Načrtovanje, sinteza in vrednotenje lipofilnih N-aciliranih derivatov glukozamina kot zaviralcev encima MurA

Povše, Rebeka (author), Frlan, Rok (mentor)

Zaskrbljujoče povečanje in širjenje mehanizmov odpornosti bakterijskih patogenov je danes eden od največjih svetovnih problemov zdravstvenega sistema, zato je raziskovanje na tem področju nujno. UDP-N-acetilglukozamin enolpiruvil transferaza (MurA) je encim, ki katalizira prvo stopnjo sinteze peptidoglikana. MurA je ključen za rast in preživetje tako grampozitivnih kot tudi gramnegativnih bakterij, ni pa prisoten v evkariontskih celicah, zato je kot tarča zelo zanimiv, hkrati pa je kot tarča validiran s fosfomicinom. Namen magistrske naloge je bila sinteza potencialnih zaviralcev encima MurA iz N-acetilglukozmina z modifikacijami na mestu N-2 in s COOH skupino na mestu C-6. Produkte smo načrtovali na podlagi molekulskega modeliranja in s pomočjo posnemanja naravnega substrata. N-acetilglukozamin smo uporabili zato, ker posnema substrat UDP-GlcNAc. Z njim lahko dosežemo pravilno usmerjenost funkcionalnih skupin in lažje prehajanje skozi celično membrano bakterij. Hidroksilni skupini na mestih C-3 in C-4 pa omogočata pomembne interakcije z aktivnim mestom. Sintezo spojin smo izvedli v šestih stopnjah. Začeli smo z zaščito hidroksilnih skupin na mestih C-1, C-3 in C-4. C-1 hidroksilno skupino smo zaščitili z O-metiliranjem, drugi dve pa s tvorbo benziliden acetala. Pri tretji stopnji smo izvedli bazično hidrolizo amidne vezi. Na prosto aminsko skupino smo v naslednjem koraku pripeli lipofilne fragmente in odščitili benziliden acetalno zaščito s kislinsko katalizirano hidrolizo. V zadnji stopnji smo hidroksilno skupino na mestu C-6 oksidirali do karboksilne kisline. Spojinam iz zadnje in predzadnje stopnje sinteze smo določali rezidualno encimsko aktivnost (RA) na encimu MurA iz bakterije E. Coli. Spojinam, ki so imele RA manjši od 50 % smo določali IC50. Le dve spojini (spojini 6b in 7d) sta pokazali dovolj zaviralnega učinka, da smo jima določili tudi IC50. Spojini sta šibka zaviralca encima MurA z IC50 371 in 375 µM, ki predstavljata potencialno izhodišče za razvoj močnejših in selektivnejših zaviralcev encima MurA.

• Language: Slovenian
• Keywords: encim MurA, N-acetilglukozamin, peptidoglikan, protibakterijske učinkovine, rezistenca
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-143857
• Publication date in RUL: 15.01.2023

Secondary language

• Language: English
• Title: Design, synthesis and evaluation of lipophilic N-acylated glucosamine derivatives as MurA inhibitors

Abstract

The alarming increase and spread of resistance mechanisms of bacterial pathogens is one of the biggest problems of the global health system today. Therefore, research in this area is of critical importance. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an enzyme that catalyzes the first step of peptidoglycan synthesis. MurA is critical for the growth and survival of both Gram-positive and Gram-negative bacteria, but is not found in eukaryotic cells, making it a very attractive target. The aim of the master thesis was to synthesize potential inhibitors of MurA enzyme from N-acetylglucosmin with modifications at the N-2 position and with a COOH group at the C-6 position. The products were designed based on molecular modeling and by mimicking the natural substrate. N-acetylglucosamine was used because it mimics the substrate UDP-GlcNAc. With it, we can achieve the correct alignment of the functional groups and it passes the bacterial cell membrane more easily. Hydroxyl groups at the C-3 and C-4 positions allow significant interactions with the active site. The synthesis of the compounds was carried out in six steps. We began by protecting the hydroxyl groups at C-1, C-3, and C-4. The C-1 hydroxyl group was protected by O-methylation and the other two by benzylidene acetal formation. In the third step, basic hydrolysis of the amide bond was carried out. In the next step, lipophilic fragments were attached to the free amino group and the benzylidene acetal protection was removed by acid-catalyzed hydrolysis. In the final step, the hydroxyl group at the C-6 position was oxidized to a carboxylic acid. The residual enzyme activity (RA) of the MurA enzyme from the E. coli bacterium was determined for compounds from the last and penultimate synthesis steps. The IC50 was determined for compounds that had a RA of less than 50%. Only two compounds (compounds 6b and 7d) showed sufficient inhibitory activity to determine their IC50. These compounds are weak inhibitors of the MurA enzyme with IC50 of 371 and 375 µM. Nevertheless, they represent a potential starting point for the development of stronger and more selective inhibitors of the MurA enzyme.

• Keywords: MurA enzyme, N-acetylglucosamine, peptidoglycan, antibacterial drugs, resistance