High Throughput Screening to Identify Glut1 Modulators
Hello and a Happy New Year to everyone!
This year has already begun with a new and exciting publication in our research community!
The paper I would like to share with you this time is called “Identification of glucose transport modulators in vitro and method for their deep learning neural network behavioral evaluation in Glut1 deficient mice”. This project was led by Dr. Jason Park and Dr. Juan Pascual at UT Southwestern Medical Center in Dallas, Texas with funding support from the Glut1 Deficiency Foundation and the Million Dollar Bike Ride/Penn Medicine Orphan Disease Center.
What was the motivation to pursue this project?
According to the authors, Glut1 Deficiency has proven incompletely or inadequately treatable. Most of the antiseizure drugs make the seizures worse or are ineffective when seizures are present. The ketogenic diet (the current standard of care treatment), despite helping to reduce seizures in most of the patients who tolerate the diet, does not help with all the symptoms in patients with Glut1 Deficiency, in some cases it is not beneficial, or it even makes seizures worse for a small number of patients. This information confirms that additional and better treatments are needed.
Additionally, the authors knew through personal experience and publications done by others that only small increases in glucose transport such as the ones experienced after a meal are beneficial for Glut1 Deficiency patients. This suggested to them that a relatively small change in net glucose transport could significantly improve brain dysfunction in Glut1 Deficiency; therefore, for a drug to be potentially useful for patients, it might only need to induce just a small increase in glucose transport.
Their goal for this study was to identify compounds that modulate glucose transport, either as activators (activate or increase the transport of glucose through Glut1 – useful for Glut1 Deficiency) or inhibitors (block the transport of glucose through Glut1 – useful for cancer treatment).
To approach this goal, they tested 9,646 compounds to evaluate their effect on accumulation of a fluorescent glucose analog reporter in cultures of a cancer cell line which expresses more Glut1 (glucose transporter type 1) than any other type of glucose transporter. This reporter is a compound that is similar to glucose but it cannot be metabolized, and in addition is it labeled with fluorescence, so it can be traced.
How does this test work?
Cultured cells are grown in media that helps them get nourished; then the compounds to be tested are added to the cell culture and after a while, the fluorescent glucose analog is added to the culture. The culture cells can then be analyzed for the amount of fluorescence present inside the cells, showing how much glucose was transported through Glut1 to the inside of the cell where it can be used.
After the cells in culture have been treated (or not) with drug and glucose has been added, they are processed, so that the amount of fluorescence can be determined.
What were the results?
Many different compounds showed a glucose transport modulatory effect.
From these compounds, they identified 5 drugs currently approved and prescribed for other conditions or that have been characterized at the pre-clinical level that had a positive effect on glucose transport. They also found 37 novel activators and 9 inhibitory compounds, meaning that these novel compounds have not yet been described before and lack any type of characterization. Among the activators, they found four that had been used in neurologic diseases, including acetazolamide and baclofen. Their publication contains tables with the list of activators or inhibitors of Glut1 that they identified.
There was no information for those compounds regarding Glut1 or what their effect was on the transporter. To sort those according to their effectiveness on Glut1 Deficiency, Dr. Pascual and Dr. Park’s team decided to study the effect of those compounds on motor dysfunction in their mouse model of the disease (motor dysfunction is present in Glut1 Deficiency patients).
Glut1 transporter activity in their mouse model is reduced to about 50 % (similar to observations in humans). Due to the fact that ataxia is one of the symptoms shown in Glut1 Deficiency, their team decided to evaluate the effect of two drugs, acetazolamide and baclofen, which showed positive results in the cell experiments and have been previously reported as having been used in patients with Glut1 Deficiency. They used the mouse model to assess spontaneous locomotion and measure body part spatial relations during movement. In addition, they used a machine learning assisted method that they had previously designed to expand the analysis.
These two methods allowed them to identify 49 locomotor parameters that differentiate Glut1 Deficiency mice from control mice. Acetazolamide and baclofen showed positive results in the locomotion and movement of Glut1 Deficiency mice treated with these two drugs, however, the authors report that when doing this type of investigation, further studies are necessary to elucidate the impact of a drug on humans when the effect cannot be directly inferred from mouse data. Additionally, the authors want to emphasize that drugs such as acetazolamide and baclofen have not yet been systematically studied in Glut1 Deficiency patients and therefore estimating dosage, efficacy and side effects will need significant more work or expertise.
According to their team, their results suggest that numerous prescription and other unsuspected drugs can exert glucose transport stimulation or inhibition and that locomotor testing in Glut1 Deficiency mice is a good way to sort the drugs’ effectiveness.
Finally, Dr. Pascual, Dr. Park and their teams report that future work for this project will study the compounds at the mechanistic level, meaning that their goal is to understand exactly how the activators are activating the increase in glucose transport. Since it is known that many different mutations on the slc2a1 gene lead to Glut1 Deficiency, and that depending on the mutation there are different effects on the Glut1 protein (for example, localization of the protein or its function), understanding the specific mechanism of action of the compounds can help determine which mutations will have a positive effect by specific drugs.
You can find the publication by clicking here. It will be free, open access for a period of time.
I would like to thank Dr. Pascual and Dr. Park for taking the time to review this document.
Sandra Ojeda, PhD
Glut1 Deficiency Foundation