Analysis of structure and function of the serotonin type-3 receptor using site directed mutagenesis, structure activity relationship and chimeric constructs

Thesis (Ph.D.) University of Alaska Fairbanks, 2005The serotonin type-3 receptor (5-HT₃R) is a cation conducting ligand gated ion channel that mediates fast synaptic transmission. The 5-HT₃R belongs to the Cys loop superfamily of ligand gated ion channels that also includes the nicotinic acetylcholine, glycine and GABAa receptors. The 5-HT₃R has been implicated in several processes such as emesis, gastrointestinal motility, drug abuse, alcoholism and nociception. Studies involving the ligand-binding domain will thus aid in development of new drugs that modulate these physiological and pathophysiological processes. The ligand-binding site of this receptor is comprised of six putative loops, viz. loop A-F. The focus of this thesis was to study the interactions of both agonists and antagonists with the 5- HT₃R. Interactions of two agonists, 5-HT and mCPBG, with the loop C region of the receptor were studied employing biochemical and receptor modeling studies. These studies identify novel determinants of 5-HT and mCPBG interactions with the 5-HT3 receptor. Similar studies involving granisetron, a competitive 5-HT₃R antagonist also reveal novel amino acids that interact with this antagonist. In order to further understand antagonist interactions with this receptor, the approach of structure activity relationship (SAR) studies was also employed to study the functional group interactions of lerisetron, a novel 5-HT₃R antagonist. Taken together with data from loops A, B, D and E, these data reveal an emerging picture of ligand interactions with the 5-HT₃R

The structural basis of function in Cys-loop receptors

Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT_3, GABA_A and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT_3) and some are anion selective (e.g. GABA_A and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience. In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT_3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them

Novel Analogs of m-Chlorophenylguanidine as 5-HT3 Receptor Ligands

Serotonin receptors play a variety of functional roles in the body. Some indications and treatment claims for one of the classes of serotonin receptors, the 5-HT3 receptor family, include: anxiety, depression, chemotherapy- and radiation-induced emesis, constipation, irritable bowel syndrome, pain, drug addiction, and satiety control. A 5-HT3 receptor partial agonist, MD-354, served as a lead compound in the development of new 5-HT3 receptor ligands. Using halogenated analogs the study investigated their effect on binding to the 5-HT3 receptor. Conformationally-constrained analogs (quinazolines) were shown to be a novel class of 5-HT3 receptor antagonists. The log P values were determined for several analogs, and indicated that these ligands should be able to penetrate the blood-brain barrier. A homology model of the 5-HT3 receptor was built and the docking modes were assessed for these two series. Quinazolines were investigated for antidepressant properties using the mouse tail suspension test, and were shown to possess antidepressant-like activity

α2-ADRENOCEPTOR AND 5-HT3 SEROTONIN RECEPTOR LIGANDS AS POTENTIAL ANALGESIC ADJUVANTS

There continues to be a need for more effective analgesics. The α2-adrenoceptor (AR) agonist clonidine is an analgesic whose use is severely limited by undesirable side effects. meta-Chlorophenylguanidine (MD-354), an agent developed in our laboratory, selectively potentiates the antinociceptive effects of clonidine in a biphasic manner. Mechanistic studies suggest that both 5-HT3 receptor and α2-AR mechanisms are involved. To further evaluate mechanisms underlying the analgesia-potentiating effect of clonidine by MD-354, pharmacological studies using more established 5-HT3 receptor agonists: meta-chlorophenylbiguanide (mCPBG) and centrally-acting SR57227A, and non-selective α2-adrenoceptor ligand TDIQ, administered alone and in combination with clonidine, were conducted in mouse antinociceptive assays. None of the examined analogs produced an antinociceptive effect when administered alone. Nevertheless, mCPBG potentiated the antinociceptive actions of clonidine in a monophasic manner and the effect was antagonized by the 5-HT3 receptor antagonist tropisetron but not by tropisetron methiodide, suggesting that potentiation is, at least in part, due to a central 5-HT3 receptor mechanism. SR57227A did not alter the antinociceptive actions of clonidine. TDIQ was found to potentiate the analgesic actions of clonidine in a synergistic manner (as determined by an isobolographic analysis) and the effect was blocked by α2-AR antagonists (BRL-44408, imiloxan, ARC-239; α2A-, α2B-, and α2C-AR antagonists, respectively). This supports the hypothesis that MD-354 could be potentiating the analgesic actions of clonidine via an α2-AR agonist mechanism. In order to explore the role of the ring nitrogen atoms and the chloro substituent of conformationally-constrained rotamers of MD-354, analogs of 2-amino-7-chloro-3,4-dihydroquinazoline, with a varying number of nitrogen atoms in the ring were synthesized. Preliminary binding affinity results indicated that the ring nitrogen atoms are essential for 5-HT3 receptor binding. In attempt to explain the varied binding and functional activity of MD-354 at α2-ARs, 3D homology models of α2A-, α2B- and α2C-AR were generated and docking studies of the low-energy rotamers of MD-354 were conducted. The present studies support a role for the involvement of 5-HT3 receptors and α2-ARs in antinociception. Analgesic adjuvants with a dual mechanism of action such as MD-354 might represent a promising avenue to pain treatment

