Modeling Cysteinyl Leukotriene Receptor Antagonist KNW for Possible Optimized Asthma Treatment

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.https://nsuworks.nova.edu/protein_modeling_reports/1012/thumbnail.jp

Binding of Beta-site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) Inhibitor Aminoquinoline (68K) for Possible Treatment of Alzheimer\u27s Disease

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.https://nsuworks.nova.edu/protein_modeling_reports/1011/thumbnail.jp

Identifying the Binding Residues on CYP3A4 to Naringin using Protein Modeling and Docking

Cytochrome (CYP) enzymes are a superfamily of monooxygenase hemoprotein enzymes that are found throughout the body but are heavily concentrated in the endoplasmic reticulum and mitochondria of liver cells. These enzymes catalyze reactions that modify a wide range of substrates into more hydrophilic and, therefore, more readily excreted forms. Cytochrome enzymes are heavily involved in the detoxification process of many medically relevant drugs. As such, the inhibition and activation of these enzymes can substantially alter the effective bioavailability of medications and can introduce additional variables or modifiable variables into a pharmacological-based treatment. Cytochrome p450 3A4 (CYP3A4) is one of the most abundant cytochrome enzymes and can target a wide range of substrates, including many medically relevant drugs. Inhibition of CYP3A4 can increase the bioavailability and duration of the availability of a medication in the bloodstream. This makes the factors associated with the inhibition and activation of CYP3A4 of great medical interest and importance. CYP3A4 has been noted to be inhibited by naringin, a flavanone found in grapefruits and other citrus fruits. However, the characteristics of naringin binding to CYP3A4 are unknown. The residues at which naringin binds to CYP3A4 were identified using 3-D protein modeling and computerized molecular docking simulations. The PDB file for the CYP3A4 enzyme (8DYC) was obtained from the RCSB Protein Data Bank and was manipulated to remove the substrates, leaving only the prosthetic heme group. The binding between CYP3A4 and different inhibitors was studied in the literature to identify specific features of the CYP3A4 protein. The residues that form the opening of the protein cleft, the area where the substrates bind and the residues that form the active site of the enzyme were noted for reference for the docked position of naringin. PyRx was used to conduct molecular docking simulations to predict the location of naringin binding in the CYP3A4 structure. However, due to computational limitations, the docking did not predict interactions with the HEME group. Rather the final docked structure of naringin to the CYP3A4 enzyme was produced using the coordinates of the docked naringin and the HEME-containing structure of CYP3A4. The Contacts/Clashes functionality in USCF Chimera was used to identify the specific residues on the surface of the protein and these residues were compared to those identified through the literature consulted. The residues of CYP3A4 that are involved in binding naringin that were identified through this process were: Glu 374, Arg 372, Arg 106, Arg 105, Ala 370, Phe 215, Arg 212, Phe 304, Leu 482, Ser 119, Ile 223, Thr 224. Of these, Arg 372 and Thr 224 were also noted in the literature to be involved in forming the opening of the cavity in the protein. The residues Glu 374, Arg 106, Arg 105, Ala 370, Phe 215, Arg 212, Phe 304, and Ser 119 were also noted in the literature and are likely involved in forming the active site. The residues Ile 223 and Leu 482 were not noted in the works consulted and may represent additional or potentially novel residues that may be involved in the inhibition of CYP3A4. Overall, the results of the molecular docking suggest that naringin inhibits CYP3A4 by binding and blocking both the opening of the cleft within which the substrate binds and binding to residues in the active site of CYP3A4.https://nsuworks.nova.edu/protein_modeling_reports/1015/thumbnail.jp

Using 3D Modeling to Describe the Electromotility of the Outer Hair Cell Protein Prestin, and its Role in Sound Perception Among Mammals

