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  Modeling Cysteinyl Leukotriene Receptor Antagonist KNW for Possible Optimized Asthma Treatment

  Shalet James, Sreejani Jonnalagadda, Emily Schmitt Lavin, and Arthur Sikora

  Underdiagnosed and under-treated, particularly in low- and middle-income countries, asthma has affected 262 million people globally. Current anti-asthmatic medications such as pranlukast inhibit cysteinyl leukotriene receptor 1 (CysLT1R), yet many patients do not respond to this drug. CysLT1R is associated with bronchoconstriction, inflammation, and mucus production in the airways of the lungs and bronchial tissues. When cysteinyl leukotrienes bind to CysLT1R, these effects are triggered contributing to the symptoms of asthma. The potential role of the related receptor CysLT2 in asthma remains poorly understood. To better understand this process, CysLT2R has been identified as a promising drug target for not only asthma but also other conditions such as brain injury and cancer. Students of the Honors Protein Modeling course at Nova Southeastern University modeled the interaction between a dual antagonist of CysLT1R and CysLT2R, KNW. A 3D model of KNW in complex with CysLT2R was based on PDB ID 6RZ6, modified using JMol, and 3D printed to showcase key interactions between drug and receptor. In this model of CysLT2R, we highlighted the ligand binding pocket, helix 8 (H8), and mutation residue interactions. The antagonist forms crucial interactions within the ligand-binding pocket (cyan). The N-linked carboxypropyl moiety forms salt bridges with Lys 37 and His 284 specific to CysLT2R (cpk). Mutating these residues to their CysLT1R counterparts decreases inhibition by antagonists. The key anchoring residue Tyr 119 interacts with benzoxazine, carboxylic groups, and amide linkers of the ligand (violet). The cleft opening residues include Leu 165, Val 208, and Tyr127 (light cyan). Unlike its counterpart, CysLT2R exhibits a wider cleft opening to the lipid membrane, enhancing ligand selectivity. Helix 8, a unique and flexible alpha-helix on the cytoplasmic side of the cell membrane, plays an important role in the regulation of G-protein activation and subsequent intracellular signaling cascades (pink). H8 conformation affects the binding site accessibility, signaling pathways, and receptor stability. Specifically, the salt bridge with Glu 310 stabilizes the junction between H8 and TM7 and the inactive state of the receptor when bound with the antagonist (cpk). Notably, the atopic asthma-associated mutation Met to Val in position 201 of CysLT2R results in a mildly impaired hypomorphic protein, with reduced ligand binding and inositol phosphate (IP) production (plum). Based on previously reported structure-activity relationship analysis, we developed a novel molecular inhibitor, Finlukast, aimed to have high affinity to both classes of receptors. Using SwissDock, we determined that this novel inhibitor molecule has high affinity binding to CysLT1 and CysLT2 receptors. Through the exploration and modeling of KNW, we gained further insight into the key structural interactions of dual antagonist KNW for similar receptor targets responsible for mediating the inflammation and bronchoconstrictive effects of cysteinyl leukotrienes.

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  Binding of Beta-site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Aminoquinoline (68K) for Possible Treatment of Alzheimer's Disease

  Juhi Dalal, Shreya Averineni, Pranav Madadi, Emily S. Lavin, and Arthur Sikora

  Alzheimer’s Disease (AD), affecting approximately 24 million people worldwide, is characterized by the formation of amyloid-β plaques within the brain. Alzheimer’s research has been focused on limiting amyloid-β production through developing inhibitors for the enzymes needed within the amyloid cascade. This project focuses on the aminoquinoline class of inhibitors, of which 68K (PDB: 5i3Y) is the most effective because of its strong Kd and IC50 values. The students of the Honors Protein Modeling class at Nova Southeastern University modeled the interaction between Beta-site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) and 68K. Using Jmol a model was developed, and 3D printed to show how the inhibitor (68K) fit into the enzyme’s active site. This model highlights important aspects of the interactions between the ligand and the BACE-1 enzyme. 68K has strong interactions with 32 amino acid residues in BACE1, some of which are intertwined with one another. For example, BACE-1’s residues Val69, Pro70, and Tyr71 are known collectively as “the flap”. “The flap” is a β-hairpin loop structure that is positioned directly over BACE-1’s catalytic dyad, a group of amino acids within the active site of the enzyme. “The flap” is also responsible for regulating access to the enzyme’s catalytic dyad (Asp 32 and Asp 228) by a given substrate (or inhibitor). Researchers found the inhibitor 68K to have interactions with the flap which maximizes the strength of the interaction with BACE-1 residues, thus minimizing the distance between the inhibitor’s various functional groups and accommodating their specific polarities. Being able to visualize the protein structure using a 3D model aids in the understanding of how the ligand inhibits this enzyme leading to the progression of AD.

