doktorska disertacija
Peter Molek (Author), Tomaž Bratkovič (Mentor)

Abstract

Leptin je adipocitni hormon proteinske narave (adipokin), ki ima pomembno vlogo pri uravnavanju energijskega ravnovesja organizma. Njegova koncentracija v plazmi je odvisna od količine belega maščevja in sprememb energijskega statusa organizma. Ob prekomernem vnašanju hrane namreč koncentracija leptina naraste, ob stradanju pa upade, kar preko izraţenih leptinskih receptorjev zaznajo specifični nevroni v hipotalamusu. Ti preko projekcij v številne ostale centre v moţganih aktivirajo različne kompenzatorne mehanizme za ponovno vzpostavitev energijskega ravnovesja. Tako leptin predstavlja najpomembnejši del povratne zanke uravnavanja količine energijskih zalog v obliki maščobnega tkiva. Pri večini debelih ljudi so plazemske koncentracije leptina povišane, kljub temu pa ne opazimo opisanih centralnih učinkov leptina za spodbujanje porabe energijskih preseţkov, kar razlagamo s pojavom neodzivnosti na leptin (leptinske rezistence) ob trajajoči debelosti. Povišane plazemske koncentracije leptina (hiperleptinemija) vodijo v prekomerno signalizacijo leptina na periferiji, kjer številne celice izraţajo funkcionalne leptinske receptorje. Vse več je dokazov, da prekomerna periferna aktivnost leptina pomembno vpliva na pojav ali poslabšanje resnih, z debelostjo povezanih obolenj, kot so nekatere oblike raka, avtoimunskih ali kardiovaskularnih bolezni. Zaradi tega narašča zanimanje za razvoj antagonistov leptina, ki bi jih kot popolnoma novo skupino učinkovin lahko uporabili pri zdravljenju omenjenih bolezni. Po drugi strani bi bili tudi agonisti leptina uporabni za nadomestno zdravljenje lipodistrofije, hipotalamične amenoreje in splošne imunske pomanjkljivosti zaradi podhranjenosti, zadnje raziskave pa nakazujejo tudi moţnost uporabe agonistov v podporni terapiji sladkorne bolezni tipa 2.Ker ima tako hiperleptinemija na eni kot pomanjkanje učinkov leptina na drugi strani škodljive posledice, smo se v okviru doktorskega raziskovalnega dela odločili s presejanjem bakteriofagnih predstavitvenih knjiţnic poiskati biološko aktivne peptidne ligande leptina ali leptinskega receptorja, ki bi predstavljali vodnice za razvoj potencialnih terapevtsko uporabnih antagonistov ali agonistov leptina. Tudi ligande leptina ali leptinskega receptorja brez biološke aktivnosti bi lahko uporabili kot detekcijske molekule v analiznih ali diagnostičnih metodah.S pomočjo presejanja bakteriofagnih knjiţnic krajših naključnih peptidov nismo uspeli izolirati ligandov leptina ali leptinskega receptorja. V afinitetnih selekcijah smo s ciljanjem nevtralizacijskih protiteles proti leptinu sicer identificirali krajše mimotope leptina, a ti niso izkazovali navzkriţne reaktivnosti z leptinskim receptorjem. Glede na razširjeno analizo rezultatov predhodnih afinitetnih selekcij, opravljenih v našem laboratoriju, sklepamo, da ima tarčna molekula, ki jo uporabimo v selekcijskem postopku, pomemben vpliv na izid slednjega v smislu uspešne izolacije krajših peptidnih ligandov. Po naših ugotovitvah so najbolj primerne tarče za selekcijo krajših peptidnih ligandov tiste, katerih naravni vezavni partnerji so peptidi oz. proteini in ki imajo na površini