Researchers from the University of Texas Southwestern Medical Center in Dallas have developed and used a cell-based assay incorporating cultured mouse neurons to evaluate the protective effects of five clinically relevant compounds on glutamate-induced apoptosis in Huntington’s disease.
The research is important in the near term for Huntington’s disease research because it provides in vitro characterization of drugs that have thus far primarily been investigated only in Huntington’s mouse models or clinical trials. It also provides additional proof that memantine and riluzole might be promising Huntington’s treatments.
In addition, the scientists hope to parlay their work into a higher-throughput assay suitable for industrial-scale drug discovery, and will likely incorporate high-content screening methods to do so, Ilya Bezprozvanny, an associate professor in Alzheimer's research at UT Southwestern, told CBA News this week. To this end, the team has already formed a loose alliance with Cellomics, Bezprozvanny said.
The research, which appears in the Oct. 30 issue of Neuroscience Letters
, was supported by the HighQ Foundation, a non-profit organization dedicated to Huntington’s disease research.
The HighQ Foundation and sister organization CHDI have the shared mission of “bringing together academia, industry, governmental agencies, and other funding organizations in the search for Huntington disease treatments,” according to the organizations’ website.
While CHDI pursues more of a biotechnology-oriented approach to Huntington’s drug discovery through industrial and academic partnerships, HighQ more often supports research aimed at target identification and validation, the development and use of animal models, drug delivery, and finding disease progression markers.
Both organizations, however, have recently heavily supported the use of cell-based and high-content strategies for Huntington’s research. For example, in August CHDI said that it had chosen Evotec as a strategic partner for its drug-discovery programs, including assay development and high-content screening.
In addition, CHDI has licensed Redistribution assay technology from Fisher’s BioImage unit for use in its drug-discovery efforts; partnered with Cellumen to develop cell-based models for Huntington’s disease; and is supporting Duke University researchers in their efforts to use HCS to identify neurodegenerative disease targets in live rat-tissue explants.
‘Do Something Useful’
Bezprozvanny and colleagues are in the midst of a two-year HighQ Foundation grant, for which there are two goals. First, the scientists wanted to use their cell-based assay, in its present format, “to do something useful” for Huntington’s drug discovery.
“Basically I decided that one of the most useful things we can do, when limited by the number of compounds, is to test compounds that are seriously being considered for clinical trials or are already in clinical trials, and provide people with some kind of quantitative and unbiased information,” Bezprozvanny said.
The scientists chose to work only with drug candidates that target the glutamate pathway in cells, which many believe is implicated in the development of Huntington’s. They specifically chose five glutamate pathway inhibitors – folic acid, gabapentin, lamotrigine, memantine, and riluzole – each of which has been previously evaluated in Huntington’s mouse models or human clinical trials.
Bezprozvanny said that some in vitro information for these drugs as potential Huntington’s treatments exists, but each of the models is very different from one another.
“This was just trying to provide useful information to people planning these clinical trials, to tell them that some drugs work and some don’t in a side-by-side in vitro comparison,” Bezprozvanny said. “People are already interested in these drugs anyway in the context of Huntington’s, but this may help them make a decision between these four or five drugs as to which one goes forward into clinical trials.”
The researchers used TUNEL staining and manual microscopic imaging to screen the compounds for their ability to inhibit apoptosis in glutamate-treated cultured mouse neurons. They found that memantine, which is better known as a treatment for Alzheimer’s disease, and riluzole exerted a significant neuroprotective effect, while the other three compounds did not. Memantine and riluzole have also shown more promise than the other compounds in Huntington’s mouse models and clinical trials.
The second and more important goal for the scientists is to develop their cell-based Huntington’s assay into something compatible with high-throughput screening methods, Bezprozvanny said. To do this, they are considering using high-content screening or an alternative caspase-3-based apoptosis assay.
“People are already interested in these drugs anyway in the context of Huntington’s, but this may help them make a decision between these four or five drugs as to which one goes forward into clinical trials.”
“We understand that if we want to discover new drugs and we want to use this as, say, a primary screen … it will probably never be high throughput enough to screen 100,000 compounds,” Bezprozvanny said. “My goal is to get it to the point where we can screen between 1,000 and 5,000 compounds.
“We are actually trying to get away from TUNEL staining,” he added. “I really wanted to focus on changes in cell morphology, because this is something you can do with live cells. Before the cells die, they change their shape. Can we use that as a readout?”
The scientists have already been in discussions with Cellomics about the possibility of such an assay. And although the cost of an HCS instrument is a bit too high for the scientists’ current budget, Bezprozvanny said that they are working with Cellomics “long distance” to evaluate its HCS platforms.
Interestingly, Bezprozvanny recently returned from a trip to Institut Pasteur Korea, where he discussed a potential collaboration on adapting the cell-based Huntington’s assay to a high-content format. Cellomics rival Molecular Devices just announced the sale of an HCS instrument to IPK, but the institute also has in-house an Opera confocal reader from Evotec, which has a co-development and co-marketing agreement with Cellomics.
Bezprozvanny said that he and his colleagues hope to demonstrate to the HighQ Foundation the value of applying HCS to their cell-based Huntington’s model.
“[HighQ] funded us to make this assay medium-throughput,” Bezprozvanny said. “If we set up the assay, and it looks like it’s working, one option is that they will let us do the screen. Or, [they] might say that they know a company that can take over … to run an industrial-scale screen.
“I hope that the foundation will be interested enough in this to support it further,” he added. “It will be their call at the end to say either ‘A company should really do this now,’ … or, ‘Here’s the money. Buy an instrument and go for it.’ I would be happy either way, but maybe a little more [with] the second one.”