Načrtovanje, sinteza in vrednotenje novih benzotiazolnih zaviralcev DNA-giraze B in njihovih konjugatov s siderofori

Marič, Gregor (author), Tomašič, Tihomir (mentor)

Vse večja pojavnost odpornosti bakterij proti protibakterijskim učinkovinam je posledica široke uporabe antibiotikov in predstavlja zdravstveni problem na svetovni ravni. Odkrivanje spojin z novimi mehanizmi protibakterijskega delovanja ter razvijanje pristopov s katerimi bomo zaobšli mehanizme rezistence, je tako ključnega pomena za obvladovanje bakterijskih okužb. Velik potencial pri iskanju novih protibakterijskih učinkovin so izkazali benzotiazolni zaviralci DNA-giraze, katerih tarčno mesto predstavlja vezavni žep za ATP, ki se nahaja na podenoti GyrB. Kot pristop za obhod mehanizmov rezistence z izvorom na celični membrani se je za uporabnega izkazala tvorba konjugatov med protibakterijsko spojino ter mimetikom siderofora (strukturni element molekule, ki tvori kompleks z železom). Princip izvira iz narave, kjer nekateri sevi bakterij izločajo v tekmovanju za železo konjugate s toksini, ki jih druge bakterije privzamejo z aktivnim transportom, namenjenim privzemu kompleksov med sideroforiin železom. Z namenom iskanja protibakterijskih spojin z novimi mehanizmi delovanja ter pristopov za obhod bakterijske rezistence smo v okviru magistrske naloge, izhajajoč iz že poznanih benzotiazolnih zaviralcev DNA-giraze, načrtovali in sintetizirali benzotiazolne zaviralce z modificiranim osnovnim skeletom ter tvorili konjugate z mimetiki sideroforov. Sintetizirali smo pet končnih spojin, ki smo jim ovrednotili zaviralno aktivnost na encimu ter protibakterijsko delovanje. Od testiranih končnih spojin sta najboljšo zaviralno aktivnost na encimskem nivoju pokazali spojini 22 in 32. Protibakterijsko delovanje proti po Gramu pozitivnim bakterijam je najmočnejše pri spojini 22, proti po Gramu negativnim bakterijam pa pri nobeni končni spojini aktivnosti nismo opazili. Pri tvorbi konjugatov smo predpostavili, da cepitev distančnika ni potrebna, saj je v primeru spojin 7 in 13 mimetik siderofora usmerjen proti topilu, pri spojini 32 pa v hidrofobni žep vezavnega mesta, kjer tvori interakcije, ki ugodno vplivajo na jakost vezave. Glede na rezultate biokemijskega testiranja je razvidno, da so sintetizirani benzotiazoli močni zaviralci DNA-giraze v nanomolarnem območju. Pri določanju protibakterijskega delovanja nismo opazili pozitivnega učinka uporabe konjugatov z mimetiki sideroforov. Sintetizirani benzotiazolni zaviralci s hidroksilno funkcionalno skupino na osnovnem benzotiazolnem obroču pa ponujajo odlično izhodišče za nadaljnje načrtovanje ter optimizacijo delovanja zaviralcev.

• Language: Slovenian
• Keywords: bakterijska rezistenca, benzotiazolni zaviralci, DNA-giraza, konjugati, protibakterijske spojine, siderofori
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-108663
• Publication date in RUL: 11.07.2019

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of novel benzothiazole DNA-gyrase B inhibitors and their siderophore conjugates

Abstract

The increasing incidence of bacterial resistance against antibacterial agents, which is a consequence of the wide use of antibiotics, is becoming a global health problem. For the control of bacterial infections discovery of antibacterial agents with novel mechanism of action and the development of therapeutic approaches to overcome resistance mechanisms is of great importance. Benzothiazole-based DNA gyrase inhibitors, which target the binding pocket for ATP located at the GyrB, have shown high potential in the search for new antibacterial agents. One of the possible approaches to surpass the resistance mechanisms with the origin in the cell wall is the use of siderophore-antibacterial agent conjugates. The principle comes from nature, where such conjugates are produced by some bacteria and are in competition for iron released into the environment, where they are taken up by other bacteria via specific ATP depended iron uptake system. In order to search for new antibacterial compounds with novel mechanisms of action and approaches to overcome bacterial resistance, starting from already known benzothiazole DNA gyrase inhibitors, we designed and synthesized benzothiazole inhibitors with a modified scaffold and formed conjugates with siderophore mimetics. Five final compounds were synthesized and tested for their enzymatic inhibitory and antibacterial activity. Compounds 22 and 32 displayed the best enzymatic activity of the tested compounds. The most potent antibacterial activity against Gram-positive bacteria displayed compound 22, however, compounds were devoid of activity against Gram-negative bacteria. We made the assumption that there was no need for cleavage of the siderophore mimetic, because in the case of compounds 7 and 13 the siderophore fragment points towards the solvent and in the case of compound 32 it is directed into the hydrophobic pocket of the binding site, where it forms interactions that favorably affect the binding affinity. According to the results of the biochemical assays, it is evident that the synthesized benzothiazoles inhibit DNA gyrase in the nanomolar range. In the antibacterial activity evaluation, no positive effect of the use of conjugates with siderophore mimetics was observed. Synthesized benzothiazole inhibitors with a hydroxy group on the scaffold are an excellent starting point for further design and optimization of DNA gyrase inhibitors.

• Keywords: antibacterial compounds, bacterial resistance, benzothiazole inhibitors, conjugates, DNA gyrase, siderophore