reţe ali ţepe, primerne za sidranje krajših peptidov. Proteini, udeleţeni v interakcije protein-protein, za katere so praviloma značilne večje in ploščate stične površine, so se namreč izkazali kot manj primerne tarče za selekcijo peptidnih ligandov. V nadaljevanju smo zato v afinitetnih selekcijah proti leptinskemu receptorju uporabili sintezne bakteriofagne predstavitvene knjiţnice enoveriţnih variabilnih fragmentov protiteles (scFv). Izolirali smo štiri različne fragmente scFv, ki specifično prepoznajo nativno obliko zunajcelične regije človeškega leptinskega receptorja, pri vezavi nanjo pa tekmujejo z leptinom. Eden od fragmentov scFv (3L5) izkazuje zmerno selektivnost do človeškega leptinskega receptorja v primerjavi z mišjim, poleg tega je v preliminarnih testih z uporabo monocitne celične linije THP-1 zaviral z leptinom spodbujeno proliferacijo celic in tako deloval kot antagonist leptina. Ocenjeni vrednosti konstant IC50 za inhibicijo vezave leptina na receptor s 3L5 in Kd za interakcijo 3L5 z receptorjem v raztopini se nahajata v nanomolarnem območju. Na podlagi tega menimo, da fragment scFv 3L5 predstavlja vodnico za nadaljnji razvoj terapevtsko uporabnih antagonistov leptina ali molekularnih sond za zaznavanje leptinskega receptorja. V zadnjem delu smo se lotili razvoja modelnega sistema za predstavitev dveh različnih proteinov na površini nitastih bakteriofagnih delcev. Pripravili smo tri različne pomoţne bakteriofage tipa 88 in primerjalno ovrednotili njihovo primernost za uvedbo zelenega fluorescenčnega proteina na kapsido fagmidnih virusnih delcev, ki obenem na površini izraţajo tudi fragmente scFv. Slednji so namenjeni prepoznavi določene tarče, zeleni fluorescenčni protein pa bi omogočal detekcijo na tarčo vezanih virionov. Uspešno predstavitev obeh funkcionalnih proteinov na rekombinantnih virusnih delcih smo potrdili s pomočjo fluorescenčne mikroskopije, pri čemer smo pod mikroskopom opazovali sefarozne delce s kovalentno vezano tarčno molekulo po inkubaciji z bifunkcionalnimi virioni. Pripravljeni pomoţni bakteriofagi ne omogočajo uvedbe zadostnega števila kopij fluorescenčnega proteina na virusne delce, da bi bili ti uporabni kot detekcijske komponente pri fluorescenčnoimunskem testu. Vseeno opisan modelni sistem predstavlja osnovo za razvoj bolj občutljivih detekcijskih komponent na osnovi bakterifagnih delcev, ki bi jih lahko vključili v nove analizne in diagnostične metode, na primer za določanje nivoja izraţanja leptinskega receptorja na celicah z imunofluorescenčnimi ali imunokemijskimi tehnikami. V okviru doktorske disertacije smo torej odkrili kompetitivne fragmente scFv proti leptinskemu receptorju in ovrednotili njihovo aktivnost v pogojih in vitro ter s tem v nabor kandidatov za razvoj terapevtsko uporabnih antagonistov leptina prispevali novo vodnico. Obenem lahko opisani fragmenti protiteles postanejo del molekularnih sond za detekcijo leptinskih receptorjev na površini celic, kar bi predstavljalo osnovo za razvoj novih diagnostičnih testov na principu določanja stopnje izraţanja leptinskega receptorja. Slednji je prekomerno izraţen na celicah določenih vrst tumorjev, v aterosklerotičnih plakih in prizadetih tkivih pri avtoimunskih boleznih.

