BRET NSAID’s Exciting New Novel: My unconventional Aunti Sereotin

Recent Submissions

  • Design, Synthesis, and Anti-inflammatory Studies of NSAID Hybrid Conjugates

    Honkanadavar, Hitesh; Department of Chemistry & Physics; Augusta University; Panda, Siva (2019-02-13)
    Non-steroidal anti-inflammatory drugs are one of the most common drugs administered worldwide as highly effective analgesic, antipyretic and anti-inflammatory agents. The drugs function by inhibiting the COX-2 enzyme system which leads to a decrease in inflammation; however, the drugs also inhibit the COX-1 enzyme system which is critical to normal renal function, gastric mucosal integrity, vascular homeostasis, and the autocrine response to circulating hormones which can lead to gastric ulcers and renal dysfunction. Hybrid conjugates of existing non-steroidal anti-inflammatory drugs (NSAIDs) have already been synthesized with Ibuprofen, Acetaminophen, and amino acids to increase potency and decrease toxicity. Computational chemistry studies of these compounds show that the free phenol moiety in acetaminophen plays a greater role in the inhibition of the COX 2 enzyme system than the amine moiety. The previous compounds utilized the phenol moiety to form the product. New hybrid conjugates of Ibuprofen and Acetaminophen via amino acid linkers have been synthesized, leaving the phenol moiety free. The compounds have been characterized by NMR and IR. Biological studies indicate that some of the synthesized compounds are showing improved potency when compared to Ibuprofen alone. Computational chemistry studies and molecular modeling will be used to support the�in-vivo�biological activity.
  • Investigating the Interaction Between G Proteins and the 5-HT1E and 5-HT2C Serotonin Receptors Using BRET

    Little, Lauren; Department of Biological Sciences; Department of Chemistry & Physics; Augusta University; Spencer, Angela (2019-02-13)
    G protein-coupled receptors (GPCRs) are important mediators in cellular signaling and are common targets of drug action. GPCRs are responsible for the transduction of extracellular signals into intracellular signals, mediated by G proteins of four types: Gs, Gi, Gq, and G12/13. A thorough understanding of a signaling pathway involves determining which G protein is coupled to a signal-activated GPCR. In this project, a technique called Bioluminescence Resonance Energy Transfer (BRET) was used to measure the interaction between an activated GPCR from the serotonin (or hydroxytryptamine, 5-HT) receptor family, and G proteins from each subtype. The cDNA for serotonin receptors 5-HT1E�and 5-HT2C�was fused with the gene for a luminescent protein called Nanoluciferase (Nluc). Then, the receptor-Nluc DNA along with DNA containing a G protein tagged with a fluorescent protein (Venus) was transfected into mammalian cells for expression. Data from BRET assays suggest that the 5-HT1Ereceptor couples to the Go and Gi subclasses of G proteins upon serotonin activation while the 5-HT2C�receptor couples to the Gq class of G proteins. Profiling serotonin receptors will deepen our understanding of serotonin receptors, associated diseases, and the drugs that target them.
  • The Identification of Novel Genes in Normosmic Hypogonadotropic Hypogonadism (nHH)

    Smith, Hannah; College of Education; Department of Obstetrics & Gynecology; Augusta University; Layman, Lawrence (2019-02-13)
    Characterized by delayed or absent sexual development, idiopathic hypogonadotropic hypogonadism (IHH) is a disorder that includes the deficient production, secretion, or action of gonadotropin-releasing hormone (GnRH). Producing neurons in the brain, GnRH directly controls sexual development during puberty. Misplacement of the GnRH producing neurons leads to hypogonadotropic hypogonadism, which is divided into two categories: Kallmann syndrome (KS) and normosmic IHH (nHH). While both KS and nIHH, defined as the absence or delay of puberty, low gonadotropins and sex steroids, are similar, KS also includes the absence or impairment of smell. Whole exome sequencing (WES) is used to examine protein-coding regions of the human genome in order to detect genetic variants that could be causative. Sanger sequencing is used to confirm variants identified by WES. Using WES and Sanger sequencing, we were able to identify new genetic variants within the nHH and KS patient populations. In this study, our goal was to identify pathogenic variants in known and novel nHH/KS genes, focusing efforts on rare, loss-of-function variants in: WDR11, GLI2, CTNNA1, ANKHD1, SEMA6A, PRRC2C, EHBP1, and RIF1 genes. This study broadens our understanding of pathogenic variants in known and novel IHH genes that may contribute to the disease phenotype.
  • The Study of 5ht-1d and 5ht-1f Receptor Interactions with Mini G Proteins via Bret Analysis