Prestin is one of the key motor proteins that has been identified in enabling auditory perception in mammals. By modulating its electromotility in response to changes in environmental voltage, prestin contracts and elongates in the plasma membrane of cochlear outer hair cells (OHCs). This allows different frequencies of sound to be processed quickly and precisely. Belonging to the SLC26A5 family of anion transporters, prestin is especially adept at binding anions in order to facilitate its oscillation through a series of unique conformations. Previous research has demonstrated that prestin is reversibly inhibited in the presence of salicylate. However, the broader mechanisms by which prestin senses and transduces voltage into cellular movement are not yet well understood. Previous studies have described the electromotility of prestin in terms of non-linear capacitance (NLC), wherein conformational changes in the protein are not linearly related to the voltage applied. High NLC is imperative for sound amplification in the cochlea, as this property enables OHCs’ selective response to different frequencies of incoming sound. 3D protein modeling was employed to better visualize the electromotility of this OHC protein by manipulating models of bottlenose dolphin (Tursiops truncatus) prestin available in the Protein Data Bank. Using the software PyMOL, Chain A of prestin in the inhibited state (7S9E) and Chain B of the compact, sensor-up state (7S8X) were spliced together into a novel merged model that depicts the fluctuation in the cross-sectional area of the transmembrane regions. This was possible because prestin is a protein homodimer whose peptide subunits could be swapped and replaced accordingly upon manipulation in the program. Key elements of prestin’s topology were then highlighted on this nascent model to emphasize the locations of the 14 gate and core transmembrane (TM) helices, the site of anion binding, and the cytosolic STAS domain. The colors corresponding with these regions include salmon, light blue, dark violet, and lavender, respectively. Helices TM3 and TM10 play a pivotal role in facilitating the movement of prestin’s dimers when bound to specific ligands. Likewise, the pocket formed by residues Gln97, Phe101, Phe137, Leu397, Ser398, and Arg399 has been identified as the anion binding site in the homodimer. The binding stability of ligands is further enhanced via additional noncovalent forces present in the active site, including pi stacking between salicylate and Phe137, and Ser393’s participation in hydrogen bonding. Arg399, the only positively charged residue in the cavity, is known to rotate up and down while the protein is moving, and neutralization of this key residue has also been found to eliminate prestin’s NLC entirely in vitro. Other residues of note that have been described by researchers include a series of 13 amino acid replacements (depicted in gold) that appear to be shared by several echolocating mammals, indicating convergent evolution between bats, whales, and dolphins.https://nsuworks.nova.edu/protein_modeling_reports/1013/thumbnail.jp

Modeling Calcium Binding to Yersinia pestis Type III Secretion Needle

A type III secretion system (T3SS) is utilized by a variety of bacteria to inject toxins into host cells to cause disease, including Yersinia pestis, the bacterium that causes the bubonic plague. The Y. pestis T3SS needle subunit is a polymer made up of YscF proteins. Calcium is thought to play a role in regulation of toxin secretion by the YscF needle. High levels of Ca2+ binding inhibit secretion while low levels of Ca2+ binding cause secretion to be stimulated. According to a study done in 2005 by Torruellas et. al, different mutations of YscF can interfere with secretion regulation, most likely due to altered calcium interactions with the needle. Some details regarding which mutations lead to constitutive secretion (CS), no secretion (NS), or regulated secretion (RS) of the Yersinia outer protein (Yop) toxin are known. The goal of our research is to understand how the mutations present in previous wet lab experiments affect calcium binding to the YscF hexomer subunit. To understand if and how calcium interacts with the YscF needle to inhibit Yop secretion, an accurate model representation of the needle-calcium interactions is required. To model this phenomenon, the interactive accelerated protein prediction tool, CollabFold, was used to create probable hexamer subunits of the needle with the mutations from the previous experiment (Torruellas et. al, 2005). MIB2, a binding prediction modeling server, was used to predict binding with Ca2+, Fe2+, Fe3+, Zn2+, Cu2+, Mg2+. Alternative divalent cations were used to compare to Ca2+ binding to the hexamers. Ca2+ bound to the wild type (WT) hexamer at 12D, 15D, 16L; 28D, 29D; 36D, 37A with an average binding potential of 2.4. The binding sites contain aspartic acids that lead to CS when mutated to alanine, suggesting those binding sites play a role in calcium regulation of the needle. Zn2+ and Fe2+ bound to the WT hexamer at 56, 60 with an average binding potential of 4.0. Zn2+ and Fe2+ bound with higher binding potentials to the WT mutant hexamers on average compared to Ca2+. The hexamer structures of the different mutations were compared to look for structural differences between the mutations. Arginine 73 creates a ring in the center of the WT hexamer. The arginines at position 73 are further apart compared to the WT hexamer in the double mutant (D28A, D46A), I13A, and D17A CS hexamer mutations. The arginines at position 73 were obstructing the center passage in the N31A and D77A NS hexamer mutation. The arginine ring structural comparison between hexamers suggests that the R73 ring may play a role in regulating Yop secretion by obstructing the central needle passage. A 3-d printed model was created of a possible hexamer of the YscF Y. pestis needle subunit with the residue mutations causing constitutive secretion of Yop, and corresponding mutations indicating no Yop secretion, each highlighted in designated colors. The mutations indicating constitutive secretion are I13A, D17A, D28A, D46A. The mutations leading to no secretion are N31A, V34A, D77A, D77C, I82A, I82C. The results determined what mutations lead to CS, NS, or RS of the Yop toxin in the presence or absence of calcium. Torruellas, J., Jackson, M., Pennock, J., Plano, G. (2005). The Yersinia pestis type III secretion needle plays a role in the regulation of Yop secretion. Molecular Microbiology, Vol 57(6), 1719-1733, https://doi.org/10.1111/j.1365-2958.2005.04790.xhttps://nsuworks.nova.edu/protein_modeling_reports/1016/thumbnail.jp