Keywords

biotehnologija;biokemija;organizmi;energijska ravnotežja;adipokini;leptin;plazemska koncentracija;povratna zanka;leptinska rezistenca;maščobno tkivo;debelost;hiperleptinemija;biološko aktivne učinkovine;ligandi;receptorji;antagonisti receptorjev;bakteriofagi;bakteriofagne predstavitvene knjižnice;doktorske disertacije;

Data

Language: Slovenian
Year of publishing:
Typology: 2.08 - Doctoral Dissertation
Organization: UL FFA - Faculty of Pharmacy
Publisher: [P. Molek]
UDC: 602.3:578.347(043.3)
COBISS: 277758976 Link will open in a new window
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Other data

Secondary language: English
Secondary title: Development of peptide modulators of leptin activity and evaluation of their biological activity
Secondary abstract: Leptin is an adipocyte-derived protein hormone (adipokine) with important role in energy balance regulation. Its plasma concentration depends on the amount of white adipose tissue and is also influenced by changes in the energy status of the organism. During the periods of excessive food intake plasma leptin levels increase while they decrease with fasting. Such changes are sensed by specific leptin-receptor expressing hypothalamic neurons, which project to many other centers in the brain to activate several compensatory mechanisms that rebalance organism’s energy status. Therefore, leptin represents the most important factor of the feedback loop which regulates the amount of energy reserves in the form of fat. Despite its elevated plasma concentrations in most obese people leptin fails to activate central compensatory mechanisms to reduce food intake and increase energy expenditure due to central leptin resistance. Increased plasma leptin concentration (hyperleptinemia) leads to excessive peripheral leptin signaling as leptin receptors are also present on diverse cells outside the central nervous system. There is accumulating evidence that excessive peripheral leptin activity contributes to the onset and progression of serious obesity-related conditions, such as certain types of cancer, autoimmune and cardiovascular diseases. In light of these findings, there is a growing interest in the development of leptin antagonists as potential novel class of drugs for use in therapy of aforementioned diseases. On the other hand, leptin agonists also hold potential as therapeutics for lipodystrophy, hypothalamic amenorrhea and general immune deficiency due to starvation. Moreover, recent studies also support the use of leptin agonists in therapy of type 2 diabetes. Because both, hyperleptinemia and leptin deficiency can have harmful consequences, we decided to screen phage display libraries with the main goal to identify biologically active peptide ligands of leptin or leptin receptor. Such peptides would be considered leads for development of therapeutically useful leptin agonists or antagonists. Even biologically inactive leptin or leptin receptor ligands might still find use as detection molecules in analytical or diagnostic techniques. We were unable to isolate any leptin or leptin receptor ligands from phage display libraries of random short peptides. While affinity selections towards anti-leptin neutralizing antibodies did yield binders, none of the mimotopes showed cross-reactivity with leptin receptor. Based on extended analysis of results from previous affinity selections performed in our laboratory, we conclude that the nature of target molecule critically affects the outcome of biopanning in terms of successful isolation of specific short peptide ligands. According to our findings, the most suitable targets for selection of short peptide ligands are those whose natural binding partners are peptides or proteins, and that contain specific topological features (e.g., clefts or pockets) allowing anchoring of short peptides. In contrast, proteins engaged in protein-protein interactions, which are generally characterized by relatively large and flat contact surfaces, proved to be less suitable targets for selection of short peptide ligands from phage display libraries. Therefore, we focused on screening phage display libraries of synthetic single-chain variable fragments (scFvs) instead. Through biopanning we isolated four different scFvs which specifically recognize the native form of extracellular region of the human leptin receptor, and compete with leptin for binding to the receptor. One of them (3L5) showed moderate selectivity for human leptin receptor as compared to its murine orthologue. In addition, 3L5 scFv inhibited leptin-stimulated proliferation of THP-1 cells in preliminary assay indicating it acts as leptin antagonist. Estimated values of the constants IC50 (for 3L5 scFv-mediated inhibition of leptin binding to the leptin receptor) and Kd (for 3L5 scFv-leptin receptor interaction in solution) are in the nanomolar range. Therefore, we believe that 3L5 scFv represents a promising lead for further development of therapeutically useful antibody-based leptin antagonists and molecular probes for leptin receptor detection. Last but not least, we developed a model system for simultaneous presentation of two different proteins on the surface of filamentous phage virions. We designed and constructed three different type 88 helper phages and evaluated their ability to introduce green fluorescent protein into the capsid of phagemid-based viral particles simultaneously expressing scFv fragments on their surface. scFvs were chosen to allow the recognition of specific target molecules, while green fluorescent protein would allow detection of target bound virions. Successful presentation of both functional proteins on recombinant viral particles was confirmed by fluorescence microscopy, where sepharose particles with covalently bound target protein were observed under the microscope after incubation with recombinant bifunctional virions. However, helper phages at this stage do not allow for the introduction of fluorescent protein onto the phage particles in sufficient copy number that would enable their use as a detection component in fluorescence-linked immunosorbent assay (FLISA). Nevertheless, the described prototype represent the basic platform for further development of more sensitive phage particle-based detection probes for analytical and diagnostic purposes, such as determining the abundance of leptin receptor on cells by immunofluorescence and immunochemical techniques. Taken together, we have identified four competitive scFv fragments targeting leptin receptor, thereby contributing new leads to the collection of candidates for the development of therapeutically useful leptin antagonists. In addition, the described scFvs can be used as components of molecular probes for detection of leptin receptors on cell surface, thus forming the basis for development of novel diagnostic tests based on determining expression level of leptin receptor. The latter is overexpressed on cells of certain types of tumors, atherosclerotic plaques and the affected tissues in autoimmune disorders.
Type (COBISS): Dissertation
Thesis comment: Univ. v Ljubljani, Fak. za farmacijo
Pages: XVI, 195 str.
ID: 17675842