Sinteza in protimikrobno vrednotenje kemijske knjižnice, pripravljene z bakrom(I) katalizirano azid-alkin 1,3-dipolarno cikloadicijo

Kvartuh, Anja (author), Pajk, Stane (mentor), Hrast Rambaher, Martina (comentor)

Odkrivanje novih zdravilnih učinkovin je drag, dolgotrajen in zahteven proces, ki se začne s prepoznavanjem in validacijo tarče, temu pa sledi izbor ustrezne metode rešetanja, s pomočjo katere odkrijemo spojino zadetek in spojino vodnico. Rešetanje visoke zmogljivosti (HTS) je metoda, ki v relativno kratkem času omogoča odkrivanje velikega števila spojin zadetkov iz nabora obsežne kemijske knjižnice spojin. V skupino HTS metod spada pulzirajoča ultrafiltracija z afinitetno selekcijsko masno spektrometrijo (PUF AS-MS), pri kateri po inkubaciji kemijske knjižnice s tarčo identificiramo spojine, ki se nanjo vežejo. Enostavna in hitra metoda za pridobivanje kemijske knjižnice spojin je klik kemija oz. z bakrom(I) katalizirana azid-alkin 1,3-dipolarna cikloadicija. V sklopu magistrske naloge smo se osredotočili na sintezo kombinatorične knjižnice spojin s pomočjo klik kemije. Cilj je bil v čim krajšem času pridobiti čim več spojin, njihovo prisotnost potrditi s pomočjo tekočinske kromatografije sklopljene z masno spektrometrijo (LC-MS) in v nadaljevanju z biokemijskimi testi oz. s PUF AS-MS ovrednotiti njihovo delovanje na različnih tarčah. S klik reakcijo smo pripravili okrog 800 različnih 1,2,3-triazolnih produktov, ki so se nahajali v 74 reakcijskih zmeseh. Z LC-MS analizo smo identificirali vse produkte reakcij med monoazidi in monoalkini. Težavnejša je bila identifikacija večjega števila spojin, ki so nastale po reakciji med monoazidi in dialkini. V tem primeru nismo uspeli identificirati vseh spojin, signali pa so bili v splošnem šibkejši, zato bi bila nujna avtomatizacija procesa generiranja produktov in iskanja zadetkov in omejitev na manjše število spojin v eni reakcijski zmesi. Biokemijske teste smo izvedli na bakterijskih encimih MurA in DdlB, virusnem encimu 3CLpro, encimih hMAO-A in hMAO-B in ovrednotili protibakterijsko aktivnost na E. coli in S. aureus. Rezultate smo večinoma podajali v obliki rezidualnih aktivnosti. Med testiranimi spojinami ni bilo zaviralcev encimov MurA, DdlB in 3CLpro, reakcijska zmes 55 pa je zavirala rast bakterije S. aureus. 39 reakcijskih zmesi je izkazovalo zmerno zaviralno aktivnost encimov hMAO-A in/ali hMAO-B. V teh reakcijskih zmeseh bi v nadaljevanju s pomočjo PUF AS-MS lahko poiskali aktivne spojine, primerne za optimizacijo.

• Language: Slovenian
• Keywords: odkrivanje novih zdravilnih učinkovin, klik kemija, LC-MS, protimikrobno vrednotenje, zaviralci encimov
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-147310
• Publication date in RUL: 30.06.2023

Secondary language

• Language: English
• Title: Synthesis and antimicrobial evaluation of chemical library synthesized by copper(I)-catalysed azide⠒alkyne 1,3-dipolar cycloaddition

Abstract

The discovery of new active substances is a costly, time-consuming, and demanding process. It starts with target identification and validation, followed by the selection of an appropriate screening method to reveal the hit and lead compounds. High-throughput screening (HTS) is a method that allows the detection of a vast number of hit compounds in a relatively short time from a set of a large chemical library of compounds. HTS methods include pulsed ultrafiltration affinity selection-mass spectrometry (PUF AS-MS), which, after incubation of a chemical library with a target, identifies the compounds that bind to it. A simple and rapid method for obtaining a chemical library of compounds is click chemistry or copper(I)-catalysed azide-alkyne 1,3-dipolar cycloaddition. Within the framework of the master's degree, we created a chemical library of monoalkynes, diakynes and monoazides based on click chemistry. The aim was to obtain as many compounds as possible in the shortest possible time, to confirm their presence by liquid chromatography coupled with mass spectrometry (LC-MS) and to further evaluate their activity on different targets by biochemical assays or PUF AS-MS. Around 800 different 1,2,3-triazole products were prepared in 74 reaction mixtures by click reactions. All the products of the reactions between monoazides and monoalkynes were identified by LC-MS analysis. More difficult was the identification of a larger number of compounds formed after the reaction between monoazides and dialkynes. In this case, not all compounds could be identified, and the signals were generally weaker, so it would be necessary to automate the process of generating products and finding hits and to limit to a smaller number of compounds in one reaction mixture. Biochemical tests were performed on the bacterial enzymes MurA and DdlB, the viral enzyme 3CLpro, the enzymes hMAO-A and hMAO-B, and the antibacterial activity against E. coli and S. aureus was evaluated. The results were mainly reported as residual activities. Among the compounds tested, MurA, DdlB and 3CLpro enzyme inhibitors were absent. Mixture 55 inhibited the growth of S. aureus. 39 reaction mixtures showed moderate inhibitory activity of hMAO-A and/or hMAO-B enzymes. In these reaction mixtures, active compounds suitable for optimisation could be further identified by PUF AS-MS.

• Keywords: drug discovery, click chemistry, LC-MS, antimicrobial evaluation, enzyme inhibitors