The acetylcholine binding protein of Lymnaea stagnalis as a biosensor and model for ligand gated ion channel proteins

Thesis (M.S.) University of Alaska Fairbanks, 2008The structural similarity of the Acetylcholine Binding Protein (AChBP) from Lymnaea stagnalis to the extracellular ligand binding domain of Ligand Gated Ion Channel (LGIC) receptors suggests that the AChBP could be used to mimic cys-loop ligand-gated ion channel (LGIC) receptors. An LGIC mimic could be used as a sensor molecule in a range of biotechnology applications including high throughput drug screening as well as in vivo and in vitro sensing of biologically active compounds. It could also be used as a lead molecule for engineering novel proteins with binding characteristics similar to non-acetylcholine receptor LGIC's. The soluble AChBP is easily expressed and purified and can be produced in reasonably large amounts. This thesis explores the potential for using the AChBP and related proteins as biosensors by evaluating their action on three key medium and high throughput systems: Scintillation Proximity Assay (SPA), Surface Plasmon Resonance (SPR), and Microcantilevers (MC). As a preliminary step to developing a 5-HT₃R-ligand binding protein, by altering the ligand specificity of the AChBP, the interaction of 5-HT₃R ligands with the AChBP is also evaluated. The work presented in this thesis contributes to improved methods of drug design and testing, and to a better understanding of LGIC structure.1. Introduction -- 2. Materials and methods -- 3. The AChBP as a molecular biosensor -- 4. Binding of serotonergic ligands to the AChBP -- 5. Summary and future directions -- References -- Appendix

GlyBP: A structural model of the extracellular domain of human ¦Á1 glycine receptor

The Glycine receptor (GlyR) is the major inhibitory neurotransmitter receptor in the spinal cord and brainstem. Dysfunction of GlyR causes hyperekplexia, a neurological disease characterized by an excessive startle response. However, limited structural information about this physiologically important receptor is available. Therefore, direct structural analyses at high resolution of truncated ligand binding domains, and possibly full-length GlyR, are required for further understanding of this important neurotransmitter receptor.This study is focused on purifying and characterizing glycine binding protein (GlyBP), a mutant form of the ligand binding domain of the GlyR, in which two hydrophobic loops were replaced with corresponding hydrophilic residues in AChBP. GlyBP was overexpressed in Sf9 insect cells. GlyBP was found in both cytosolic and membrane-bound fractions after subcellular fractionation. The cytosolic fraction was misfolded. In contrast, the membrane-bound form is functional as shown by its ability to reversibly bind to 2-aminostrychnine resin. After affinity purification, membrane-bound GlyBP could be isolated in an aqueous form and a membrane-associated vesicular form. Radiolabeled binding assays showed both forms of GlyBP retained abilities to bind to its ligands, with affinities comparable to those of full-length GlyR. Furthermore, studies using chemical crosslinking, light scattering and luminescence resonance energy transfer (LRET) showed that both forms of GlyBP are oligomeric, and are very likely pentameric. The LRET studies also showed GlyBP undergoes conformational changes upon glycine binding equivalent to changes in full-length GlyR. Further studies using chemical crosslinking coupled with mass spectrometry were conducted to probe the low resolution three-dimensional structure and inter-subunit interactions. A number of intramolecular and/or intermolecular Lys-Lys crosslinks were identified. Those crosslinks provided useful information about protein folding and validated our computationally-derived model of GlyBP.Results from this study indicate that GlyBP adopts a native-like structure and is a structural and functional homolog of the extracellular domain of GlyRs and other members in Cys-loop receptor family. Further detailed structural studies will lead to further understanding of function of the ligand binding domain of GlyRs. In addition, efforts on resolving a high-resolution structure of GlyBP might result in detailed structural information about this physiologically important receptor and also other Cys-loop receptors

Structural and Functional Characterization of Haditoxin, a novel neurotoxin isolated from the venom of Ophiohagus Hannah (King Cobra)

