A team from the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, collaborating with scientists from the University of Oxford, the Weizmann Institute of Science and the University of Dundee, has identified an unexpected factor in this response: a protein called NUDT5.

The discovery builds on recent research by the Kubitschek Institute and the Hoover Institution. That initial study showed that NUDT5 plays an important cellular role independent of its normal enzymatic activity. NUDT5 functions not only as a catalyst that promotes chemical reactions, but also as a molecular scaffold that helps organize cellular metabolism.

This non-catalytic function appears to be important in determining how cells respond to 6-TG.

“We initially expected that NUDT5 would affect 6-TG through its enzymatic activity,” said co-first author Tuan-Anh Nguyen of CeMM. “Rather, we found that inhibiting the enzyme had little effect. What mattered was whether the protein itself was present.”

Removing NUDT5 changes the effects of 6-thioguanine

Many drugs that target enzymes are designed to block the chemical reactions those enzymes perform. The researchers wanted to determine whether inhibiting NUDT5 in this way would also change the effects of 6-TG.

To test this, they used a strategy known as targeted protein degradation. Instead of simply blocking the protein’s activity, this approach prompts cells to remove the protein entirely.

“We developed a cell-based platform to accelerate the discovery of NUDT5 degraders. This platform helped guide the medicinal chemistry efforts that ultimately produced our most active degrader, dNUDT5,” said Anne-Sophie Marquez, first author of the paper, whose research at the University of Oxford contributed to the discovery.

A medicinal chemistry program led by the Huber Laboratory at the University of Oxford produced a collection of highly selective NUDT5 degraders. The researchers also created a control compound that binds to NUDT5 without destroying it.

They then compared the effects of these compounds with those of traditional NUDT5 inhibitors.

The difference was clear. Blocking NUDT5’s enzymatic activity did not significantly change how cells responded to 6-TG. However, removing NUDT5 protected the cells from the drug’s toxic effects. Genetic experiments produced the same result.

“Chemical degraders give us a way to distinguish between what a protein does as an enzyme and what it physically exists inside a cell,” said Professor Killian Huber of the University of Oxford’s Center for Drug Discovery and co-leader of the study. “In this case, the distinction was decisive. Removing NUDT5 revealed biology that traditional inhibitors missed.”

NUDT5 has a hidden role beyond its enzyme activity

The findings indicate that NUDT5 influences sensitivity to thiopurine drugs through a mechanism independent of its catalytic activity. In other words, understanding the chemical reactions performed by an enzyme may not be enough to explain how it affects the response to 6-TG.

“Once the results arrived, it quickly became clear that dNUDT5 protected cells from 6-thioguanine toxicity in a dose-dependent manner. That was an incredibly exciting moment,” said Ludwig Bauer, first author of the paper.

The researchers also identified an important connection between NUDT5 and NUDT15, another protein already known to influence patient responses to thiopurine drugs.

The two proteins appear to have opposing effects. Loss of NUDT15 makes cells more sensitive to 6-TG, whereas reducing NUDT5 makes cells more resistant to treatment. This suggests that the proteins influence thiopurine responses through different mechanisms that push cells in opposite directions.

“Our results show that proteins can have important biological functions that are completely independent of their enzymatic activity,” said Stefan Kubicek, corresponding author and principal investigator at CeMM. “By removing NUDT5 rather than simply inhibiting it, we were able to uncover hidden layers of biology that help determine how cells respond to clinically important drugs.”

What the discovery means for leukemia drug research

Although the discovery does not immediately lead to a new treatment, it reveals an unexpected mechanism that helps control the effects of a long-used leukemia drug.

By showing that NUDT5 affects 6-TG through a non-catalytic function, the study could help scientists better understand why cells respond differently to thiopurine treatment. It also demonstrates how targeted protein degradation can expose biological functions that may remain hidden when researchers rely solely on traditional enzyme inhibitors.

This research was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program, the Austrian Science Fund (FWF), the Vienna Science and Technology Fund (WWTF), the Marie Skłodowska-Curie Actions Postdoctoral Fellowship Program, the Innovative Medicines Initiative 2 Joint Undertaking (IMI2 JU), the Wellcome Trust, Merck Sharp & Dohme, and Janssen Pharmaceutica NV.