Sinteza konjugatov zaviralca DNA-giraze in mimetika siderofora z različnimi aminokislinskimi distančniki

Vogrinec, Urška (author), Tomašič, Tihomir (mentor)

Rezistenca bakterij proti protibakterijskim učinkovinam je v porastu in posledično zdravljenje bakterijskih okužb predstavlja velik izziv v sodobnem zdravstvu. Zaradi tega je raziskovanje novih pristopov k zdravljenju bakterijskih okužb nujno. DNA-giraza in topoizomeraza IV sta postali zanimivi tarči za razvoj učinkovin za zdravljenje bakterijskih okužb. Strukturno sta si encima podobna, kar izkoriščamo pri načrtovanju učinkovin z dvojnim delovanjem, s čimer zmanjšamo možnost razvoja odpornosti. Glavno oviro pri transportu protibakterijskih učinkovin v celico predstavlja celična stena bakterij. To oviro smo skušali premagati s strategijo trojanskega konja, pri kateri z vezavo učinkovine na siderofor, majhno molekulo z veliko afiniteto do železovih ionov, olajšamo transport protibakterijske spojine v bakterijsko celico. Konjugati, ki delujejo po principu trojanskega konja, so sestavljeni iz mimetika siderofora, distančnika in zaviralca DNA-giraze. V sklopu te naloge smo sintetizirali nove spojine, ki se razlikujejo v vrsti distančnika med zaviralcem DNA-giraze in mimetikom siderofora. Kot distančnike smo uporabili tri aminokisline: ?-alanin, L-valin in L-alanin. Kot zaviralec DNA-giraze je bil uporabljen derivat (S)-4,5,6,7-tetrahidrobenzo[d]tiazol-2,6-diamina s pripetim 3,4-dikloro-5-metilpirolovim obročem, kot mimetik siderofora pa derivat 3-hidroksipiridin-4(1H)-ona. Pripravili smo tri končne spojine 5, 10 in 14, ki smo jim ovrednotili zaviranje DNA-giraze iz Escherichia coli. Vrednosti IC50 za vse tri spojine so bile v nanomolarnem območju, in sicer med 259 in 636 nM. Vsem trem spojinam smo vrednotili protibakterijsko delovanje na Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa in na dveh mutiranih sevih E. coli. V splošnem so spojine pokazale šibko zaviranje rasti bakterijskih sevov. Največje zaviranje rasti bakterij smo dosegli pri spojini 14 (83% zaviranje), na mutiranem sevu E. coli, pri katerem manjka gen z zapisom za efluks črpalke. Spojine smo testirali tudi na Acinetobacter baumannii, kjer smo z odsotnostjo železa skušali posnemati pogoje in vivo. Spojine niso izkazale boljšega zaviranja kot v pogojih, ko je železa dovolj. Rezultati magistrske naloge dajejo pomembne informacije o povezavi med strukturo in delovanjem in tako osnovo za nadaljnjo optimizacijo konjugatov zaviralcev DNA-giraze s siderofori.

• Language: Slovenian
• Keywords: ATP-vezavno mesto, DNA-giraza, protibakterijske učinkovine, siderofor
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-108631
• Publication date in RUL: 10.07.2019

Secondary language

• Language: English
• Title: Synthesis of DNA gyrase inhibitor and siderophore mimetic conjugates with different amino acid linkers

Abstract

The prevalence of bacterial resistance is increasing, which is why the treatment of bacterial infections currently represents one of the greatest challenges in medicine. Therefore, continuous discovery of new approaches to overcome bacterial resistance is of great importance. DNA gyrase and topoisomerase IV are interesting targets for antibacterial drug discovery. Because of their structural similarities, we can design dual targeting inhibitors, which reduces the probability for target-based resistance development. The main barrier for the penetration of bacterial drugs into the bacterial cytoplasm is the bacterial cell wall. One possible strategy to overcome this issue is the use of the Trojan horse approach, in which drug, linked to a siderophore mimetic, is transported into the bacteria. Conjugates that work on the Trojan horse principle consist of a siderophore mimetic, linker and DNA gyrase inhibitor. In the context of this thesis we synthesized new inhibitors of DNA gyrase B that differentiate in linker between DNA gyrase inhibitor and siderophore mimetic. As linkers we used three different amino acids: ?-alanine, L-valine in L-alanine. Inhibitors were based on the (S)-4,5,6,7-tetrahydrobenzo[d]thiazol-2,6-diamine scaffold with attached 3,4-dichloro-5-methylpyrrole ring. The siderophore mimetic was the 3-hydroxypyridin-4(1H)-one. We managed to synthesize three final compounds 5, 10 and 14, which we tested for their Escherichia coli DNA gyrase inhibition. The IC50 values for all three analogues were between 260 and 640 nM. We determined antibacterial activity for all three compounds against Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and on two E. coli mutants. In general, all of them displayed weak antibacterial activity. Conjugate 14 showed the highest rate (83%) of bacterial growth inhibition of the mutated E. coli strain. We also tested final compounds against Acinetobacter baumannii in iron-depleted conditions, but the inhibitory effect did not increase compared to the iron-supplemented media. The results of this Master's thesis provide important information about the structure-activity relationship and therefore the basis for further optimization of DNA gyrase inhibitors and their conjugates with siderophores.

• Keywords: antibacterial drugs, ATP-binding site, DNA gyrase, siderophore