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STEM

STEM is definitely one of the more unique classes I have taken at MAMS, and more so for its nature. In this class Dr. Crowthers guides us in indulging in an independent research project. With this guidance, we are led to come up with a solution to any type of problem in the world no matter what the topic may be. We perform experiments in local labs, 3D print what was once a sketch, and prepare for our December Fair in which we present all the hard work we out into or various projects. I specifically did a project on hematology and Sickle Cell Anemia, so feel free to scroll down to learn more about it!

Modifying Ribonucleotide Reductase and Hydroxyurea to Increase Binding Affinity

This year, I have had the opportunity to indulge in a research project focusing on Sickle Cell Anemia. The reason I indulged in this topic is because the disease is actually something that runs through my family, and it is something I have grown up with for a majority of my life. Therefore, it has always been in my best interest to learn more about the disease and to come up with way to alleviate it as effectively as possible. This project has shown me that fluorination (a method further discussed below), is a method that has the potential to shift the way medicine is viewed. With more exposure, fluorination may have a large impact on the bolstering of drugs for the future. Feel free to reach me at Kakese@wpi.edu

Abstract

Studies show that out of every 365 African American childbirths in America, one has the red blood cell modifying Sickle Cell Anemia disease. Hydroxyurea (HU), the most cost-efficient treatment for individuals with the disorder, attacks the red and white blood cell proliferation agent, ribonucleotide reductase. This allows for fewer sickle cells to be created through the DNA synthesis of deoxynucloeside triphosphates (dNTPs). Through the stifling DNA polymerase, red blood cells (RBCs) and white blood cells (WBCs) experience a pause in creation. However, due to the excretory nature of ribonucleotide reductase (RR), HU leaves the cell soon after ingestion, which poses the issue of individuals having to take medicine every day with no faults in consistency. Due to the recommended consumption of HU being daily, fluorinating hydroxyurea may increase the binding affinity of HU to RR. Using computational protein modeling software, PyMOL, and SWISS Modeling, protein interaction modeling simulates the effects of fluorinating HU on the increase of hydrogen bonds on the ribonucleotide reductase-hydroxyurea binding complex. By comparing the number of hydrogen bonds formed in each iteration, the binding affinity of the drug and its synthetic viability is affirmed. Increased binding affinity ensures that individuals who take hydroxyurea now can wait longer intervals before consuming the medicine, allowing them to focus on other aspects of their health. Using protein modeling to simulate the modification of drug-binding relationships may play a prominent role in the development of future drugs to come, ensuring optimal treatment for all blood-related diseases alike.

Research Proposal

Proposal Link!

Phrase 1

If hydroxyurea is fluorinated to make it permanently bind to ribonucleotide reductase, how may this increase the efficiency of binding in the system?

Phrase 2

If hydroxyurea were to be fluorinated, this would allow for permanent binding of HU to ribonucleotide reductase due to the nature of the fluorine atom. This will also allow for more leeway for individuals who take hydroxyurea as treatment, as instead of having to take it every day, they would instead take it for longer increments allowing them to focus on other aspects of their health than their medicinal intake.

Background

Sickle Cell Anemia is an anemic disorder that causes an individual's red blood cells to be produced from red blood cell progenitors as sickle-shaped cells. It has run rampant, affecting the lives of many African Americans, including individuals in my family. However, with the help of protein modeling, drugs in the hematological industry can go through the process of becoming refined for a more practical purpose. Whether this is through fluorination methods to increase binding affinity, or other means of mutation, the usage of protein modeling may prove to benefit the pharmaceutical and medical industry's need to create medicine with the most optimal treatment capability. Sickle Cell disease is most common amongst those of African and Middle-eastern descent, as its recessive trait allows for protection against malaria: which is highly prevalent in such areas of the world. Affected cells have a significantly lower life span than the typical red blood cell—10-20 days as compared to 110-120 days (Johns Hopkins Medicine, 2019)—and carry certain properties that cause blood-related hardship for its recipients. Such blood cells, due to the hemoglobin associated with them (HbS), cause the development of sticky membranes and decreased flow through the body. This translates to a lack of oxygen flow throughout the body leading to depleting health events that cause severe pain: a sickle cell crisis. Sickle cells also affect one's ability to fight certain diseases properly due to the inability of lymphocytes to migrate throughout the body effectively. Due to the insufficiency of hemoglobin-S (sickle) to benefit one’s oxygen affinity, many individuals with sickle cell, or any anemic disease for said matter, have higher levels of hemoglobin-f (fetal) in their blood system due to its high affinity for oxygen induction. Introspectively, such a disease causes not only physical distress but mental stress as well. Due to its effect on red blood cells, many males with the disease tend to have a lower sperm count (91% of male sickle cell patients over 25) depriving many of them of having children of their own (Smith-Whitley, 2014). Not only does the disease affect males, but females as well. Due to sickle cell disease, female pregnancy may harbor more painful episodes due to irregular blood flow. Individuals may also run a high risk of giving birth to a baby under the optimal birth weight of 5 pounds and 8 inches—consequently, treatment for such a disease is highly sought-out.

