Hello, I'm Mokako Yoshioka. Thank you for giving me the opportunity to introduce my project. Today, I'd like to talk about the activation of endogenous mutated GNE product by small compounds. Now, I am talking from Tokyo, Japan. I am studying GNE myopathy at the National Center of Neurology and Psychiatry under Dr. Nishino and Dr. Inouye. This red-marked place is our center, and the Department of Neuromuscular Research is here in the red mark. In spring, we can see cherry blossoms, and in fall, the autumn colors of leaves, and they are so beautiful. So, please visit us when you come to Japan. So, these are the key points: activation of GNE would enable continuous sialic acid supply, and we believe that it would be very effective to treat GNE myopathy. So, today, I'd like to introduce our brand new approach to find small molecules that activate the GNE enzyme, and this project is funded by NDF. As you know, GNE myopathy is an adult-onset distal myopathy caused by allelic variants in the GNE gene.

the first appearance of symptoms typically occurs in around the third decade of life, and these two muscles are preferentially affected and developed to involve other muscles, leading to loss of ambulation. And as you know, by Allied mutations in DNA, Gene would lead to hypothalalization, and this is known to be the cause of muscle weakness and muscle atrophy in this disease. So, uh, let's take a close look at the pattern mechanism. There are two Pathways in salicylic acid biosynthesis, and one is the newly producing pathway called the noble pathway. And in this is a cell, and in this cell cytosol, cells use UDP group knob from glucose. It makes salic acid using enzyme reactions. And the other service pathway, and this is the pathway to take up silic acid from Foods or drugs, is here and encoding the two key enzymes of salic acid, the noble pathway. And if there are biolytic mutations in this DNA Gene, it would lead to low enzyme activity and then result in hypothalalization.

so the decrease of salicylic acid production is the essential cause of the muscle weakness in gene myopathy. So because it's a system of salicylic acid used in animal experiments or clinical trials in Newark, it's salicylic acid. It's so in New York, and solid lactose would be taken up using this Salvage pathway and Monarch, which is now used in the clinical trials or using this noble Pathway to make cervic acid. We hope the concept has been acquired using modernized studies. So when you treat a symptomatic model mice with orosilic acid, skeletal muscle atrophy and muscle weakness have been totally prevented. And even if you treat symptomatic mice with oral salicylic acid, skeletal muscle atrophy and weakness improved. So you can see it from these results that the treatment of salicylic acid supplementation was very effective in modern mice. But one of the major concerns to develop extrinsic salicylic acid supplementation therapy for humans was the rapid metabolization of salicylic acid.

so these are data of serum salicylic acid concentration in three patients. The upper graph showed serum salicylic acid concentration after a single administration of salicylic acid itself, and each color represents each of the three patients. You can see that there is no obvious increase in the salicylic acid concentration after a single administration. The figure below shows salicylic acid concentration after three-time administration of salicylic acid itself for five days. Also, you can see that serum salicylic acid concentration did not elevate in all three patients. I did not show in this figure, but urine salicylic acid concentration elevated immediately after administration. Salicylic acid is metabolized in a few minutes, but in my experiments, serum salicylic acid concentration was elevated. This is because it was diluted in water, and mice were drinking it very frequently throughout the day long. So, this enabled continuous salicylic acid supplementation, and I think this was one of the key points of high efficacy in mice. But as you know, humans cannot take salic.

acid so frequently all day long. So, extended-release tablet form of salicylic acid was developed for clinical trial to make salicylic acid last longer in patient's body. This figure shows serum salic acid concentration after oral supplementation of salicylic acid extended-release tablet. You can see in the upper figure that serum salicylic acid concentration increased in all three patients immediately after administration, and also you can see in the period below that salic acid concentration elevated in three times administration for seven days, even though there is a big individual difference. Extended-release topless form could elevate serum salicylic acid concentration, and these are the results of human clinical trials conducted using salicylic acid extended-release tablets. Serum salic acid concentration elevated in all three studies, higher in Phase 2 trial in U.S., Europe, and in Phase 2-3 trial in Japan compared to Phase 3 trial in U.S., Europe. And regarding to the efficacy, improvement of upper extremities was shown in Phase 2 trial but not in Phase

3 trial of U.S. Europe so regrettably autogenics had to terminate the development of salicylic acid extended release tablet for gmaopathy in Japanese study. The results are yet still under analysis, but it was similar to the study results of phase 2 trial in U.S. Europe, and they are still working to obtain drug approval. But anyway, you can see that supplementation of salicylic acid extended release tablets could be effective, but efficacy was not enough to improve symptoms as it modernize. So let's see the figure of the change in serum Civic acid concentration using salic acid extended release tablets again. So extended release of it for elevated serum salicylic acid concentration very well, but if you carefully see that at the same time it was decreased in a few hours. So we think if there we could provide more stable and continuous Supply, it might be more effective to treat this disease. And how to achieve more stable and continuous supplementation of salicylic acid? We think the best way is to activate GNE.

