HSCI researchers use patient-derived motor neurons to screen for clinically relevant targets

Harvard Stem Cell Institute (HSCI) researchers led by Clifford Woolf, M.D., Ph.D., have developed a high-throughput platform for discovering drug targets to treat amyotrophic lateral sclerosis (ALS), a severe neurodegenerative disorder that causes the loss of motor neurons and voluntary muscle action. One of the targets identified by the platform is currently being developed as a therapy by QurAlis, a biotechnology company co-founded by Woolf and fellow HSCI faculty member Kevin Eggan, Ph.D.

While previous mouse studies have identified potential treatments for ALS, these drugs have typically done very poorly in human clinical trials. “One of the most difficult challenges in drug discovery is identifying a target that has a key role in the disease process,” said Woolf, the director of the F.M. Kirby Neurobiology Center at Boston Children’s Hospital.

In the new study, Woolf collaborated with Pfizer to develop a high-throughput platform for discovering such targets. Published in the journal Cell Reports, the platform uses motor neurons made from ALS patients themselves, coupled with imaging to measure the neurons’ response to different drug candidates. The platform was able to confirm two known ALS drug targets, and then identify a new potential target that is amenable to treatment with existing drugs.

The researchers had previously showed that human motor neurons with ALS mutations are more excitable than normal motor neurons and tend to “fire” excessively. That hyperexcitability — and the prospect of calming it — is at the heart of the discovery platform.

“Hyperexcitability makes the motor neurons more susceptible to degeneration and ultimately death,” Woolf said. “By doing a screen aimed at reversing this hyperexcitability, we were able to discover new targets and disease mechanisms for ALS and confirm others in an unbiased way.”

To create the platform, the researchers started with induced pluripotent stem cells from the Eggan lab, made using tissue samples from patients with ALS who carried a particular genetic mutation. They then differentiated the stem cells into motor neurons and exposed them to a variety of drugs.

To measure excitability, the researchers used a type of imaging that scans for a fluorescent marker of calcium levels. This is an indicator of how frequently the neurons are firing, whether regularly or not, and how strong their signal is.

In all, the researchers screened a library of 2,900 drugs from Pfizer with known mechanisms of action. After three screening rounds, they found 67 compounds that reduced hyperexcitability of the patient-derived motor neurons without causing toxicity.

Further investigations homed in on 13 potential drug targets with the greatest effects. Seven of the target proteins belonged to two classes already known to be associated with ALS hyperexcitability: AMPA receptors and Kv7 potassium channels. Indeed, QurAlis is currently developing a therapy that targets Kv7 channels.

The researchers also found a promising new class of candidate drugs, which targeted the dopamine D2 receptor (DRD2). These receptors’ role in motor neuron hyperexcitability had not previously been recognized. Moreover, some DRD2 drugs are already commercially available, opening the possibility of testing them in patients with ALS.

“Our results show that neuronal excitability screens are a powerful platform for discovery of relevant, druggable targets,” Woolf said. “We believe it can be applied to other neurological diseases that involve neuronal excitability, such as epilepsy and other neurodegenerative disorders like Alzheimer’s disease.”

In the meantime, Woolf is continuing to use human neuron screening platforms to identify novel targets for treating pain and neuropathy. “Using patient-derived neurons to model disease and test large sets of compounds whose targets are known can be the driver for developing new therapies,” he said.

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This story is adapted from an article originally published on the Boston Children’s Hospital website on June 8, 2021.

Source article: Huang, X., Roet, K. C. D., et al. (2021). Human amyotrophic lateral sclerosis excitability phenotype screen: Target discovery and validation. Cell Reports. DOI: 10.1016/j.celrep.2021.109224

This research study was funded by Target ALS, Pfizer Centers for Therapeutic Innovation, an ALSA Milton Safenowitz postdoctoral fellowship, and the National Institutes of Health. The authors declare the following competing financial interests: Kasper Roet, Clifford Woolf, and Kevin Eggan are founders of QurAlis Corporation. Roet is CEO of QurAlis. Coauthors Liying Zhang, Amy Brault, Allison Berg, Anne Jefferson, Jackie Klug-McLeod, Karen Leach, Fabien Vincent, Hongying Yang, Anthony Coyle, and Lyn Jones are current or previous employees of Pfizer Inc.