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Proteomic Study Suggests Potential Approach for Targeting "Undruggable" Proteins

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Using a combination of proteomic approaches, researchers from Boston's Brigham and Women's Hospital, the Broad Institute, and the Dana-Farber Cancer Institute have identified the mechanism of action of the multiple myeloma drug lenalidomide.

Their results, detailed in a study published last month in Science, demonstrate that the drug acts by increasing the binding of a pair of transcription factors, IKZF1 and IKZF3, to a ubiquitin ligase complex, leading to their degradation.

While elucidating the mode of action of lenalidomide is, of course, interesting in and of itself, the findings are perhaps even more significant in that they suggest a strategy for targeting transcription factors, a class of molecule previously considered essentially undruggable, Broad researcher Steven Carr, an author on the paper, told ProteoMonitor.

"It's essentially paradigm shifting," he said, noting that by and large pharma has avoided efforts to target transcription factors with small molecules "because of the perceived difficulty" of this challenge.

The study also demonstrates the potential of proteomics as a tool for drug screening and target deconvolution.

Carr and his colleagues began their effort with a quantitative chemoproteomic experiment aimed at identifying targets of lenalidomide in multiple myeloma cells. Using SILAC mass spec, the researchers measured binding of proteins to an immobilized lenalidomide, identifying as a likely target the protein DDB1, part of the CRBN-CRL4 ubiquitin ligase complex.

That finding, said Broad researcher Monica Schenone, also an author on the paper, led the team to look to protein ubiquitination as a possible mechanism.

Conveniently, ubiquitination has been an area of focus for Carr and the Broad's proteomics platform. The modification has traditionally been difficult to measure via mass spec due to its relatively low abundance and a lack of effective affinity reagents. In recent years, however, work by labs including Carr's has led to improved workflows for its detection.

One key to ubiquitination analysis is upfront enrichment using K-ε-GG antibodies from Cell Signaling Technology.

The modification "is sub-stoichiometric, and so we need a way to enrich them," said Namrata Udeshi, an author on the Science paper and a research scientist in Carr's lab. "And so our and other labs have been using this antibody, which is extremely good at recognizing [ubiquitination]."

The antibody does not, however, have particularly good specificity, Udeshi told ProteoMonitor, and so she and her colleagues have taken a number of steps, including offline fractionation and crosslinking of the K-ε-GG antibodies to the beads used in the enrichment process, to decrease the levels of background binding.

Also critical, Carr said, is use of SILAC to ensure good quantitation. This, he noted, makes "it much easier to discriminate against non-specific binding and background so that without having to do [a large number of replicates] and having lots of additional data to peruse you can readily ascertain what is a true interactor."

Applying their ubiquitination workflows on a Thermo Fisher Scientific Q Exactive instrument, the researchers followed up their target identification work by examining total protein expression and global ubiquitination in multiple myeloma cells both untreated and treated with lenalidomide. This analysis identified IKZF1 and IKZF3 as the most highly lenalidomide-regulated proteins, at both the level of protein expression and ubiquitination.

"We saw that the levels of these transcription factors were going down upon treatment with lenalidomide and that their ubiquitination levels were going down as well," Udeshi said.

At the same time, the researchers were also investigating lenalidomide-dependent protein interactors of the CRBN-CRL4 ubiquitin ligase complex identified in the initial target ID experiment. These experiments identified IKZF1 and IKZF3 as interactors of the ligase complex whose binding was enhanced by the addition of lenalidomide.

In sum, these findings suggested that lenalidomide increases binding of IKZF1 and IKZF3 to the ligase complex – as shown by the protein interaction data – leading to increased degradation of these two transcription factors – as indicated by the global protein expression and ubiquitination analysis.

"We ended up realizing that [lenalidomide] was working to stabilize the [CRBN-CRL4 ubiquitin ligase] complex," Schenone told ProteoMonitor, noting that this was the opposite of how small molecule drugs were typically presumed to work.

"You normally think of small molecules as disrupting a protein interaction, she said. "But in this case it was the way that lenalidomide was increasing the affinity of these two transcription factors to this complex that was the key."

The finding was bolstered by a paper in the same issue of Science led by Dana Farber researchers that similarly showed that loss of IKZF1 and IKZF3 are behind lenalidomide's effect in multiple myeloma.

The identified mode of action suggests new possibilities for drug development, Schenone said.

"These two transcription factors are essential for these leukemias," she said. "So what is interesting is that there is a small molecule acting on a transcription factor, [a class of protein] that has traditionally been thought of as undruggable. This was a nice case showing how a small molecule could actually impact this undruggable space [via] protein-protein interactions."

Carr said that so far as he knows only one other molecule, the plant hormone auxin, appears to operate in a similar manner, increasing binding between a ubquitin ligase and a particular substrate. His study co-authors, Brigham and Women's researchers Benjamin Ebert and Jan Krönke, identified this molecule in a literature search following their unraveling of lenalidomide's mode of action, he said.

Nonetheless, the team's findings present researchers with the opportunity to begin searching for similarly functioning systems, Schenone suggested.

"What it does is open up a whole new way of screening small molecules," she said. "So now you can start thinking about different screening paradigms looking for these kind of effects."

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