Origins of hydroxyurea

The approval of hydroxyurea to be used for Sickle Cell Disease began in 1995 when the food and FDA approved its optimal usage (Charache et al., 1992). With its original use being for cancer treatment, due to its cell-division-inhibiting and coaxial nature, many healthcare officials and researchers saw it fit to be used for other excessive organ proliferative diseases, such as Sickle Cell Anemia. Hydroxyurea is now widely used as one of the primary treatments for individuals with sickle cell anemia, being used by many individuals with Sickle Cell today. Because of its nature, many patients who take the medicine report success after a couple of weeks to months of taking the medicine. For some individuals, change is not as apparent due to either infrequent consumption of the drug, or a non-optimal drug dosage prescription by one’s doctor; however, the drug has not been seen to be ineffective in individuals for any other reason. Nevertheless, the drug continues to change a menagerie of lives, inducing the congregation of hemoglobin-f in one’s bloodstream, allowing for a large decrease in vaso-occlusive events (pain in the body due to lack of blood flow). In light of the disease, individuals forgetting to take hydroxyurea daily (which is the most optimal frequency for the medicine to work), or them being prescribed a dosage not suitable for their disease severity allows for such vaso-occlusive events to arise once more(Charache, 1992). In order to combat this, one effective atom is capable of taking the nature of hydroxyurea and allowing it to bind more effectively to its respective sight on ribonucleotide reductase, changing the biological properties of the drug. This atom is none other than fluorine—an atom with highly enriching properties that will continue to be used as more awareness is drawn to its optimality.

Fluorination

As a result of measuring the electronegativity of fluorine and its small size (18 atomic mass), scientists have been able to prove its efficiency in the medical realm. Small molecules such as fluorine, tend to have a higher mobile affinity, thus being more effective when passing through cell membranes. This proves to be highly advantageous to individuals, as when the drug is consumed, the ligand will most likely bind to its protein due to the high permeability of the fluorine atom. Apart from its size, such a molecule has a high electron-withdrawal property that allows it to attract electrons easily, thus increasing its binding affinitive properties. Being able to withdraw electrons and bind to certain molecules, serves as an intrinsic baseline for altering the biological properties of certain ligands. Adding fluoride to certain drugs allows for the oleophobicity (repelling of liquids) to decrease, allowing for drugs to safely pass through non-polar liquids in our bodies. Such liquids and molecules, such as lipids, and oils, tend to prohibit molecules that have a high oleophobicity (oil-repelling nature) from entering the cell. Therefore, having a molecule that can alter this biological property of a compound will allow for the drug to effectively pass through one’s bodily systems, thus increasing the chances of effective binding of the drug. Due to there being two types of fluorine reagents, nucleophilic (relatively negative) and electrophilic (more positive), it is important to know what molecule one intends to fluorinate before assessing what subsection of fluorination one would like to adopt. In this regard, fluorination may very well be one of the most influential aspects to the altering and bettering of a melange of drugs in the world today, and continues to do so even now (Yerian, et. al, 2016). Hydroxyurea, a highly prevalent drug in the anemia and cancer world, requires frequent dosages in order to work effectively. A study on the effects of hydroxyurea on individuals with Sickle Cell Anemia, facilitated by the Johns Hopkins Pharmaceutical company shows how even with a prescribed dosage, many individuals either forget to take the medicine or are not prescribed the most optimal dosage of the drug (Charache, S, et.al, 1992). These faults can lead to a mélange of vaso-occlusive events as mentioned previously, which include pain crisis, acute chest syndrome, organ damage, and an overall lack of airflow through the blood system of the affected patient. Therefore there must be a way to allow hydroxyurea to permanently bind to its targeted protein, in order for such cell “killing”. In addition, potentially mutating the active site at which the drug binds, may allow for increased binding affinity if such a mutation facilitates the binding of hydroxyurea more than normal. I intend to implement this method using various protein editing software such as PyMOL, SwissDock modeling, and Maestro, to model the outcomes of mutation inputs. Through modeling different iterations of hydroxyurea using SwissDock AME, the pharmacokinetics and pharmacodynamics of each iteration may be compared. After such iterations are simulated using Maestro, the number of hydrogen bonds that form due to the addition of the fluorine atom will be comparable as well. These two factors serve to allow for the most viable and attainable drug to be synthesized, overall benefitting the consumer.

