Most drugs are designed to inhibit a particular protein or interfere with its activity. But in diseases involving widespread cellular dysfunction, targeting a single protein may not be enough. What if, instead, researchers could activate the cell’s own machinery to remove several problematic proteins at once?
In a study published in Science, researchers describe a strategy called stimulated protein degradation (SPD), which activates a cellular protein-clearance system to accelerate the breakdown of multiple proteins. In cellular models of mitochondrial disorders, the approach improved mitochondrial function and helped neurons survive.
The findings suggest that SPD could provide a different way to tackle diseases driven by multiple dysfunctional cellular processes.
Turning protein degradation on
Cells continually produce and break down proteins to maintain normal function. One of the systems responsible for this process involves E3 ubiquitin ligases, enzymes that mark proteins for destruction by attaching a molecular tag called ubiquitin. The tagged proteins are then directed to the cell’s protein-degradation machinery.
Drug developers are already harnessing this system through targeted protein degradation (TPD). Approaches such as proteolysis-targeting chimeras (PROTACs) bring an E3 ligase into contact with a specific protein, prompting its degradation. Rather than simply blocking a protein’s activity, these drugs aim to remove the protein altogether.
TPD has attracted interest for diseases in which a particular protein drives pathology. But most conditions involve dysfunction across multiple proteins and cellular pathways. As a result, removing one protein isn’t sufficient to restore normal cell function.
SPD takes a different approach. “SPD activates the native protective function of quality control E3 ligases to promote degradation of multiple pathological factors. It therefore stimulates a native protective activity and allows us to degrade many proteins at a time,” Michael Rapé, Professor of Molecular Therapeutics at UC Berkeley Research and senior author of the study, told DDN.
What E3 ligase did they use?
The researchers focused on an E3 ligase called CUL2FEM1B, which helps regulate the movement of proteins into mitochondria and also plays a role in cellular stress response.
The complex is particularly sensitive to changes in reactive culoxygen species (ROS), molecules produced during mitochondrial energy production that also act as cellular signals. When activity in the electron transport chain drops, ROS levels fall, prompting CUL2FEM1B to break down proteins that might interfere with mitochondrial protein import. This helps keep the pathway open for proteins needed to maintain mitochondrial function.
In the absence of reductive stress, CUL2FEM1B is restrained by proteins from the BEX (brain-expressed X-linked) family, which engage its substrate-binding pocket without getting ubiquitylated.
To find a way around this inhibition, the researchers screened approximately 120,000 compounds for molecules that could displace BEX proteins without blocking substrate recognition. They identified an initial compound, BD1 (BEX displacer 1), and subsequently developed a cell-active analogue called BD2.
By freeing CUL2FEM1B from its inhibitors, BD2 increased the degradation of multiple natural substrates, including FNIP1 (Folliculin-Interacting Protein 1) and COA4 (Cytochrome c Oxidase Assembly Factor 4). The result was improved mitochondrial protein import and electron transport chain function.
Restoring mitochondrial function
Mitochondrial dysfunction occurs in a range of inherited disorders and neurodegenerative diseases. In some conditions, faulty proteins can prevent mitochondria from assembling the electron transport chain, compromising energy production and contributing to cell damage or death.
The researchers first tested SPD in cellular models of cytochrome c oxidase (COX) deficiency syndrome, a group of disorders associated with defects in the electron transport chain. In neurons derived from induced pluripotent stem cells (iPSCs), these defects reduced levels of complex IV, a key part of this machinery, and increased cell death.
Treatment with BD2 restored levels of complex IV subunits and improved neuronal survival. The compound also improved cell health in models of several other mitochondrial disorders, including Friedreich’s ataxia, an inherited condition caused by a deficiency of the protein frataxin. The researchers found that treatment with BD2 had comparable effects to those seen with optimal concentrations of omaveloxolone, an approved treatment for Friedreich’s ataxia, in their experimental model.
However, BD2 did not rescue every type of mitochondrial dysfunction tested. It failed to correct certain defects affecting other electron transport chain complexes, suggesting that activating CUL2FEM1B may be useful only when the underlying disease mechanism is compatible with the pathway it regulates. “We see this limitation as a hallmark of specificity, though,” said Rapé.
From cellular models to potential treatments
The team is now working to improve BD2’s potency, stability, and ability to reach the brain, Rapé said. These steps will be important in determining whether the compound can progress beyond cellular models towards a potential treatment.
Despite the potential, degrading multiple proteins at once does also raise concerns about selectivity and safety. Although BD2 improved mitochondrial function in cellular models, researchers will need to establish which proteins it affects, whether their degradation disrupts other cellular processes, and how its effects change with different doses and treatment durations.
This proof of concept could mark the beginning of an entirely new approach for drug discovery. If researchers can find a way to release the brakes on other E3 ligases, they could potentially target entire pathways involved in conditions like heart disease or neurodegeneration. The next challenge will be to establish how broadly this strategy can be applied, whether its effects can be achieved safely, and whether its benefits can be reproduced in more complex models of disease.
