Simulation of Amyloid Beta Peptide Vaccine to Treat Alzheimer’s

Alzheimer's disease is the sixth and leading common cause of death in the United States. The seventh most frequent reason behind death for people 65 years of age or older. It is estimated that 5.8 million Americans who are 65 years of age or older will be diagnosed with Alzheimer's by 2020.1The cost of providing for them will reach $360 billion by 2024, With the cost only expected to increase. . Medicaid and Medicare, the federal and state governments pay for two-thirds of Alzheimer's disease-related expenses.2 Alzheimer's disease is becoming more fatal than heart disease and cancer, which are both seeing decreases in death rates while Alzheimer’s is seeing an increase. Research has shown that underreporting of Alzheimer's disease and other dementias on death certificates occurs often so the number of older people living with this disease may be much greater.1 Alzheimer's disease is a brain condition that progressively reduces one's ability to function, think clearly, and remember moments or people. For most people with Alzheimer's, the symptoms develop later in life.3

The idea that too much amyloid beta peptide formation in the brain is what causes Alzheimer’s is the most scientifically supported explanation. 42 amino acids make up the amyloid beta peptide, which comes from the precursor protein, amyloid beta precursor protein (APP). This is expressed in numerous organs, particularly in the synapses of neurons as a key player in the pathophysiology of Alzheimer's disease (AD). On chromosome 21, the gene encoding of the amyloid beta precursor protein. The beta-secretase and gamma-secretase break apart the amyloid beta precursor protein to produce amyloid beta peptide, which has 40 or 42 amino acids and is designated as Ab 1-40 or Ab1-42. The peptide's N and C terminals are produced by beta-secretase and gamma-secretase on amyloid beta precursor proteins. Ab1-42 the primary pathogenic peptide is hydrophobic and has a propensity to form fibrils as well as oligomers. The hydrophobicity of the peptide is imparted to its C terminus by the hydrophobic amino acids. The amyloid plaques that are visible with the standard H and E staining of amyloid-specific staining are subsequently formed by these fibrils arranged in a beta-pleated sheet.4-5 Additionally, the buildup of amyloid beta protein causes protein clusters to develop. The main focus for the development of Alzheimer’s treatment has been the Aβ Peptide.6-8 Many studies have been done manipulating the amyloid beta peptide in mice with varying success rates. But most show that amyloid beta peptide does play a role.9-11the creation of a vaccine to treat Alzheimer’s has been discussed widely and shown to be a promising idea.12-15 However, the idea of reducing the amyloid beta in the brain via the use of a vaccine has not been looked into specifically using an endopeptidase (an enzyme that can break peptide bonds). This fact has inspired the idea of creating a 3d model and simulation that shows a vaccine injecting an endopeptidase to break apart amyloid beta clusters in the brain. The reason a 3d model and simulation are most appropriate at this time is that they are the perfect foundation for future creation and research into an amyloid beta vaccine. A 3d model demonstrates how a vaccine like this would look and be used. A simulation proves whether or not the endopeptidase would be able to successfully break apart and reduce the formation of the amyloid beta clusters. By giving a starting point for a physical model of the vaccine and showcasing that a vaccine like this would in fact be successful allows for valuable future research/advancements on a amyloid beta vaccine. Also, even though my hypothesis is that the simulation will show that the endopeptidase is able to successfully break apart the amyloid beta, if it instead shows that an endopeptidase is not able to successfully break apart amyloid beta clusters in the brain. This project is still beneficial because it ensures that there is no time, effort, or resources wasted on creating an AB vaccine similar to the one created in this project as it will not work or will need changes in order to. 

Methods 

This project is a non-experimental creation project. As I am making a product not holding an experiment. The methods are mixed and this is because while my simulation will yield quantitative results my 3d model will not as I am not testing/counting anything. The reason I chose to make a product rather than hold an experiment is because at this stage of my academic career I am unable to hold any experiments on real Alzehimer’s Patients. Also, I wanted to focus my project on advancing the research for vaccine creation. To create my 3d model I began by looking at common protein vaccine structures in order to ensure that my model would be accurate. I used the website Tinkercad (Tinkercad [Internet]; San Francisco: Autodesk, Inc.; c2024) to construct my model. I chose this website because I was familiar with it from secondary school. I began by creating an account with the website so as not to lose my progress then I watched the website tutorial video to refresh my memory. After I watched the video I created a new project. I then started creating my model, I used the photos of vaccines, specifically protein vaccines, as a foundation and created a model I thought would get the job done. Here is it below. 

As you can see there are two grips on the side allowing the vaccine to be held between the pointer and middle finger and a button on the back that can be pressed with the thumb. There is a sharp point in the front of the vaccine which is intended to be the needle. The vaccine would be 

much thicker and larger than the average flu or COVID vaccine. This is because this vaccine would have to hold much more product than the average vaccine as the amount of endopeptidase needed would be larger than the amount of medicine in other vaccines. Also this vaccine would have to be injected in the brain (as stated the amyloid beta protein is in the brain) meaning that the structure of it would have to be much more complicated. 

