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Investigating PYY3-36 and PYY3-36 Analogues in the Development of an Obesity Therapy

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Investigating PYY3-36 and PYY3-36 Analogues in the Development of an Obesity Therapy Investigating PYY3-36 and PYY3-36 analogues in the development of an obesity therapy A thesis submitted for the degree of Doctor of Philosophy from Imperial College London Mélisande L. Addison 2010 Section of Investigative Medicine Division of Diabetes, Endocrinology & Metabolism Imperial College London 1 For my parents, and Barry 2 Abstract Obesity, defined as a body mass index greater than 30kg/m2, has reached the scale of a pandemic. The increasing prevalence of obesity, and its associated morbidity and mortality, together with limited treatment options, underscores an urgent need for effective therapeutic interventions. Gut hormones have been identified as integral factors in the regulation of appetite. One such gut hormone is Peptide YY (PYY), a postprandial satiety hormone that communicates nutritional status to the central nervous system. PYY is processed to generate the principle circulating form PYY3-36, which acts on Y2 receptors in the brainstem and hypothalamus to reduce appetite. Chronic intravenous infusion of PYY3-36 induces weight loss in rodents and acute intravenous infusion to obese humans reduces food intake. Furthermore, obese humans have been reported to display a blunted postprandial rise in PYY3-36, suggesting PYY3-36 is a potential anti-obesity drug target. However, exogenous PYY3-36 is rapidly cleared and has a short circulating half-life. Additionally, at supraphysiological levels PYY3-36 can produce nausea in humans. The administration of long-acting PYY3-36 analogues to the obese may overcome these limitations. This thesis investigates the design and development of PYY3-36 analogues, and their potential in the treatment of obesity. I have investigated modifications to different domains within the primary structure of PYY3-36 in order to elucidate Y2R affinity, susceptibility to proteolytic degradation and biological activity. I demonstrated that a combination of substitutions in different domains can create a long acting analogue. I have examined sites at which PYY3-36 is susceptible to degradation by specific proteases and identified that inhibition of meprin metalloendopeptidases can prolong the plasma longevity and anorectic actions of PYY3-36. I also found that substitution of a section of the α-helix of PYY3-36 with a conserved α- helical epitope creates a long-acting analogue, that is more efficacious than PYY3-36 in chronically reducing food intake and body weight in a diet-induced obese rodent model. Finally, in investigating a slow-release delivery system for PYY3-36 analogues, I have shown that substituting His residues into the α-helix-substituted analogue promotes chelation with Zn in vitro, and facilitates a slow-release pharmacokinetic profile in vivo, that may circumvent the side effects associated with administering high levels of PYY3-36. 3 Declaration of contributors The majority of work described in this thesis was carried out by the author. All collaboration and assistance is described below. Chapter 2 Feeding studies were carried out with assistance from members of the PYY group. Chapter 3 The feeding studies and plasma levels studies were carried out with assistance from Dr James Minnion, Dr Keisuke Suzuki and Joy Shillito. The nephrectomy studies were carried out with assistance from Samar Ghourab and Klara Hostomska. Chapter 4 The behavioural study was carried out in collaboration with Samar Ghourab and Jordan Baxter. The solubility and pharmacokinetic studies were done in collaboration with Joy Shillito and Samar Ghourab. The 1H-MRS was carried out in collaboration with Mohammed Hankir. 4 Acknowledgements Firstly, I would like to thank Professor Stephen Bloom for providing me with the opportunity to carry out my work, and for his support, and Professor Mohammad Ghatei for his guidance. Special thanks go to Dr Kevin Murphy for his invaluable advice and guidance during his supervision of my PhD. I would like to thank the members of the PYY group, in particular Mohammed Hankir, Dr Samar Ghourab, Jordan Baxter, Dr James Minnion and Joy Shillito for all their help and friendship over the past 3 years. I would like to thank Dr Mike Patterson for his advice regarding radioimmunoassays. I am grateful to my parents for their unconditional encouragement and advice, and Claudia, for being there for me. Finally, I am grateful to Barry; for his energy, understanding, calming presence and sense of humour, which has kept me going throughout my time as a student. 5 Contents Abstract ................................................................................................................................ 3 Declaration of contributors .................................................................................................. 4 Acknowledgements ............................................................................................................... 5 Index of Figures ................................................................................................................... 9 Index of Tables ................................................................................................................... 15 Abbreviations used in thesis ............................................................................................... 19 Chapter 1: General Introduction ........................................................................................ 21 1.1 Introduction ....................................................................................................... 22 1.1.1 Obesity ............................................................................................................ 22 1.1.2 Cause and origin of obesity .............................................................................. 22 1.2 The Physiology of Appetite and Metabolism .................................................... 23 1.2.1 Appetite control centres: overview ................................................................... 23 1.2.2 The hypothalamus ............................................................................................ 26 1.2.3 Hypothalamic neuropeptides regulating appetite control .................................. 33 1.2.4 Peripheral adiposity signals .............................................................................. 46 1.2.5 Hormones secreted from the GI-tract regulating energy balance ....................... 48 1.3 Design and development of PYY3-36 anti-obesity therapies ........................... 65 1.3.1 Current licensed obesity treatments .................................................................. 65 1.3.2 Use of PYY3-36 as a potential antiobesity therapy ........................................... 67 1.3.3 Structural design of PYY analogues ................................................................. 68 1.4 Overview ............................................................................................................ 70 Chapter Two: Alterations to the primary sequence of PYY3-36 in elucidating structure/activity and for the design of long acting analogues ........................................... 72 2.1 Introduction and aims ....................................................................................... 73 2.1.1 Use of analogues to investigate peptide properties and structure activity relations.................................................................................................................. .........74 2.1.2 Structural properties of PYY and PYY3-36 ...................................................... 75 2.2 Aim of studies..................................................................................................... 85 2.3 Materials and Methods ...................................................................................... 85 2.3.2 Peptide design: investigating the importance of specific regions of PYY3-36 in receptor binding and bioactivity................................................................................... 87 2.3.3 Production of hY2R overexpressing cell line.................................................... 91 2.3.4 Cell Culture ..................................................................................................... 98 2.3.5 Receptor binding studies ................................................................................ 101 2.3.6 Degradation assay using KBB membrane ....................................................... 103 2.3.7 In vivo studies ................................................................................................ 105 2.3.8 Statistics ........................................................................................................ 106 2.4 Results .............................................................................................................. 107 2.4.1 Investigations into the role of the C terminus of PYY3-36 in structure activity relations at the Y2R and in its in vivo bioactivity ....................................................... 107 6 2.4.2 Investigations into the role of the mid-section of PYY3-36 in structure activity relations at the Y2R and in vivo bioactivity ................................................................ 117 2.4.3 Investigations into the role of the N-terminal of PYY3-36 in structure activity relations at the Y2R and in vivo bioactivity ................................................................ 124 2.4.4 Effect of modifications to improve longevity on receptor binding, degradation and in vivo bioactivity ..............................................................................................
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