Ph.DDOCTOR OF PHILOSOPH

Neurotransmitter-Related Molecular Modeling Studies

This book focuses of the neurotransmission phenomenon. By definition, neurotransmitters are chemicals that enable communication, i.e., the flow of nerve impulses between nerve cells or between nerve cells and muscles and glands. Recently, one can distinguish excitatory and inhibitory mediators, both of which are endo–exogenous compounds that control the function of the whole organism. From a chemical point of view, neurotransmitters belong to many different structural groups, such as amino acids (such as glycine), peptides (such as substance P, somatostatin), monoamines (such as noradrenaline or dopamine), purine derivatives (such as adenosine), gases (such as nitrogen, NO, carbon monoxide CO), and acetylcholine. From a medical point of view, disturbances in the concentration of neurotransmitters in the body result in the occurrence of mental disorders and diseases (such as depression, schizophrenia, Parkinson’s disease) and contribute to the occurrence of dementia (including Alzheimer’s disease), among other diseases. However, the problem is much wider. These disorders can lead to a number of cardiovascular diseases and can lead to the development of vascular diseases of the brain as well as in many other organs. Therefore, pharmacological intervention is a therapy that tries to interfere with regulatory processes year after year. Such treatments improve survival, reduce the frequency of readmission, and improve patients' quality of life

Die Serotoninhypothese der Zwangsstörung : Assoziation genetischer Varianten des serotonergen Systems mit der Zwangsstörung und kognitiven Endophänotypen

Vor dem Hintergrund einer möglichen Dysfunktion des serotonergen Systems als mögliche Ursache von Zwangsstörungen wurden in der vorliegenden Untersuchung häufige genetische Varianten serotonerger Gene als Suszeptibilitätsgene erforscht. Der angenommenen genetischen Heterogenität der Zwangstörung wurde durch Auswertungen von gut erforschten Subtypen der Erkrankung begegnet. Zusätzlich wurden bei Zwangserkrankten vorbeschriebene kognitive Dysfunktionen als potentielle Endophänotypen validiert und mit den genetischen Varianten assoziiert. Insgesamt wurden N=238 Zwangspatienten, N=310 Kontrollprobanden und N=137 Angehörige in die Studie eingeschlossen. Neuropsychologische und okulomotorischen Daten lagen in einer kleineren Substichprobe vor. In der Fall-Kontroll-Stichprobe, sowie in Kombination dieser mit einer Stichprobe von N=58 Eltern-Kind-Trios, fand sich eine signifikante Assoziation der HTR3D-Variante Arg52His (rs1000952, p=0.024). Spezifisch für männliche Zwangspatienten zeigte sich eine signifikante Assoziation der funktionellen HTR3C-Variante Asn163Lys (rs6766410, p=0.007) und eine nominal signifikante Assoziation der HTR6-Variante 267C>T (rs1805054, p=0.023), während sich keine geschlechtsspezifischen Zusammenhänge für weibliche Probanden ergaben. Die Auswertung dimensionaler Subtypen ergab eine signifikante Rolle der HTR3E-Variante Ala86Tyr (rs7627615, pHTR3C-Variante Gly405Ala (rs6807362, p=0.021), des 5-HTTLPR (p=0.039) und der TPH2-Promotorvariante -703G>T (rs4570625, p=0.023) bei der Entstehung von Waschzwängen. Für den Subtyp mit starken Zwangsgedanken zeigte sich eine nominale Assoziation der HTR6-Variante 267C>T (p=0.023). Bei spät erkrankten Patienten (>21 Jahre) fand sich im Vergleich zu früh erkrankten Patienten (≤21 Jahre) eine mögliche Rolle der HTR1A-Promotorvariante -1019G>C (rs6295, p=0.014). Die Endophänotypanalysen zeigten, dass sowohl Patienten als auch deren gesunde Angehörige signifikant mehr Fehler in der Antisakkadenaufgabe machten als gesunde Kontrollprobanden. Vorbeschriebene Endophänotypen in visuell-räumlichen und exekutiven Funktionen konnten nicht bestätigt werden. Schließlich modulierte die kodierende HTR3E-Variante Ala86Tyr die Verarbeitungsstrategie von visuellen Stimuli (p=0.007), einem in anderen Studien validierten Endophänotypen der Zwangsstörung. Die Ergebnisse werden in Bezug auf die Serotoninhypothese der Zwangsstörung, die Eignung von Subtypen der Zwangsstörung für molekulargenetische Studien und die Bedeutung kognitiver Endophänotypen der Zwangsstörung diskutiert