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  Comparing Effectiveness of Two Antibodies (Aducanumab and Gantenerumab) on Reducing Amyloid-Beta Plaques

  Nikhila Paleati, Pranav R. Neravetla, Akhil B. Godbole, Emily S. Lavin, and Arthur K. Sikora

  Alzheimer’s disease (AD) is a degenerative neurological disorder that destroys memory and other important cognitive functions. As time progresses, brain cell connections, as well as the brain cells themselves, atrophy and die. AD is caused by a missense mutation in the amyloid-beta peptide within the amyloid precursor protein (APP). The mutation results in glutamine being replaced with glutamic acid. Previously conducted studies showed that mutated forms of the amyloid-beta peptide fragment have a greater tendency to stick together and form protein clumps or aggregates. The abnormal build-up of aggregates in and around the brain cells has been found to be strongly associated with the development of Alzheimer’s disease, therefore, it appeared crucial to study the methods that reduce these build-ups.

  Attempts to treat this disease have produced antibodies that bind to the mutated amyloid-beta peptide and clear the aggregated amyloid precursor protein out of the brain. The overall goal of this project is to use 3D printed protein models to show interactions leading to a clearer explanation of the efficacy variations between antibodies. One antibody, Aducanumab, is currently in Phase 3 clinical trials and has been fast-tracked by the U.S. Food and Drug Administration. Aducanumab functions by specifically binding to the mutated amyloid-beta peptide and clearing aggregates out of the brain. This antibody binds to a smaller linear epitope formed by amino acids 3-7 of the amyloid-beta peptide. Using Jmol, protein visualization software, the Aducanumab (6CO3) PDB was manipulated to highlight multiple hydrophobic interactions, shown in a dark salmon color, and 2 hydrogen bonds, shown in white. The small binding location, flexibility provided by fewer strong interactions, and high affinity for aggregates at a high density make the antibody ideal for clearing out large aggregates.

  Another antibody, Gantenerumab, is still undergoing testing in order to ensure safety and efficacy. This antibody functions by binding to a longer linear epitope formed by amino acids 3-11 of the amyloid-beta peptide. Unlike Aducanumab, Gantenerumab interacts with peptides through 2 salt bridges in addition to 3 hydrogen bonds and multiple hydrophobic interactions. Along with hydrogen bonds in white and hydrophobic interactions in dark salmon, the Gantenerumab (5CSZ) PDB was manipulated to show negative side chains of the salt bridge, labeled in red, while the positive side chains were labeled in blue. The increased number and strength of interactions reduces the flexibility of this antibody, thus making it difficult to easily bind and clear aggregated peptides. While both antibodies bind to a similar region of the amyloid-beta peptide and function to remove aggregates, they vary in the amount and type of interactions made with the amyloid-beta peptide.

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  Investigating The Mechanisms of Active Site Mutations to the 1T9G WT MCAD Protein to Better Understand Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCADD)

