Background

Homeostasis describes the healthy state. It is regulated by tightly controlled feedback loops which adjust in response to changes in the internal or external environment. While a lot has been learned about feedback and homeostasis over the last century, many feedback loops likely remain to be discovered – and we don’t know which are the most important ones. While a number of different triggering events can lead to disease, “sickness” can be fundamentally understood as departure from homeostasis. Likewise, manipulating feedback loops to restore homeostasis could treat currently incurable diseases such as ALS, a devastating neurodegenerative disease. Thus a fundamental goal of therapeutics ought to be restoration of homeostasis.

Unfortunately, it has not been known how to restore homeostasis. Therefore, most drugs today focus on targeting the cause of the disease – and hope that homeostasis will restore itself, once the disease is stopped. Usually it does, but sometimes, like the “colored wheel of doom” when your computer operating system is frozen, it does not – and it doesn’t work to try and undo the keystroke that caused your computer to freeze. As with your computer operating system, when that happens to homeostasis, you need a reboot. We hypothesized that ALS occurs when there is precisely such a failure to restore homeostasis after certain central nervous system injuries.

Enter viruses: they have been manipulating homeostasis by evolution through natural selection, most likely since the origin of life. They have figured out the best ways to disrupt homeostasis while silencing our defenses. If we could get them to tell us how they do that, and what those targets are, drugs with unprecedented therapeutic potential could be developed. That is what we believe we have done, and, courtesy of viruses, as described here, https://cassyni.com/events/88ZRW6kuvE9gZ8WXQPwJCm, our assembly modulator compound provides the necessary reboot of the operating system governing homeostasis, as it relates to ALS.

Our studies revealed first, that catalyzed protein assembly is a crucial weak link in biology that viruses exploit. In effect, we used viruses as “trufflehounds” to reveal those high value targets that are inaccessible to conventional drug discovery tools. For example, powerful tools such as CRISPR are unable to parse small subsets of a gene product that do different functions based on differences in protein-protein interactions, e.g. due to covalent post-translational modification, intrinsically unfolded domains or different pathways of biogenesis and assembly, all of which have been demonstrated to occur.

While advancing these drugs against viruses, we discovered that some of them also work on particular non-viral diseases. Thus, when we learned of work by others revealing a relationship between ALS and retroviruses (for example, HIV), we assessed our anti-retroviral assembly modulator compounds -- and found several of them active on cellular models of ALS. Advanced analogs not only became safer and more potent against ALS, but some actually lost their anti-viral activity, becoming specific for ALS.

In this paper, we sought to further explore the relationship between ALS-selective assembly modulator compounds and homeostasis using blood samples from ALS patients versus healthy individuals. The results are important not only for what they reveal about previously unappreciated feedback loops relevant to ALS, but also because they provide an extremely early signature of ALS by which it can be diagnosed prior to significant disability – using a drug that is strikingly therapeutic for ALS.

Key Findings- Identification of a protein signature in ALS patient PBMCs and a related  feedback response upon treatment of ALS patient blood samples

When ALS assembly modulator compounds are used as “fish hooks” to detect their targets, they bind to distinctly different protein complexes in PBMCs (white blood cells) from healthy vs ALS patients. The proteins RanGTPase and p62/SQSTM1 were found to have an inverse relationship in the small amounts found in the ALS drug target. P62 is a crucial regulator of autophagy (a fundamental process of host defense), and is lost from the drug target with disease progression. Ran GTPase regulates protein transport in and out of the nucleus, long suspected defective in ALS, specifically with regards to the protein TDP-43.  A distinctive fragment of RanGTPase increases with ALS progression. Furthermore, our data reveals new feedback loops implicated in ALS upon which these compounds act.

Next Steps

This work provides foundation for important next steps, including development of an ALS therapeutic, an ALS biomarker, and a way to understand ALS as a disease of homeostasis.

ALS Therapeutic

The ex vivo response we observed is that when ALS patient blood samples are treated with the same compound that is efficacious in animal models, degradation of RanGTPase– likely through feedback– is reduced. Efficacy on a novel target in ALS patient blood, distinctive to ALS, and related to the target seen in ALS mouse brain where the compound is proven efficacious, is encouraging and mandates further exploration.

ALS Biomarker

This signature was present and detectable early in the course of ALS, including even before the patient was disabled. The observed ALS signature in blood could both enable a diagnostic test to detect ALS early, prior to disability, and provide a biomarker for ALS clinical trial design, monitoring and objective assessment. Any treatment for ALS will be more effective if it can be administered to rescue motor neurons before they die. This creates the possibility of a future where ALS patients can be treated early in the disease to prevent disability and achieve a cure.

Tools for Understanding ALS as a disease of homeostasis

ALS is viewed as a disease of motor neurons. However, our findings, derived from ALS patient blood, suggest ALS may actually be a systemic disease, manifest most severely in motor neurons. Further studies using the compounds as molecular probes could provide insight into ALS pathogenesis.

Conclusion

Drugs of the future must do more than just stop primary diseases. They need to restore homeostasis. Evidence, including some presented in this work, suggest assembly modulators do just that for ALS.  We look forward to following up these studies, developing assembly modulators as diagnostics and therapeutics for ALS, and using them as tools to better understand the molecular basis for homeostasis, the best hope for superior ALS drugs in the future.