular data more,” she said. “In vitro assays are great in that you can very cleanly control what you’re adding, but I think they also need to be complemented by cellular assays.”

The researchers treated MM.1S cells with the allosteric ligand and used a Western blot-based CETSA®. After heating the samples, they measured how much soluble cereblon remained. The ligand stabilized cereblon in a dose-dependent manner. Stabilization was more pronounced in the presence of lenalidomide, consistent with cooperative engagement of the allosteric and orthosteric sites.

CETSA supported cellular target engagement by demonstrating compound-induced thermal stabilization of native cereblon in cells. It did not establish direct binding or locate the pocket by itself; the biochemical and crystallographic experiments supplied that evidence. Its role was to show that the observation made with purified protein could be carried into a cellular setting.

Dippon called CETSA “the first leaping-off point for these other assays”. It was the earliest experiments she performed in the project and the first major checkpoint for the cellular project.

“That was the first checkpoint that enabled me to continue doing this project. So that was definitely really exciting and relieving to get those data.”

A result that complicated the original hypothesis

With cellular engagement established, the project could move forward. NanoBRET provided a quantitative cellular measure of enhanced orthosteric-site binding. Western blots and reporter cell lines examined neosubstrate degradation, while global proteomics looked beyond the selected reporters.

The initial expectation was straightforward: stronger orthosteric binding might increase degradation. It did for some neosubstrates. For others, the effect ran in the opposite direction.

“We can enhance degradation of certain neosubstrates while also inhibiting the degradation of others.”

Across more than 100 orthosteric ligands and 10 neosubstrates, the outcome depended on the ligand and substrate combination. Cryo-EM studies at Scripps Research helped explain why. Allosteric engagement altered cereblon’s conformational distribution and could reposition ligands within the orthosteric pocket, favoring neosubstrate recruitment in some contexts while weakening it in others.

For Dippon, this was the point at which the discovery became more than the identification of another binding site. It showed that engaging the pocket could change functional outcomes in ways that were not apparent from enhanced orthosteric binding alone.

The finding suggests several directions for future research. Allosteric ligands might eventually help tune the activity or selectivity of cereblon-directed compounds, limit unwanted neosubstrate degradation, or provide another surface for degrader design. These remain possibilities, not demonstrated clinical benefits. The discovery also invites researchers to ask whether comparable allosteric control has been overlooked in other E3 ligases.

From a first experiment to a five-year study

By the time Dippon discussed the work, she was preparing to graduate. The timing gave the project a full-circle quality.

“It was the first go/no-go of the project and the first thing that I ran in this lab.”

Her experience illustrates why cellular target engagement can become an early project decision rather than simply another measurement. Biochemical and structural studies can build a persuasive molecular hypothesis. In this study CETSA was used as one of a range of orthogonal techniques, but it answered the question that needed to come first: whether there was enough cellular evidence to justify going further.

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