  Omar E. Saleh, Rhea Khatiwala, and Jeremy Ignatius

  Medium-chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is a human disorder that hinders β-oxidation, affecting approximately 1 in 17,000 people in the United States. Once mutated, the Acyl-CoA Dehydrogenase Medium-Chain (ACADM) gene, which is solely responsible for MCADD, is unable to produce enough MCAD enzymes to metabolize medium-chain fatty acids. As a result, fats are not catabolized, causing symptoms of lethargy and hypoglycemia, as well as damage to the brain and liver due to a buildup of unused fatty tissue. The purpose of this project was to investigate the possible and known effects of different amino acid mutations on the human MCAD protein and produce a 3D-printed model to explain the molecular story of MCADD. This model builds on previous bioinformatics and in vivo experiments aimed at revealing the underlying enzymatic mechanisms of MCADD. Using PyMOL, the human wild-type MCAD (PDB ID: 1T9G) had its electron transferring flavoprotein (ETF) complex removed and a single chain from its homotetramer portion isolated for clarity. PyRx was used to dock the substrate, Octanoyl-CoA (PDB ID: CO8) into the slightly mutated enzyme, referencing PDB ID 1EGC. Known mutations from the PDB files and related literature were then compared and analyzed on the modified 1T9G to determine known and possible effects the mutations had, such as helix-helix stability and ligand hydrogen bonding. LigPlot+ was then used to analyze ligand-active site interactions. Jmol was used to cosmetically enhance the modified 1T9G to produce a 3D model for printing. In the model, the mutations were ranked according to known K_M range values (0.4, 0.6), (0.7, 0.9), and 0.9+, which were highlighted in the colors “lightskyblue”, “royalblue”, and “midnightblue”, respectively; unknown K_M values were colored “chartreuse”. All mutations had their side chains shown for further clarity. E376, the catalytic base, was colored “magenta”, the backbone was colored “dimgray”, and the support struts of the model were colored “lightseagreen”. The use of the 3D model was beneficial, enabling model viewers to locate, determine, and hypothesize the mutations and their effects on MCAD, in addition to providing a visual and physical learning aid for researchers, professors, students, and other biomedical professionals. Furthermore, the clarity produced by a physical model ultimately enables further research for MCADD and may assist in the development of a cure for those who unfortunately suffer from this rare condition.

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  Modeling the binding of ω-conotoxin and other toxins to the N-type voltage-gated calcium channel

  Serena Sha, Sophie Welch, and Ashley Guillen-Tapia

  Approximately 1.5 billion people in the world suffer from chronic pain, persistent pain that carries on for longer than 12 weeks despite medication or treatment. Management of chronic pain typically includes the use of non-steroidal anti-inflammatory drugs (NSAIDs) or prescription pain medications, including opioids. An alternative therapy derived from conotoxins, toxins released from marine predatory snails in the family Conidae, was approved for the treatment of severe chronic pain in 2004. This pharmaceutical has the trade name Prialt and is also known as ziconotide. Once ziconotide is in the human body, it acts as a channel blocker of the N-type voltage-gated calcium channels, also known as Cav2.2. The literature and related PDB files were manipulated using PyMol and Jmol to create a 3D-printed model to explain the molecular story behind how a particular conotoxin binds to a calcium-gated ion channel. Additionally, computer visualization tools were used to show how several related toxins from other organisms would be expected to dock to the calcium ion channel. The 3D-printed model highlights specific features that contribute to the ω-conotoxin (MVIIA) binding to the calcium channel alpha 1B subunit as described in the literature (PDB: 7MIX). These conotoxins have a very characteristic disulfide bond linkage pattern which plays a role in the correct folding of the peptide and stabilization of its structure. In MVIIA, the non-cysteine amino acids form unstructured loops affecting binding affinity and calcium channel-blocking activity. Of particular interest is the second loop located between Cys8 and Cys-15. It appears to be exceptionally important in directing selectivity toward N-type calcium channels and away from P/Q-type calcium channels. Ziconotide does not directly seal the entrance to the vestibule of the selectivity filter, but it blocks ion entrance by neutralizing the outer electronegativity and sterically hindering the ion access path to the entrance of the selectivity filter. Salt bridges are formed between Arg10 and Tyr13 on ziconotide and Asp664 of the channel. Four of the eight ziconotide-coordinating residues, Thr643, Asp1345, Lys1372, and Asp1629 in Cav2.2 are not conserved in other calcium channels which may explain the subtype specificity of pore blockage by ziconotide. The EEEE motif consisting of Glu314, Glu663, Glu1365, and Glu1655, determines the Ca2+ selectivity. Also included in the model are the receptor's alpha helices and bound calcium ion. The N terminus and C terminus of the receptor are labeled in blue and red respectively to orient the model.

