Načrtovanje zaviralcev bakterijskih avtolitičnih encimov N-acetil glukozaminidaz

doktorska disertacija

Tjaša Tibaut (Author), Marjana Novič (Mentor), Marko Anderluh (Co-mentor)

Abstract

V sklopu doktorske disertacije smo študirali encime odgovorne za metabolizem peptidoglikana, ki je glavna sestavina bakterijske celične stene. Osredotočili smo se na naslednje encime:  avtolizin E (AtlE) – N-acetil glukozaminidazo, ki je odgovorna za razgradnjo PG,  encime Mur, ki sintetizirajo osnovne gradnike peptidoglikana. Izgradnja in razgradnja PG sta v bakterijski celici v ravnotežju, porušenje tega ravnotežja v eno ali drugo smer pa vodi v odmiranje bakterij ali nezmožnost razmnoževanja. Prvi dve poglavji v disertaciji opisujeta delo v okviru prve hipoteze, ki predpostavlja, da obstajajo podobnosti med vezavnimi mesti med encimov, ki sodelujejo pri metabolizmu PG. Uvodoma smo s pomočjo različnih računalniških programov za napovedovanje vezavnih mest poiskali žepe na površini AtlE ter definirali katalitično vezavno mesto. Nadalje smo v raziskave vključili tudi človeški lizocim, encime Mur in enega od encimov, ki spada med »proteine, ki vežejo peniciline«. Za vse naštete encime je značilno, da so njihovi substrati zelo podobni ligandom iz kompleksa kristalne strukture encima AtlE. Zaporedja in strukture lizocima in slednjih smo primerjali z encimom AtlE. Primerjava ni pokazala podobnosti med encimi, ki izgrajujejo in encimi, ki razgrajujejo PG (avtolizini), je pa zelo podobna struktura in sestava katalitičnega vezavnega mesta pri avtolizinih iz različnih vrst bakterij. Podobnost je opazna tudi med samimi encimi Mur, na katerih je bilo testiranih dovolj spojin, da smo za Mur A in Mur B pripravili klasifikacijske modele za napoved jakosti zaviralcev. Poleg tega, da imajo vsi encim Mur rešene 3D strukture, so nekateri izmed njih dobro poznane že validirane tarče. Izgradnjo klasifikacijskih modelov opisuje tretje poglavje v disertaciji. V zadnjih dveh poglavjih je opisana uporaba strukturno podprtega načrtovanje ligandov za encim AtlE. Pri tem smo uporabili dva pristopa: virtualno rešetanje knjižnice majhnih molekul (fragmentov) ter načrtovanje novega razreda ligandov (de novo) na podlagi karakteristik vezavnega mesta. Obetavne spojine iz virtualnega rešetanja smo kupili, de novo načrtovane pa sintetizirali. Vezavo pridobljenih spojin z encimom smo izmerili s površinsko plazmonsko resonanco (angl. surface plasmon resonance ali SPR), biofizikalno metodo za določanje medmolekulskih interakcij. Pridobili smo dva fragmenta z afiniteto vezave v mikromolarnem območju, medtem ko večina ostalih fragmentov kaže trend nestehiometrične vezave, kar pomeni, da se spojine vežejo na več kot eno mesto na proteinu. Tekom študija smo uspeli pridobiti uporabne informacije o vezavnih mestih ter ligandih encimov, ki so odgovorni za metabolizem peptidoglikana. Informacije o vezavnih mestih AtlE ter fragmenti, katerim smo uspeli dokazati interakcije z encimom, lahko pripomorejo k nadaljnjemu razvoju zaviralcev avtolizinov. Klasifikacijski modeli za napovedovanje jakosti zaviralcev encimov Mur so uporabno orodje za razvoj učinkovin, ki zavirajo sintezo peptidoglikana. Povzamemo lahko, da smo z opisanimi študijami naredili korak naprej pri razvoju zaviralcev encimov, ki so dogovorni za metabolizem peptidoglikana.

Keywords

protibakterijske učinkovine;načrtovanje;bakterije;celična stena;peptidoglikan;metabolizem;encimi;avtolizini;avtolizin E;N-acetilglukozaminidaza;encimi Mur;encimski inhibitorji;knjižnice spojin;virtualno rešetanje;vezavna mesta;disertacije;

Data

Language:	Slovenian
Year of publishing:	2019
Typology:	2.08 - Doctoral Dissertation
Organization:	UL FFA - Faculty of Pharmacy
Publisher:	[T. Tibaut]
UDC:	615.281.9(043.3)
COBISS:	300519680
Views:	189
Downloads:	0
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Design of inhibitors of bacterial autolytic enzymes N-acetyl glucosaminidases
Secondary abstract:	During PhD study enzymes responsible for peptidoglycan (PG) metabolism were examined. Our focus was on the following enzymes:  autolysin E (AtlE) - a N-acetylglucosaminidase responsible for degradation of PG  Mur enzymes, responsible for synthesis of PG. PG is major component of the bacterial cell wall, where synthesis and decomposition are in constant balance. If the balance is disturbed the cell growth and and division are impeded, which can be crucial for bacterial survival. Fist and second chapter in the thesis encompass first hypothesis about similarity of the binding sites and ligands among PG metabolising enzymes. Initially, we used in silico tools for prediction of binding pockets of not yet validated target AtlE and determination of its catalytic binding site. We also defined binding sites of the lysozyme, Mur enzymes, and penicillin binding protein (PBP), which bind substrats with similar substructures. Sequences and structures of the lysozyme and aforementioned PG synthesizing enzymes were compared with the aminoacid sequence and structure of the AtlE. The only similarities were in binding sites of different bacterial autolysins and in binding sites of the Mur enzymes. Additionally, based on publicly available experimental measurements and chemical structures of designed inhibitors, we developed classification models for prediction of the activity of compounds towards Mur enzymes. The developement of the clssification models is presented in the third chapter. Last two chapthers describe the structure-based drug design of AtlE ligands. Two approaches were selected for this study: fragment-based virtual screening and design of new class of ligands (de novo) based on the binding site features. Promising hits from the virtual screening were purchased, while de novo designed compounds were synthetized. The binding of compounds with the AtlE was measured using surface plasmon resonsnce (SPR), a biophysical method for the detection of intermolecular interactions. Based on SPR measurements, two fragments with the binding affinity in low micromolar range were obtained. Majority of the tested compounds exhibit nonstoichiometric binding, which is common for compounds that bind to more than one site on the protein surface. The thesis represents the following contributions to the science: detailed information about binding sites and ligands of the PG metabolising enzymes, classification models for the prediction of affinity with Mur enzymes and novel designed fragments with affinity toward AtlE. Such contributions represent a step forward in the development of inhibitors of PG metabolising enzymes.
Type (COBISS):	Dissertation
Thesis comment:	Univ. v Ljubljani, Fak. za farmacijo
Pages:	146 str.
ID:	15595308