Scientists find a molecule that cells activate to destroy a protein

Scientists find a molecule that cells activate to destroy a protein

On August 5, a team from Dana-Farber and Harvard described in Nature a method for finding small molecules that direct the cell’s disposal system toward a selected protein. The researchers screened seven E3 ligases, enzymes that mark proteins for degradation, against 5 000 compounds and identified M12. A cellular enzyme attaches a glutathione molecule to M12, and the resulting product brings the ligase DCAF11 into contact with the protein DDX18.

Many proteins lack a suitable site for a drug to bind. A molecular glue temporarily connects a target protein to an E3 ligase. The ligase tags the protein with ubiquitin, and the proteasome, the cell’s disposal system, breaks it down.

Only a small number of well-studied E3 ligases have been used with such glues, although humans have more than 600 E3 ligases. The authors began with seven ligases and a compound library to find a new pairing. They immobilized the ligases on particles, then added a library of 5 000 compounds and cell lysate, which contains the components of disrupted cells, including active enzymes. Mass spectrometry, which determines the composition of a mixture, detected DDX18 in one of these mixtures. Experiments with each immobilized ligase identified DCAF11, and screening progressively smaller groups of compounds identified M12.

The lysate proved to be an essential part of the screening method. Purified DCAF11 and DDX18 did not form a complex in the presence of M12 alone. The complex appeared when lysate was added and disappeared after the lysate had been boiled. The authors found that the enzyme GST attaches glutathione to M12. The resulting compound, GSH-M12, occupies a site in DCAF11, while the exposed portion of M12 creates a surface that contacts DDX18. Synthesized GSH-M12 assembled a complex from purified components and enabled DCAF11 to tag DDX18 with ubiquitin. Cryo-electron microscopy images showed GSH-M12 positioned between the two proteins.

The authors linked M12 to fragments that bind other proteins. In cell models, the resulting two-part molecules reduced the levels of BRD4, SMARCA2, BRD9, and several kinases, which are enzymes that transmit cellular signals. For BRD4 and SMARCA2, the authors confirmed dependence on DCAF11 by deleting the DCAF11 gene. M12 can therefore serve as one part of a molecule that directs DCAF11 toward several proteins.

In the screening assay, GST converted M12 into an active molecular glue.