  No crystal structures are available for ω-conotoxins bound to several other types of N-type calcium channels. To investigate the potential calcium channel blocking properties of conotoxins MVIIC, GVIA, MoVIB (from the cone snail, Conus magus) and ω-agatoxin IVA (from the spider, Agelenopsis aperta), the computer-based tool ROSIE was used to simulate binding of these peptides to the Cav2.2 channel. As expected, the toxin shown in the crystal structure of 7MIX, bound best to the Cav2.2 channel. The toxins MVIIC, GVIA, and MoVIB bound with lower affinity. The agatoxin IVA did not have any relevant binding to this calcium channel. Overall, protein modeling allowed for a deeper understanding of how conotoxins bind to and block the calcium channel possibly leading to additional therapeutic approaches to pain relief.

  Support or Funding Information:

  This work was made possible by funding through the National Science Foundation, Division of Undergraduate Education (NSF-DUE) grant number 1725940 for the CREST Project. Nova Southeastern University’s Farquhar Honors College and Dept. of Biological Sciences also provided support. Protein model printing was made possible by 3d Molecular Designs.

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  Exploring structural differences between antagonistic peptides for the development of orally bioavailable PCSK9 inhibitors

  Bhavya Soni and Pritika Vemulapalli

  Familial hypercholesterolemia (FH) is an autosomal genetic disease that causes elevated blood levels of low-density lipoprotein (LDL). One of the leading causes of FH is gain-of-function mutations in the gene coding for proprotein convertase subtilisin/kexin type 9 (PCSK9). The PCSK9 protein binds to LDL receptors (LDLR) on the surface of hepatocytes and promotes their degradation, preventing the recycling of LDLRs and thus increasing LDL blood levels. Monoclonal antibody therapies that bind to PCSK9 inhibiting LDLR binding are currently only available as an injection. However, several orally bioavailable PCSK9 inhibitors have been formulated and are undergoing clinical trials. One such therapy contains small-molecule-peptide inhibitors that bind to a cryptic site (N-terminal groove) adjacent to the LDLR binding site located in the catalytic domain. A helical region (S153-I161) is contained within this groove with conformational flexibility leaving the area open to small-molecule peptides. The peptide must consist of two components: a helical peptide with a high binding affinity to PCSK9 and an appended extension with antagonistic properties to inhibit LDLR binding. The extension must encroach upon the LDLR binding site’s hydrophobic pocket which significantly contributes to the binding energy of LDLR. Using two known PDB structures containing the removable peptides (A (5VLP) and B (6U3I)) in the N terminal grove, a three-dimensional printed model was created to demonstrate the interactions and proximity to the hydrophobic pocket. This model highlights critical amino acids on the peptides and PCSK9 to emphasize how the interactions support certain substitutions. One significant interaction lies between PCSK9 residues Ile369, Phe379, Asp238, and Ala239 and Peptide B’s organic moiety (1-amino-phenylcyclohexane-1-carbonyl). These PCSK9 residues surround the LDLR binding region's hydrophobic pocket indicating a successful inhibition. On the other hand, Peptide A’s FPG motif added to the Trp1 anchor formed a beta-turn which was unable to reach and, therefore, interact with PCSK9 residues Ile369, Phe379, Asp238, and Ala239. Through the 3D model, it was visualized that Peptide A had a beta-turn that precluded further extension into the target site, limiting its antagonistic ability. The model also highlighted Peptide B’s attached organic moiety which reaches the hydrophobic pocket in the proximal LDLR binding region, as shown in the literature to increase the binding affinity by >100 fold with reduced overall mass for an improved oral therapeutic.

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  How can we design an inhibitor with an enhanced binding affinity that is selective for MMP12 ?

  Aisha Y. Abdool, Lyla Abbas, Tassnime Sebaei, Emily Schmitt, and Arthur Sikora

  Matrix metalloproteinase 12 (MMP 12) is one of the twenty-three members of the peptidase M10 family which are primarily responsible for the breakdown of the extracellular matrix. MMP12 plays a key factor in the degradation of elastin and is commonly studied in the lungs of smokers, where MMP12 digests the elastin and serves as a chemokine to recruit a pro-inflammatory immune response. Thus, MMP12 is a major therapeutic target in wound healing and scar formation following a myocardial infarction. Students from the Honors Protein Modeling class at Nova Southeastern University modeled the interactions between MMP12 and various inhibitors. Using the protein data bank, an MMP12 protein complexed with the inhibitor called EEG under the code 3LIK was discovered. The structure was imported into JMOL: a protein visualization software. EEG intercalates into the S1 loop of the MMP12 protein without causing any disturbance to the loop's conformation. Murine trials were found with corresponding data for another MMP12 inhibitor known as AS111793 which was shown to reduce inflammation associated with cigarette smoke. A series of inhibitors were created using key components of EEG and AS111793. The binding was modeled on Py-Rx: a screening software used to dock the inhibitors. It was found that the hybrid compound created had a higher binding affinity than AS111793, but less affinity than EEG. This may be because a majority of the solvents and elements were removed from the inhibitor which did not allow the docking to occur.