Procedure

Over the course of 5 months, I was responsible for collecting data via various computational software about ribonucleotide reductase and its relationship to hydroxyurea when binding. All of the computational simulations were performed using Maestro, a protein modeling software belonging to the UMass Chan Medical School. With the help of Diego Suchenski Loustaunau, I was able to get familiarized with the Maestro software, using it for various aspects of the experiment. Dr. Celia Schiffer was very pivotal in allowing me to have access to the UMASS Chan computer labs, where most of the simulations of the hydroxyurea iterations were conducted. Using the computer labs, the ligand preparation of each computational molecule of hydroxyurea was created, allowing for the simulation process to run as effectively as possible. Using PyMOL to extract the PDB code of the hydroxyurea (molecular formula CH4N2O2) to ribonucleotide reductase complex (access number: 3VPM), the binding properties of the drug to the protein became available without any mutation and or fluorination applied to the system. Maestro was utilized to edit the biological and chemical makeup of hydroxyurea and such a change was transferred into SwissDock for it to be rendered into code. This code was then assigned to PyMOL to be displayed in context with ribonucleotide reductase in order to view the binding pattern of the protein and the molecule to be compared with other fluorinated iterations of the relationship. Each iteration was considered using SwissDock AME molecule analysis software, where each iteration received certain criteria based on its pharmacokinetics and pharmacodynamics. Then, using Maestro to perform the simulation of the protein-to-ligand binding relationship, the number of hydrogen bonds formed as a result of the modification were considered, as well as how feasible the drug would be based on specific criteria provided by SwissDock ADME and the increase in binding affinity through the addition of hydrogen bonds, as a result. Overall, the most feasible iteration of fluorinated hydroxyurea was identified in order to prove the benefits of fluorination and its possible future use. Preparation Primarily, the structure of hydroxyurea binding to ribonucleotide reductase was converted into a PDB 3D iteration in order to view the electrostatic interactions in the system. Using PyMOL, the PDB code of the structure (3VPM), coupled with the chemical makeup of hydroxyurea, was used to create hypothetical binding complexes using SwissDock. To differentiate the colors of the hydroxyurea hypothetical clusters, the protein is colored green, with the clusters binding to separate areas of the protein. In order to decide which iteration is the correct one, the area where Tyrosyl-radical (the radical in which HU conducts electron transfer to inhibit the reduction of ribonucleotides to deoxyribonucleotides) is found. Recent studies have shown that the most optimal starting area for tyrosyl radical was near the 120-128 residue area, hence the positioning for the non-fluorinated model (Gräslund et al.). Using SwissDock AME, each iteration of hydroxyurea was created by analyzing the chemical components of the molecule. Knowing that the areas where fluorination would have the least effect on the physiochemical makeup of the molecule, replacing it with certain hydrogen the molecule was the most prudent approach. After each iteration was created, each molecule was transferred into Maestro where ligan preparation was enacted, to ensure that each molecule would be physiochemically fit to be put through simulation. The protein Ribonucleotide Reductase was then imported into Maestro to be prepared for simulation as well, being computationally solved at a pH level of 7.5. After both the ligand and the protein were prepped, an induced fit docking, using the induced fitting feature on Maestro, was executed. This allowed for each ligand to be placed at its respective protein pockets to have the best outcome when going through simulation. The protein pockets were determined by the position of tyrosyl-radical as previously identified in Gräslund et al. With the ligands located in their respective protein pockets on ribonucleotide reductase, simulation was achieved using Maestro molecular dynamics feature. Each iteration went through a simulation of approximately 200ns, translating to 15 to 20 hours of total simulation.

Analysis

Discussion/Conclusion