    Trang, Amy; Department of Chemistry & Physics; Department of Pharmacology and Toxicology; Augusta University; Spencer, Angela; Lambert, Nevin (2019-02-13)
    G protein-coupled receptors (GPCRs) are receptors involved in signal transduction, a process for converting extracellular signals into internal messages to elicit a cellular response. Signal transduction pathways involve activating various G protein subtypes (Gs, Gi/o, Gq/11�and G12/13) which typically lead to second messenger production. Traditionally, second messenger concentration assays are used to identify GPCR coupling with G protein(s), but they are not efficient in profiling GPCRs since they compare the concentrations from different downstream signals. Instead, novel tools, such as Bioluminescence Resonance Energy Transfer (BRET) and mini G (mG) proteins, can be used to profile GPCRs. BRET is a technique that provides quantitative data when protein-protein interaction occurs and requires the proteins of interest to be fused with either a bioluminescent protein or fluorescent protein. In this study, we used mG proteins representing each G protein subtype to identify 5-hydroxytryptamine (5-HT; serotonin) receptor coupling upon serotonin stimulation. Through BRET assays, we determined that both the 5-HT1D�and 5-HT1F�receptors couple primarily with the mGsiand mGo�classes of mG proteins. This supports previous studies that these receptors couple to Gi/o�proteins and suggests that the use of mG proteins in BRET assays is an effective tool for GPCR profiling.
  • Unconventional Coupling of 5HT7 receptors to Gs heterotrimers

    Adams, Elizabeth; Department of Pharmacology and Toxicology; Augusta University; Lambert, Nevin (2019-02-13)
    GPCRs play a major role in cell signaling through their interactions with heterotrimeric G proteins. In conventional models of GPCR-G protein coupling, agonist binding promotes a conformational change within the receptor, which then associates with G proteins, facilitating the exchange of GDP for GTP. GTP-bound G proteins dissociate from the receptor and exert their effects on downstream signaling molecules. Previous studies suggest that serotonin 5HT7 receptors associate with Gs�heterotrimers prior to agonist binding, and that 5HT7-Gs�complexes dissociate after the G protein is activated. Here we study this unconventional mode of coupling using bioluminescence resonance energy transfer (BRET) between luciferase-tagged 5HT7 receptors and Gs�heterotrimers labeled with Venus. Our results confirm that 5HT7 receptors interact with inactive (GDP-bound) Gs�heterotrimers in the absence of an agonist, and that this interaction is stabilized by the inverse agonist methiothepin. Stimulation with the endogenous agonist serotonin (5HT) decreased BRET between 5HT7 receptors and Gs, indicating that the activation of the receptor leads to 5HT7-Gscomplex dissociation. Interestingly, Gs�activation was not required for complex dissociation. These results are consistent with the hypothesis that 5HT7 receptors couple to Gs�heterotrimers via an unconventional mechanism involving ligand-sensitive complexes of receptors and inactive Gs.
  • Nox 1-Evoked Ros Causes Fibrosis in Caerulein-Induced Chronic Pancreatitis Through the Akt Pathway

    Chakraborty, Ananya; Department of Biological Sciences; Augusta University; Sabbatini, Maria (2019-02-13)
    Chronic pancreatitis (CP) manifests from a long-term inflammation, resulting in significant fibrosis of the pancreatic tissue and permanent organ damage. This occurs due to pro-inflammatory mediators, including reactive oxygen species (ROS). One of the sources of ROS is NADPH oxidase (Nox) enzymes, which transfer electrons across biological membranes to reduce oxygen to superoxide. The rodent genome encodes four Nox enzymes: Nox 1-4. We found that Nox 1 is implicated in pancreatic fibrogenesis in a mouse model of CP. Our next goal was to determine which intracellular pathway mediates the effect of Nox1-derived ROS. Several intracellular pathways are activated following Nox1-derived ROS, including JNK, AKT, and ERK1/2. Each pathway is also activated following caerulein, a cholecystokinin analogue. Our hypothesis was that repetitive administration of caerulein stimulates Nox1-derived ROS, which causes increased oxidative stress, leading to fibrogenesis through phosphorylation of ERK, AKT and JNK. We found the lack of Nox1 impaired the phosphorylation of AKT in a mouse model of CP. In conclusion, Nox1 mediates fibrogenesis through the AKT pathway in mice with CP.