To create my simulation I began by researching the specific endopeptidase I wanted to use. The research in the field details neprilysin as a strong candidate so this is the endopeptidase that I chose.15-16I decided to use the GROMACS application as it is free and I was slightly familiar with it unlike other simulation softwares. GROMACS is a software used for the simulation of nucleic acids, lipids, and proteins. To begin making the simulation I downloaded GROMACS on my personal computer I then watched a quick tutorial video to refresh my memory. After doing this I went into the “Protein Data Bank”, which is a website that contains the molecular structure for all proteins. I searched “Amyloid Beta Protein '' at the top right of the website. The correct photo for the protein popped up, it is below. 

(Photo of Amyloid Beta from RCSB PDB(Protein Data Bank) was used in simulation)17 After I located the correct structure for the amyloid beta protein I clicked the “download” button on the upper right hand corner sending the download to a specific file I had created for the project. I then searched for the neprilysin structure and clicked the download button again, sending it to the same file. The photo is below. 

(Photo of Substrate Free human Neprilysin from RCSB PDB(Protein Data Bank) was used in simulation)17 

After I had both structures downloaded it was time to “clean them”. “Cleaning” the structures means preparing them to be sent into the simulation; this is necessary to ensure that there are no factors including the proteins that may affect the accuracy of the simulation results. To clean the protein you begin by removing all the water molecules and ligands this can be done with the tools within GROMACS (you pull the structure up and delete the water molecules and  ligands the application indicates where these parts are). Then you ensure that the protein structure is accurate by pulling up photos of the structure and comparing. In my case the structures were accurate so I did not need to change them. It is also important to check for correct protein chain connectivity this means that the amino acids are connecting appropriately.

To check this look at the residuals and bond angles they should be accurate for the structure. The correct values can be found with a google search. My values for both of my structures were accurate so I did not need to change them, if your values are not correct use a molecular modeling software to resolve the issues. Once my structure was “clean” it was time to build the simulation box. I used the Gromacs program “editconf:” which centers the proteins. I then prepared my submit script. My script included the module preparation and the Gaussian command. I used the script “esculentin.slm:”. I then used the “esculentin.slm:” script and “bsub” command in order to submit the job. I then monitored the job to ensure that everything was running smoothly. I ran the job 100 times to ensure that my results would be valid. After the job was done I needed to retrieve the output files, I ran the appropriate commands in the terminal (this is from the local directory where I wanted to download the files) and analyzed them. The main limitation in my method was my lack of simulation knowledge while I was familiar with GROMACS I had never run a simulation like this before. So there may have been more I could have done with the simulation that someone more knowledgeable would have known to do. A certain trick I was not aware of would have made my results more favorable or allowed me to run my simulation more efficiently which many have yielded different results. Results/Analysis My hypothesis was correct: the endopeptidase (neprilysin) was able to effectively break apart the amyloid beta in the brain. The results of my simulation showcased a 80% success rate out of 100 trials. This means that out of 100 trials Nepryslin was unable to break apart the amyloid beta clusters for 20 trials. This shows a relatively high success rate but indicates that it is not 100% successful. The simulation also indicated that in the successful trials it began to break apart the amyloid beta protein after about the equivalent of 4 or 5 days. These results indicate that a amyloid beta vaccine would work in reducing the amount of AB peptide clusters in the brain but do not indicate whether or not this would reduce or cure an individual's Alzheimer’s it is simply a treatment following the theory that the over formation of the amyloid beta protein in the brain is what causes Alzheimer’s. It also does prove or disprove the overformation theory, it just gives a 

treatment for it. 

Conclusion 

The results of this project indicate that the endopeptidase, neprilysin specifically, can efficiently break apart the amyloid beta clusters in the brain. This means that if the theory that the reformation of the amyloid beta clusters is what causes Alzheimer’s is true this vaccine, if advanced upon, could be used as a treatment for Alzheimer’s. But again it does indicate whether or not the theory is true. The limitation present in this project is that the ability for neprilysin to break apart the amyloid beta clusters in the brain was run on a simulation. The human brain is complicated and not as predictable as a simulation. So it is possible that if neprilysin is used to break apart the amyloid beta clusters in a real human brain the success rate will not be exactly 80%. Also the 3d model created may not be able to be replicated physically very easily as it is just a model. The implications of this project are that more research will be done into the creation of an amyloid beta vaccine to treat Alzehimer’s as this project shows it has promise. This project also adds to the research on the over formation of AB peptide causing Alzehimer’s theory. Which in turn can help us determine where this theory is true or not. For those attempting to replicate or advance this research I recommend beginning to create a physical model of the amyloid beta vaccine that is based on the 3d model shown in this project. I also recommend running the simulation with a different endopeptidase. Even though neprilysin was the endopeptidase that was the most discussed in my field as a potential amyloid beta breaker.

There are many others. And these other endopeptidases may yield better results; examples of these other endopeptidases are Proteolysis-Targeting Chimera Strategy or an Insulin-Degrading Enzyme (IDE). 

Written by: Yasmina Kodeih 

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