Novel fluorescence-based methods for illuminating transmembrane signal transduction by G-protein coupled receptors

Abstract

Informatsiooni vastuvõtmiseks ja edasikandmiseks on rakkudes valgud, mida kutsutakse retseptoriteks. Lähtudes nende struktuurist ja toimest on retseptorid jagatud kuude klassi, millest suurima moodustavad G-valk seotud retseptorid. Inimese genoomist on tuvastatud ligi 800 erinevat G-valk seotud retseptorit, mis reageerivad väga erinevatele signaalidele alustades valgusest kuni suurte valkudeni. Samuti on need retseptorid seotud paljude erinevate elutähtsate protsessidega, mistõttu vead retseptorite talitluses on seotud tõsiste haigustega, mistõttu ligi 35% kõikidest retseptiravimitest on sihitud G-valk seotud retseptoritele. Uute ravimikandidaatide leidmiseks on oluline teada, kas ja kuidas molekulid seostuvad retseptoritega. Algselt teostati katseid loomade, organite või kudedega, kus mõõdeti otsest füsioloogilist vastust lisatud ainele, näiteks konna jala kokkutõmbumist sõltuvalt soolalahuse kangusest. Selleks, et leida uusi ja paremaid ravimeid on vaja arendada uusi meetodeid, millega uurida retseptorite reaktsiooni erinevatele ravimianaloogidele. Käesoleva doktoritöö raames arendati välja mitu uut fluorestsentsmeetodit, millega saab uurida retseptoreid nii elusrakkude pinnal kui ka nanoosakestes. Need meetodid kasutavad uudseid, Regensburgi Ülikoolis sünteesitud, fluorestsentsmärgisega ained, mis seostuvad retseptoritega tugevamalt kui varasemad ained. Need võimaldavad määrata seostumist retseptoritele elusates rakkudes, kasutatades automatiseeritud mikroskoopiat ja tehisintellekti. Nii on võimalik uurida retseptorid inimkehale sarnasemas keskkonnas ning saada retseptorite kohta täpsemat informatsiooni. Lisaks tegime uue meetodi kasudades ka nanoosakestes olevaid retseptoreid, mille puhul aine sidumist saab mõõta tema pöörlemise kiiruse vähenemise järgi või siis, ülitundliku meetodi korral pildistades klaasi pinnale seotud nanoosakesi. Sellise lähenemine muudab uued meetodid varasematest oluliselt tundlikumateks, võimaldades välja töötada uusi ravimeid, mida saaks tarbida väga väikestes kogusdes. See vähendab omakorda kõrvaltoimete riski ning kahju keskkonnale nii ravimi tootmisel kui ka utiliseerimisel.

An important role in information processing in cells play proteins called receptors. Based on their structure and functioning mechanisms, the receptors are divided into six classes, of which the G protein-coupled receptors is the largest one. Nearly 800 different G protein-coupled receptors have been identified in the human genome, and they recognise very different signals, starting from light and ending with large protein hormones. All these receptors are essential for the normal functioning of organisms, and their dysfunctions are associated with a wide variety of diseases. Therefore approximately 35% of all prescription medicines are targeted to G-protein-coupled receptors. In order to find new drug candidates, it is essential to know if and how the molecules bind to the receptors. Initially, experiments were performed on animals, organs, or tissues to measure the direct physiological response to the added substance, such as the contraction of the frog's leg depending on the strength of the saline solution. To find new and better drugs, it is necessary to develop new methods to study the response of receptors to different drug analogues. In this dissertation, several new fluorescence methods have been developed that can be used to study receptors on the surface of living cells as well as in nanoparticles. These methods use novel fluorescently labelled substances synthesised at the University of Regensburg that bind more strongly to receptors than previous substances. Thus allowing for monitoring their binding to receptors in living cells using automated microscopy and artificial intelligence. This makes it possible to study the receptors in a more human-like environment and obtain more detailed information. In addition, we also used nanoparticles to develop new methods. In one of them, the binding of a substance can be measured by the decrease in its rotational speed. The other is a hypersensitive method where nanoparticles bound to a glass surface are imaged with an ultrasensitive microscope. This approach makes the new methods much more sensitive than before, allowing new drugs to be developed that can be consumed in minimal quantities. This, in turn, reduces the risk of side effects and damage to the environment during both the production and disposal of the medicine.