Development of Sharkavir : A Hypothetical Inhibitor for HIV-1 Protease

Human immunodeficiency virus (HIV) affects approximately 37 million people worldwide and results in over 1 million deaths annually. A class of drugs first developed in 1995 inhibits the enzyme HIV‐1 protease, thus preventing the maturation of an HIV precursor protein. There are now over 10 protease inhibitors available to treat HIV. Multiple mutations in the protein have made this treatment less effective. The CREST (Connecting Researchers, Educators, and STudents) team at Nova Southeastern University modeled the interaction between HIV‐1 protease and its competitive inhibitors. Darunavir is currently one of the strongest competitive inhibitors, as it binds effectively to the substrate envelope and has yielded a lower resistance for patients. The most effective treatment is a combination of two of these inhibitors: Darunavir and Ritonavir. In order to explain how these drugs work, the active site of the non‐mutated wild‐type HIV‐1 protease was depicted as a binding box model. The protease inhibitors Darunavir, Ritonavir, and our hypothetical drug, “Sharkavir” were 3D printed to show how they fit into the protease active site. Details of the wild‐type HIV‐1 protease, as well as the drugs Darunavir, and Ritonavir, were found in the Protein Data Bank files, 1T3R, 4DQF, and 1N49, respectively. Both structures were imported into Jmol: a protein visualization software. The new protease inhibitor molecule, “Sharkavir”, was designed as a combination of Darunavir and Ritonavir using Marvin Sketch: a software used to manipulate molecular structures. The molecular structure of the hypothetical drug “Sharkavir” is shown below

The Power of Play: A Pediatric Role in Enhancing Development in Young Children

Children need to develop a variety of skill sets to optimize their development and manage toxic stress. Research demonstrates that developmentally appropriate play with parents and peers is a singular opportunity to promote the social-emotional, cognitive, language, and self-regulation skills that build executive function and a prosocial brain. Furthermore, play supports the formation of the safe, stable, and nurturing relationships with all caregivers that children need to thrive

The use of phylogeny to interpret cross-cultural patterns in plant use and guide medicinal plant discovery: an example from Pterocarpus (Leguminosae)

The study of traditional knowledge of medicinal plants has led to discoveries that have helped combat diseases and improve healthcare. However, the development of quantitative measures that can assist our quest for new medicinal plants has not greatly advanced in recent years. Phylogenetic tools have entered many scientific fields in the last two decades to provide explanatory power, but have been overlooked in ethnomedicinal studies. Several studies show that medicinal properties are not randomly distributed in plant phylogenies, suggesting that phylogeny shapes ethnobotanical use. Nevertheless, empirical studies that explicitly combine ethnobotanical and phylogenetic information are scarce.In this study, we borrowed tools from community ecology phylogenetics to quantify significance of phylogenetic signal in medicinal properties in plants and identify nodes on phylogenies with high bioscreening potential. To do this, we produced an ethnomedicinal review from extensive literature research and a multi-locus phylogenetic hypothesis for the pantropical genus Pterocarpus (Leguminosae: Papilionoideae). We demonstrate that species used to treat a certain conditions, such as malaria, are significantly phylogenetically clumped and we highlight nodes in the phylogeny that are significantly overabundant in species used to treat certain conditions. These cross-cultural patterns in ethnomedicinal usage in Pterocarpus are interpreted in the light of phylogenetic relationships.This study provides techniques that enable the application of phylogenies in bioscreening, but also sheds light on the processes that shape cross-cultural ethnomedicinal patterns. This community phylogenetic approach demonstrates that similar ethnobotanical uses can arise in parallel in different areas where related plants are available. With a vast amount of ethnomedicinal and phylogenetic information available, we predict that this field, after further refinement of the techniques, will expand into similar research areas, such as pest management or the search for bioactive plant-based compounds

A pragmatic approach to resolving technological unfairness: The case of Nike’s Vaporfly & Alphafly running footwear

Background Technology is often introduced into sport to facilitate it or to improve human performance within it. On occasion, some forms of novel technology require regulation or prevention entirely to ensure that a sport remains fair and accessible. Recently, the Nike Vaporfly and Alphafly shoes have received some concerns over their appropriateness for use in competitive distance running. Methods This paper evaluates the use of these shoes against an existing framework for sports technology discourse and adopts a pragmatic approach to attempt to resolve them. Results It is proposed that the three concerns regarding cost, access and coercion cannot be ruled out but likely remain short term issues. As a result, it is proposed that these running shoes are acceptable forms of technology but that ongoing vigilance will be required as such technologies develop further in the future. Conclusions The Nike Vaporfly/Alphafly shoes do push the perceived acceptability of running shoes to the limits of the current sports regulations. However, the alleged gains have not manifested themselves to a level that could be considered excessive when reviewing historical performances or when evaluated against a set of well-cited criteria. The sport will need to adopt a stance of ongoing vigilance as such technologies continue to develop or be optimised in the future