A new protein construct helps scientists study drugs that break down protein targets.
While most conventional drugs work by inhibiting proteins, not all proteins are easy to block in this fashion. Drug developers are investigating new classes of drugs that mark proteins for degradation in the cell. A large, barrel-shaped structure called the proteasome drives this breakdown process, and a protein called Cereblon behaves as an usher, delivering proteins to the proteasome for destruction. Some drugs act as “molecular glue”, sticking to Cereblon and altering its structure so that it binds to target proteins. Other drugs called proteolysis targeting chimeras (PROTACs) bind to target proteins and Cereblon, bridging the two together. Thus, an in-depth understanding of Cereblon’s morphology is crucial for drug investigations. However, scientists have struggled to determine high-resolution structures of this protein in the past due to complications with its synthesis and stability. David Zollman, a structural biologist and drug developer at the University of Dundee, and his colleagues developed a highly stable, easily purified Cereblon variant. Collecting X-ray crystallography data at the Diamond Light Source beamlines I04 and I24, they demonstrated that the structure of their Cereblon variant matched ones previously collected by other groups, but the new crystals achieved higher resolution. Cereblon changes shape when bound to different drugs, and the team collected small-angle X-ray scattering (SAXS) data at beamline B21 to study how shapeshifting varies between different drug candidates. Together, these findings reveal that the new Cereblon variant is amenable to structural analysis, which could facilitate future research into this promising class of protein-degrading drugs.
Most conventional drugs work by inhibiting proteins. The pain-reliever ibuprofen, for example, blocks a bodily enzyme called cyclooxygenase by stoppering its active site and preventing it from producing chemical signals that induce pain. However, Zollman said that researchers have long considered some proteins “undruggable” because they lack active sites that can be targeted by inhibitors. These include proteins that have structural roles rather than enzymatic functions. Taking an alternative approach, scientists are exploring drugs that flag proteins for degradation in the cell by protein shredders called proteasomes.
The most infamous example is the drug thalidomide, a sedative from the 1950s that pregnant women took to relieve morning sickness but led to birth defects. Today, doctors have repurposed thalidomide to treat multiple myeloma, and researchers have developed other drug candidates, like lenalidomide and mezigdomide to treat other cancers. Currently, there are over 40 drugs related to the degradation pathway in cells undergoing clinical trials. Many of them work by recruiting transcription factors to Cereblon and targeting them for destruction, thereby preventing the expression of an array of genes.
Research into these drugs has been held back by a lack of structural insight into Cereblon. Previously, scientists could only purify Cereblon coupled with an adapter protein called Damage Specific DNA Binding Protein 1 (DDB1), resulting in an unwieldy complex. Scientists also struggled to produce high yields of the protein, and they could only prepare it in insect cell expression systems. When scientists managed to crystallize the protein, they found it was unstable, hampering efforts to collect high-resolution structural data. Most experiments determined the structure to a resolution of 3 Ångströms (Å) or worse. Dr Zollman said:
It’s expensive to produce, hard to get in large quantities, and then when you do have it, it’s quite poorly behaved.
What scientists needed was a stable version of Cereblon that was easy to purify in the absence of DDB1. Dr Zollman commented:
We have cut out the part of Cereblon that binds to DDB1, and because of that, we are able to produce it stably from E. coli on its own.
E. coli are the go-to bacteria for producing proteins for purification, making it easier to achieve high yields for scientific studies.
Besides omitting the DDB1-binding domain, Zollman’s team designed 15 versions of Cereblon, some of which carried unique sets of mutations that swap out one amino acid for another in different places. They introduced these mutations to stabilize the proteins, and they discovered that version 8, complete with 12 mutations, proved most stable. “We can get it at a much higher yield, it’s much cheaper to produce, it’s much easier to produce, and then the complex does crystallize a lot better.” Zollman said version 8 is a “middle ground” between full-length Cereblon and other truncated versions trialled previously, so his team renamed it Cereblonmidi.
Next, they had to put their crystallised Cereblonmidi to the test at the Diamond Light Source. Zollman said the protein formed small crystals, and the microfocus beams at beamlines I04 and I24 enabled his team to collect high-quality data from samples of this size.
Read more on Diamond website