Načrtovanje, sinteza in vrednotenje derivatov N-acetilglukozamina kot kovalentnih zaviralcev encima MurA

Kastelic, Katarina (author), Knez, Damijan (mentor), Hrast Rambaher, Martina (comentor)

Peptidoglikan je ključnega pomena pri rasti bakterij in njihovi sposobnosti preživetja. V citoplazemski fazi sinteze peptidoglikana imajo pomembno vlogo encimi iz družine Mur (MurA–MurF), ki pretvorijo UDP-N-acetilglukozamin v UDP-N-acetilmuramil-pentapeptid. Ta stopnja tako predstavlja tudi velik potencial za učinkovito zaviranje rasti in razmnoževanja bakterij. Prvo stopnjo v citoplazemskem delu sinteze katalizira encim MurA, čigar naloga je prenos enolpiruvata s fosfoenolpiruvata na UDP-N-acetilglukozamin, kar privede do tvorbe UDP-N-acetilglukozamin enolpiruvata. Edini trenutno registriran kovalentni zaviralec MurA je fosfomicin, ki je precej učinkovit, vendar je vedno bolj zaskrbljujoča odpornost bakterij nanj. Zdravilne učinkovine s kovalentnim mehanizmom delovanja delujejo tako, da imajo v strukturi reaktivno funkcionalno skupino, ki se s kovalentno vezjo poveže s tarčno molekulo in tvori stabilen kompleks tarča–zaviralec. V magistrski nalogi smo načrtovali, sintetizirali in ovrednotiti derivate N-acetilglukozamina kot potencialne kovalentne zaviralce encima MurA. UDP-N-acetilglukozamin je osnovni substrat encima MurA, zato smo poskušali N-acetilno skupino glukozamina nadomestiti z različnimi elektrofilnimi bojnimi glavami, ki bi lahko tvorile kovalentno vez s katalitičnim cisteinom. Izhajali smo iz naravnega sladkorja D-glukozamina: najprej smo zaščitili amino skupino v obliki imina, nato pa v obliki acetata še vse alkoholne skupine, po odstranitvi imina pa smo na prosto amino skupino uvedli različne elektrofilne bojne glave z reakcijami N-alkiliranja ter N-aciliranja. V zadnjem koraku smo poskusili odščititi acetatno zaščitno skupino z alkalno metanolizo, vendar so bili pogoji reakcije preveč agresivni – želenih odščitenih produktov nismo uspeli izolirati, zato smo v biološkem testiranju ovrednotili devet O-peracetiliranih derivatov N-acetilglukozamina (spojine 4–11 ter 20), ki so vsebovali različne elektrofilne bojne glave. Časovno-odvisno je encimsko aktivnost MurA iz bakterije E. coli zaviral le kloroacetamid 20, kar nakazuje, da je to zaviranje kovalentno. Kljub nakazani zaviralni aktivnosti na izoliranem encimu pa nobeden izmed derivatov ni zavrl rasti bakterij v protibakterijskih testih na dveh bakterijah – S. aureus in E. coli.

• Language: Slovenian
• Keywords: peptidoglikan, MurA, derivati N-acetilglukozamina, kovalentni zaviralci encima MurA, elektrofilne bojne glave
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-155423
• Publication date in RUL: 31.03.2024

Secondary language

• Language: English
• Title: Design, synthesis, and evaluation of N-acetylglucosamine derivatives as covalent MurA inhibitors

Abstract

Peptidoglycan is crucial for the growth and viability of bacteria. Enzymes from the Mur family (MurA–MurF), which convert UDP-N-acetylglucosamine into UDP-N-acetylmuramyl pentapeptide, play an important role in the cytoplasmic phase of peptidoglycan synthesis. This phase also represents a great potential for effective inhibition of bacterial growth and proliferation. The first step in the cytoplasmic part of the synthesis is catalysed by the enzyme MurA, whose task is the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, resulting in the formation of UDP-N-acetylglucosamine-enolpyruvate. The only currently approved covalent inhibitor of MurA is fosfomycin, which is quite effective, but bacterial resistance to this agent is a growing concern. Drugs with a covalent mode of action have a reactive functional group in the structure reacts with the target molecule via covalent bond, forming a stable target-inhibitor complex. In our master thesis, we designed, synthesized and evaluated N-acetylglucosamine derivatives as potential covalent inhibitors of the enzyme MurA. UDP-N-acetylglucosamine is the substrate of the enzyme MurA. Therefore, we tried to replace the N-acetyl group of glucosamine with different electrophilic warheads that can form a covalent bond with the catalytic cysteine. We started with the natural sugar D-glucosamine: first we protected the amino group in the form of imine and then all alcohol groups in the form of acetate. After removing the imine, we introduced various electrophilic warheads into the free amino group by N-alkylation and N-acylation. In the last step, we attempted to deprotect the acetate protecting group by alkaline methanolysis, but the reaction conditions were too aggressive, and we were unable to isolate the desired deprotected products. Therefore, we evaluated in biological assays nine O-peracetylated derivatives of N-acetylglucosamine (compounds 4–11 and 20), which contained different electrophilic warheads. In a time-dependent manner, the enzymatic activity of MurA from E. coli was inhibited only by chloroacetamide 20, suggesting that this inhibition is covalent. Despite the indicated inhibitory effect on the isolated enzyme, none of the derivatives inhibited bacterial growth in antibacterial tests with two bacteria – S. aureus and E. coli.

• Keywords: peptidoglycan, MurA, N-acetylglucosamine derivatives, covalent inhibitors of MurA, electrophilic warheads