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  Comparing the Effectiveness of Ivacaftor and GLPG1837

  Jordan Nichole Carreras, Saimi Reyes, Vibha Sankavaram, Emily Schmitt Lavin, and Arthur Sikora

  Cystic Fibrosis is a fatal autosomal recessive genetic disease that effects more than 70,000 people worldwide. This disease is caused by a mutation within the CFTR causing an excess of mucus within the lungs, and effects other cells that produce bodily fluids (i.e. sweat and digestive fluids). The aim of this project was to identify the key differences between two similar potentiator drugs, Ivacaftor (Kalydeco®) and GLPG1837, that are used in the treatment of Cystic Fibrosis. The research question posed was: How does a model show the difference in efficacy of GLPG1837 compared to Ivacaftor? The comparison was done through the research and rendering of protein models through the Jmol software. The Jmol files of the drugs were taken from the Protein Data Bank (PDB). The files that correlate to the drugs are 6O2P (Ivacaftor) and 6O1V (GLPG1837). The files displayed the drugs within the CFTR channel attached to their binding sites. Additionally, further research was done on their respective chemical compositions through PubChem. The noticeable differences between the two drugs were their chemical composition, despite binding to the same active site within the CFTR. There is sulfur found within the hinge region (binding site) of the CFTR. Due to GLPG1837 containing a sulfur molecule and sulfur’s high binding affinity for itself, this appears to be the cause of GLPG1837’s increased efficacy over Ivacaftor. Due to the noticeable difference of the drugs displayed in the models it is concluded that an efficacy can be shown between the medications through modeling. Furthermore, it’s recommended that models such as these be used in the process of providers explaining treatments to patients who are using these medications. By providing tools to break down the science behind these medications to all age groups it can further rapport between patient and provider. As well as reduce stress of patients due to a better understanding of their treatment.

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  Limiting Iron Acquisition of E. Coli With Anti-TonB1 and AntiTonB2​

  Jose Diaz, Ryan Luib, and Seethal Doki

  The bacteria E Coli infect approximately 200,000 individuals in the United States and can lead to critical illnesses. The transfer of iron from the bloodstream to bacteria is one of the main requirements for the survival. One of the mechanisms to obtain iron by use of siderophores and the protein FhuA was investigated. Undergraduate students grouped into teams to each explain a unique molecular story- modeled in Jmol/Pymol and demonstrated through a poster and PowerPoint. Using the PDB file, 2GRX, a drug to inhibit the TonB-Ton Box interaction which provides the FhuA iron mechanism energy to operate was researched. A protein model was designed to bind to the Ton Box region of FhuA with higher affinity by altering polar and charged residues to nonpolar or aliphatic residues that were present in the original TonB protein through programs Pymol and Jmol. The presence of many nonpolar amino acids in residues 8-16 and 588-592 of FhuA and the charged amino acids in residues 166-170 and 225-235 of TonB suggest that mutations of R166F, N227L, K231A on the tonB will lead to a protein with a stronger interaction with the Ton Box. The change from Arginine 166 to Phenlyalanine facilitates a nonpolar-nonpolar interaction between the TonB and Alanine of FhuA. Similarly, mutating polar asparagine to nonpolar leucine and the change of positively charged Lysine to uncharged Alanine will allow a stronger interaction. These hypothesized interactions of the mutated Anti-TonB1 with the FhuA are based on predicted outcomes because of the limitations of programmed docking of large proteins. Through the research and design of the course, students were able to develop skills with protein interface programs such as Pymol, Jmol, and Pyrx. This project was made possible by Nova Southeastern University and the guidance from Dr. Arthur Sikora and Dr. Emily Schmitt Lavin.

