Precision Pruning with PROTACs: Selective Targeting of Toxic Huntingtin

In nature, dominant organisms are known for taking over the natural resources in an area. For example, a tree that grows tall over time is exposed to large amounts of sunlight. However, if a seedling is planted next to the tall tree, it will not have the same access to the sun and may not survive. In cells, misfolded proteins may interact with each other to form sticky clumps, also known as aggregates, which can act like a dominant organism. In diseases like Huntington’s disease (HD), these clumps can use up energy and resources in the cell, causing protein stress and, ultimately, contribute to cell death.

In a recent study, scientists created a chemical that breaks down expanded huntingtin HTT clumps in HD cells and mouse models. This chemical finds HTT clumps and targets them to be broken down or degraded. Pruning only expanded HTT clumps and leaving behind healthy HTT may ultimately improve cell health. Let’s take a closer look at what this study found.

Selecting for the seedling

In HD, a repeating stretch of the genetic letter code C-A-G causes an expanded form of the HTT protein to be made. Expanded HTT is extra-long and folds into a slightly different shape than the regular form of HTT. The new HTT shape can lead to the formation of HTT clumps, which are suspected to increase protein stress, which can lead to cell death. One idea is to get rid of these toxic HTT clumps, but this comes with its own set of challenges.

People with HD produce both regular and expanded HTT. Because regular HTT plays important roles in cell health and function, preserving some of it may be important. Imagine if a forester wants to give a special seedling more sunlight, so they spray a weed killer chemical that destroys all plants in the area. The chemical would get rid of the tall tree, but the seedling probably wouldn’t survive either. Instead, the forester needs to find a way to remove the tall tree without harming the seedling. Similarly, scientists are exploring treatments that focus on getting rid of the expanded HTT protein while keeping the healthy regular form of HTT. 

Targeting the tall tree

How do we target expanded HTT but not regular HTT? In a study from the Jen-Tse Huang lab in Taiwan, researchers designed a compound called PROTAC 2’ (pronounced pro-tack two prime) that can do just that in lab models of HD. PROTAC 2’ finds expanded HTT clumps that have a shape distinct from regular HTT, and grabs on. 

The Huang lab designed PROTAC 2’ not only to recognize expanded HTT, but also bring it to the cell’s trash disposal machinery to break it down. It’s like if the forester carried the weed killer chemicals with them when searching for the tree. This is much more efficient than locating the tree, going back to get the chemicals, and hoping to remember where they found it. Because PROTAC 2’ grabs expanded HTT clumps AND the cell’s existing trash disposal machinery, the clumps can be broken down and disposed of more easily. 

Cleaning up toxic expanded HTT clumps in cells

Expanded HTT can exist in different forms. Researchers sometimes define these forms based on their ability to dissolve in different liquids, or how they behave when labeled with colourful chemical “tags.” Clumps of expanded HTT that don’t dissolve easily or are packed tightly together are often called “insoluble.” The expanded HTT protein can also exist in a “soluble” form. There’s some evidence that the insoluble form expanded HTT might be more toxic. 

In cells in dish that were treated with PROTAC 2’, the researchers found lower levels of insoluble expanded HTT clumps, while levels of the other HTT forms stayed the same. This suggests that PROTAC 2’ is helping to the cells get rid of expanded HTT clumps, without affecting other forms of the protein – exactly as the researchers hoped! 

The Huang lab was also interested in how cells responded to the lower levels of expanded HTT clumps. In cells treated with PROTAC 2’, researchers found an increase in cell survival and less evidence of stress. This suggest that treating cells with PROTAC 2’ might help reduce some of the problems in the cell that we associate with HD.

Slowing HD progression in mice

The researchers also injected PROTAC 2’ into mice that model HD to see if PROTAC 2’ could reduce their HD-like symptoms. These mice are engineered to carry the human HD gene with an expansion of around 120 CAG repeats. This leads to the formation of expanded HTT clumps and inflammation in the brain, as well as progressive weight loss, motor decline, and a much shorter lifespan compared to mice that don’t have the HD gene.

Before working with the sick mice, the researchers learned more about the safety and actions of PROTAC 2′ by injecting this molecule into mice that did not carry the HD mutation. Next, they treated mice that model HD, and found that the mice given PROTAC 2’ had fewer HTT clumps, improvements in other brain markers and symptoms, and a longer lifespan compared to the untreated HD mice. This suggests that treatment with PROTAC 2’ might be delaying the effects of HD progression in this disease model. 

Looking forward 

Researchers from the Huang lab designed a compound that recruits both HTT clumps and degradation machinery to the same place. Like a forester protecting a prized sapling, PROTAC 2’ effectively breaks down clumps in cells while sparing regular huntingtin. This slowed the progression of HD-like symptoms in mice, and the authors suggest that PROTACs could have potential as a therapeutic in the future. 

While they were able to demonstrate the potential of these molecules in mice, there is much more to be done before we could move towards applying this approach in people. One challenge is that PROTACs are on the larger size of drug-like molecules, and it can be difficult for them to enter deep into the brain tissue, which is a bigger issue in a bigger (human) brain. Furthermore, this study did not directly compare regular and sick mice – and it’s possible that HD could alter the PROTAC’s ability to enter brain tissue. Larger studies would also be needed to better understand how PROTAC 2’ influences other processes affected by HD, like energy production.

Nevertheless, breaking down toxic protein clumps is a strategy that is being explored across multiple brain diseases, and it’s exciting to see that it can have positive effects in HD mice. The authors note that protein-based clean-up approaches could remove some of the toxic culprit with less potential for long-term harm than strategies like gene therapy. 

Summary

• PROTAC 2’ selectively targets expanded HTT clumps for the cells trash can, sparing regular HTT
• It works by recruiting trash cleanup machinery to toxic protein clumps
• In cells, this treatment lowered insoluble expanded HTT clumps and reduced stress 
• In mice that model HD, PROTAC 2’ reduced movement problems and other disease features, and increased lifespan