enzyme itself, so like in the right figure, and we think this is the most logical way, and this would enable continuous supply of silic acid for a very long time. And then how should we activate Gene amount of kindness. So traditional new drug development by pharmaceutical companies is one way, but it is not realistic in the field of open drugs, and it takes a long time for the development of new drugs and causes so much, and as the safety is not established, it is so risky. On the contrary, drug repositioning is economical, and safety is established, and it's faster for clinical use. So I decided to select drug repositioning to look for known drugs that could be a very effective therapeutic option for GMA party patients. But as you know, identification of the drug by drug repositioning is very difficult, especially in the field of finding activator used is called high throughput screening, and it is a method to read out luciferes or fluorescence of entire Wells of cells and read out by a plate leader. And we have previously

performed this kind of screening using lectin staining and used three kinds of libraries, which include 400 to 700 drugs. However, keeping cell proliferation constant was very difficult, and so density affected fluorescence, making it hard to achieve reproducibility. We had to explore another method. We decided to use in silico screening, and we are now collaborating with Dr. Yamamoto and Dr. Sekijima at the Tokyo Institute of Technology. They have a supercomputer, which is one of the world's top large-scale supercomputers. So, we asked them to collaborate with us and to perform structure-based screening. In this screening, the predicted structure of the enzyme and drug compounds are computationally matched, which is known as docking simulation. There are many clefts in the enzyme, so what we first have to do is identify which cleft to focus on as the binding target. Then, the computer will calculate how strongly these kinds of compounds, or each compound, will fit this target and provide us with the data. This supercomputer is able to dock.

the structure known compounds and calculate the binding score very very fast. So, it enabled to identify candidate compounds from 8042 structurally known compounds in drug Bank using this technology. The heat rate is known to be about 20 times higher than the previous High throughput screening. So first, we constructed the 3D structure of fullness DNA molecule, and the left figure shows the 3D structure we constructed, but it is a little bit complicated. So, I made a simple figure on the right side, and it feels that DNA molecule forms a stable dimer. And dimer means that two subunits are joined, and light blue is one subunit, and light green is the other subunit, and they are close like X. And this structure seems to be very important for enzyme reaction. And as you know, the protein Gene energy encoding have two enzymatic activities. One is UDP group two epimeros, the lower part of the enzyme in this figure, and the other is manokinase activity, the upper part of the enzyme in this figure. And there are so many cloves in these.

enzymes. So, where should we target? And this is the most key point and the most difficult point of this study. We have tried two approaches: inhibition of CMP Newark binding and stabilization of oligomerization. First, um, I will explain the first approach we took. This is a simple scheme of the dimer of GNE enzyme for producing manak. First, DNA enzyme will be open and UDP glutenant will bind to GNE enzyme. Then, GNE enzyme would be closed for enzyme reaction, and in this space, UDP Gluck knock will be changed to Monarch. And after the enzyme reaction, GNE enzyme would reopen to release Manic. And this kind of open-close, open-close formation change is always repeated to produce monarch. But when CMP New York, the final product of citric acid biosynthesis, binds to Gene e, it would act like a glue and interrupt the formation change of open-close, open-close to stop the production of mandak. So, this mechanism exists for the negative feedback process to maintain homeostasis by reducing excessive production of

salicylic acid. We first try to inhibit the binding of Simply New York using small compounds. We found the cavity in the back of the CMP New York binding site, and we thought that small compounds that fixed this cavity have a possibility to inhibit the burning of CMP New York and inhibit the interruption of formation change by Simply New York. So, I asked Dr. Love to perform in silico drug screening to discover and to select compounds which will face this cavity by not interrupting the formation change of G and E. We selected compounds that strongly bind to this cavity, and we have used 8042 compounds for screening. And in the right figure, each dot represents each compound, and we selected the compounds that have a very strong docking score. Next, we had to measure whether enzyme activity would be increased or decreased using these compounds. And previously on Jenny monokiness, enzyme activity was assessed by measuring radioactive substrates, though it is considered to be harmful nowadays. So, we established the method using Abe leveling, and this figure shows the method I used.