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  Highlighting How the Structure of Marine Bacterial Laminarinase Can Improve Biogeochemical Cycling During Global Climate Change

  Heidi Hellenbrand, Rachel Harris, and Chino Villanueva

  Marine bacterial laminarinase cuts polysaccharides in the process of remineralization, a key process in the ocean biogeochemical cycling of nutrients. We reviewed the protein model of 6JH5 in Jmol to highlight the important areas of the model that relate to thermostability and function and the effects of global climate change on the protein. The catalytic cleft of Glu135 and Glu140 was vital to the depolymerization mechanism. Substrate chain positions 130-143, specifically Trp130 being used for recognition, were important to the proficiency of the structure. The calcium ion on the opposite side of the β-sheet from catalytic cleft increased its degrading activity and thermostability. The residue interactions with Glc(−1) and Glc(−2) were unveiled to be crucial for β-1,3-glycosidic bond selectivity by the enzyme. Previous studies also showed the residue interactions were also important to the protein’s thermostability and thermophilicity. Our research found that the protein’s function will be negatively impacted by global climate changes as temperature beyond the protein’s optimal temperature (6JH5: 20°C) caused by climate change will also decline activity as hydrogen bonds between proteins weaken and being to denature. The function of our protein is important in the biogeochemical cycling of nutrients in the ocean and without its high activity, the cycling of nutrients and DOM like carbon and nitrogen won’t be as proficient.

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  Investigating the Structure of Potential New Drug to Treat Sickle Cell Anemia through Inhibition of the Polymerization of Hemoglobin S​

  Brianna M. Lacasse, Isadora R. De Abreu, and Rathika Manikandan

  Sickle cell anemia is a hematologic disorder impacting over 15 million people worldwide. It is caused by a single point mutation in the gene hemoglobin-Betha, where a glu group is replaced by val (GAG --- GTG) in the seventh codon (glu7val) of chromosome 1. In this study, we are comparing the anti-sickling properties of drugs in varied conditions in order to create a drug that is effective in an O2-independent manner and with a 1:1 stoichiometry for lower dosage purposes. We used Pymol and Jmol to compare the structures of the aldehydes GBT-440 and VZHE-039, which interact on the same binding site to treat sickle cell disease. GBT-440’s bulkiness allows it to have a 1:1 stoichiometry, while VZHE-039’s solubility is due to its interaction with the hemoglobin’s alpha cleft, allowing it to be O2-independent. We identified the pyridine and pyrazole structure from GBT-440 and the methyl hydroxy moite from VZHE-039 as key structures, and created a hypothetical new drug, a hybrid of VZHE-039 and GBT-440. The pose predicted would allow the drug to interact with the sickled hemoglobin in a 1 to 1 ratio and in an O2-independent manner.

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  Comparing the Effectiveness between Ivacaftor and GLPG 1837

  Saimi Vanessa Reyes, Jordan Nichole Carreras, and Vibha Sankavaram

  Cystic fibrosis is an autosomal disease that is caused by a defect in the CFTR, or cystic fibrosis transmembrane conductance regulator. It affects more than 70,000 people world-wide and is life-threatening to those affected. To treat this disease there are two drug classes: correctors and potentiators. Correctors aim to correct the misfolded CFTR proteins, while potentiators aid in increasing ion concentration outside of the cells by keeping the channels open for longer periods of time. The aim of this project was to identify the key differences between two similar potentiator drugs, Ivacaftor (Kalydeco®) and GLPG 1837. The research question posed was: How does a model show the difference in efficacy of GLPG1837 compared to Ivacaftor? The comparison was done through the research and rendering of protein models through the Jmol software. The Jmol files that were used were 6O2P (Ivacaftor) and 6O1V (GLPG 1837) from the Protein Data Bank (PDB), which displayed the drugs in their binding site within the CFTR protein. It was shown that GLPG 1837 contained a sulfur molecule, while Ivacaftor did not. It is believed that this difference is what causes GLPG 1837 to appear more effective than Ivacaftor (in clinical trials) because the hinge region of the CFTR protein also contains a sulfur molecule. These models can be used by providers to explain treatments to patients who are using these medications much more effectively. By providing tools to break down the science behind these medications to all age groups it can further rapport between patient and provider. As well as reduce stress of patients due to a better understanding of their treatment.