first I prepared wallpap g e WT means wall type and normal G and E and also mutated G and E and this time we use the mutation d207b and we use the cells and that would overexpressed these G and E then we incubate it so crude with UDP glutenoc and selected compounds and then measured Abe level monarch and please see the figure in the right side the below right flow figure and using this method manak production was significantly low in mutated during e compared to Wild type normal G and E so it was about 30 percent of all type Gene e so the primary goal over assay was to find compounds that increase mutant GNE enzyme activity vertical axis is the amount of Monarch produced in the tube after incubation and if it is high it means that GN enzyme activity is very high and WT means wall type normal G and E and the other words represents Mana production of mutant G and E which have mutations in D 207b you can see that Monarch production was

lower in mutant G and E. I added selected compounds to this mutant Gene E, but Monarch production did not increase. Also, please see at the right figure. I added CMP Newark in the incubation of wild type G and E, and then the small compound. So, you can see if you add CMP New York, the production of Monet decreases. I expected if it would increase after adding the compounds, but regulatively, we could not find any compounds that increase the Monarch production. So, no compound activated mutant G and E more. Worked as a competitor of CNP Newark. So, we tried the other approach. Genie is known to be active after organization, and oligomer is a molecule that consists of a few repeating units. Organization allows protein to form large structures without increasing genome size and provide stability, which would be crucial for enzyme activity. It is known that organization is required in DNA enzyme activity. In healthy people, the majority of the GNE protein forms the highest oligomer, but in GNA myopathy patients with mutant, especially in the patients with mutants.

involved in organization interaction service enzyme activity would be low by the failure of organization. The figure is immunoreactive patterns of cross-linked products. The horizontal axis represents the size of the protein. So when the GNA protein forms the highest oligomer, the peak of the mountain would be here at 400 kilos. And from this analysis, you could see that there are so many mutants that have failure in oligomerization, like these mutants which have peaks in 200 kiloton, or these mutants which are pigs in 100 kiloton. So especially in these mutants, stabilization of organization has the potential to elevate enzyme activity. And this is just a possibility, but also we believe that even for the mutants without organization failure, like this um, we think that acceleration of polymerization by small compounds has the potential to increase enzyme activity. Um, I would show ski mobile activity and oligomerization. So UDP globnock would turn to manak in the primaries domain. Then it will be passed to monocoinus domain, and monarch would turn to be mad at 6p in the

monokinase domain, and this monoc6p would become salic acid in the following other enzyme activities of the noble pathway, and we will call it the internal molecular transport pathway. And those also, there is another pathway, production of monarch. Monarch would be passed the amount of kindness of the other subunit and become 1x6p in manokinase. But if there is an oligomerization failure, like this figure, my neck will be produced in epimer's domain but could not be passed the Communist domain or the same subunit or to the other subunit. So, this structure could not produce monax 6p expectably because of the organization failure and leading to low production of salicylic acid. So, what we want to do is to fix this organization failure, like this animation, by small compounds. And we found that in the pink area, this is a very important interaction surface of the two subunits, and we found hydrogen bonds that are connecting the two subunits together, trick three, and making dimer in water Gene e.

so we selected the candy here as the bionic target and trying to find small compounds fighting this target to fix oligomerization and these are the results. So vertical axis is the manic production in the tube which represents enzyme activity and wild type WP black bar represents nomogene e and d207v is mutant Gene e and t207b is a known mutant which is very frequent in Asian and which is known to have organization failure and manic production of the 207b mutant is about 30 percent of wild type and we added candidate compounds to this mutant and as you can see some of the compounds seem to increase the Monarch production um it did not increase to the level of water type but it is said that an enzyme activity of heterozygous G and E which means that while olive oil is normal and the other have mutation are also reduced and about half of water gin e in the lowest but people with heterozygous mutation or are infected and healthy so from that maybe the

increase of the enzyme activity up to wall type might not be necessary. We are continuing to work [Music] compounds. We would repeat the experiments to confirm reproducibility and select the best three candidates, and we need to confirm that those dependent increase in the enzyme activity. Then we will perform computational screening again for structuring similar compounds to these three best candidates because we want to find the best compounds, the compounds that increase enzyme activity the highest, and also we would like to select compounds with low toxicity for clinical use, and if possible, approved drugs for faster clinical use. And also, we will confirm using patient cells with mutant of d207b and other DNA mutants and measure and conform the increase in salicylic acid production using these compounds. I would like to thank my great teachers, Dr. Nishino, Dr. Noguchi, and Dr. Hayashi, who were always giving me advice, including how to proceed with experiments or technical advice, and Miss Ogawa for her technical support, and all of the lab members like in this picture for helping me in administrations.

and also I'd like to thank Dr. Yamamoto and Dr. Sekijima for their exclusive specialized techniques using their supercomputer and also the platform project for supporting drug discovery and life science research project because this project matched Dr. Yamamoto and Dr. Sekijima to me. And also I'd like to thank NDF from the bottom of the heart for supporting this study because we could not do this work without NDF. And these are the key takeaways: so activation of DNA Mana kinase would enable continuous supply of oxalic acid, which can provide better treatment. And in circle screening, computational screening is a very effective approach to find activators from known compounds. And we found that stabilization of organization by small compounds has the potential to be a new therapeutic strategy. Thank you very, very much.

2022 Dr. Wakako Yoshioka: "Activation of Endogenous Mutated GNE Product by Small Compound"

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