Sinteza in vrednotenje novih zaviralcev DNA-giraze B z 2-amino-6-karboksi-5-hidroksi-benzotiazolnim ogrodjem

Jus, Špela (author), Zidar, Nace (mentor)

Število mikroorganizmov odpornih proti antibiotikom je iz leta v leto višje. Zaradi tega so na področju odkrivanja novih protibakterijskih učinkovin nujne vedno nove raziskave. Bakterijska DNA-giraza in topoizomeraza IV sta zelo perspektivni tarči. Encima sta si strukturno zelo podobna, zato obstaja možnost za razvoj takšnega zaviralca, ki bo deloval na oba encima hkrati. Zelo pomembno je tudi, da encima DNA-giraza pri človeku ne najdemo, medtem ko je prisoten v vsaki bakteriji. To nam zagotavlja selektivno toksičnost. DNA-giraza in topoizomeraza IV imata pomembno vlogo pri uravnavanju topologije DNA med podvajanjem, zato sta nujno potrebna za preživetje mikroorganizma. DNA-giraza je sestavljena iz dveh podenot DNA-giraze A (GyrA) in iz dveh podenot DNA-giraze B (GyrB). DNA giraza A je zadolžena za cepitev in ponovno združitev verige DNA, medtem ko je glavna vloga DNA-giraze B hidroliza ATP in s tem zagotavljanje energije potrebne za cepitev in ponovno združitev DNA verige. Pri eksperimentalnem delu smo načrtovali in sintetizirali nove potencialne ATP-kompetitivne zaviralce DNA-giraze B. Sintetizirali smo spojine z 2-amino-6-karboksi-5-hidroksi benzotiazolnim obročem, ki imajo pripet 3,4-dikloro-5-metilpirolni ali 4,5-dibromopirolni obroč. Spojine 14, 19 in 21 smo testirali na encimih DNA-giraza in topoizomeraza IV iz bakterije Escherichia coli in bakterije Staphylococcus aureus. Najbolj obetavna spojino 19, ki je kislinski derivat s 3,4-dikloro-5-metilpirolnim obročem in pripeto hidroksilno skupino na mestu 5, je izkazovala zelo dobro delovanje na encimu DNA-giraza (IC50 = 3,62 ± 0,87 nM) iz bakterije E. coli. Spojini 19 smo določili še protibakterijsko delovanje na bakterijskih sevih Acinetobacter baumannii, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, S. aureus, E. coli ter na dveh mutiranih sevih E. coli. Čeprav je spojina pri encimskih testih močno zavirala encim DNA-giraza iz E. coli, protibakterijske aktivnosti na bakterijo E. coli, ni izkazovala. Vzrok za to so verjetno izlivne črpalke, ki jih je divji sev razvil v odgovor na antibiotike. Bakterijskim sevom, ki jim je spojina 19 zavrla rast za več kot 80%, smo določili še vrednosti MIK90. Spojina 19 je izkazala zelo nizke vrednosti MIK90 na bakterije E. faecalis, K. pneumoniae ter na divji sev S. aureus in sev S. aureus odporen na meticilin.

• Language: Slovenian
• Keywords: protibakterijska učinkovina, zaviralec, DNA-giraza B, benzotiazoli
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-106373
• Publication date in RUL: 20.02.2019

Secondary language

• Language: English
• Title: Synthesis and evaluation of new DNA gyrase B inhibitors with 2-amino-6-carboxy-5-hydroxy-benzothiazole scaffold

Abstract

The number of microorganisms resistant to antibiotics is increasing each year. Because of that, new research in the field of discovering new antibacterial agents is much needed. Bacterial DNA gyrase and topoisomerase IV are very promising targets. The enzymes are structurally very similar, so there is a possibility for the development of inhibitors that would work on both enzymes at the same time. It is also very important that DNA gyrase is not found in humans, while it is present in every bacteria, which ensures selective toxicity. DNA gyrase and topoisomerase IV control topological state of DNA during replication and are therefore essential for the survival of microorganisms. DNA gyrase consists of two DNA gyrase A (GyrA) subunits and two DNA gyrase B (GyrB) subunits. Subunit GyrA is responsible for cleavage and reunion of the DNA chain, while the main role of subunit GyrB is ATP hydrolysis and thereby providing energy for the cleavage and reunion of the DNA chain. In the experimental work we designed and synthesized a series of new potential ATP-competitive DNA gyrase B inhibitors. The synthesized compounds possessed 2-amino-6-carboxy-5-hydroxybenzothiazole scaffold, with attached 3,4-dichloro-5-methylpyrrole ring or 4,5-dibromopyrrole ring. Compounds 14, 19 and 21 were tested on DNA gyrase and topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most promising compound 19, which is an acid derivative, with the 3,4-dichloro-5-methylpyrrole ring and attached hydroxyl group at position 5, was very active against DNA gyrase (IC50 = 3,62 ± 0,87 nM ) from bacteria E. coli. For compound 19 we also determined antibacterial activity on bacterial strains Acinetobacter baumannii, Enterobacter aerogenes, Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, S. aureus, E. coli and on two mutated strains of E. coli. Although the compound strongly inhibited the DNA gyrase from E. coli in the enzyme assays, the antibacterial activity on E. coli was weak. This is probably due to the efflux pumps developed by the wild strain in response to antibiotics. For compounds, that inhibited the growth of bacteria by more than 80%, MIC90 values were also determined. Compound 19 showed very low MIC90 values on E. faecalis, K. pneumonia, wild-type S. aureus strain and methicillin resistant S. aureus.

• Keywords: antibacterial drug, inhibitor, DNA gyrase B, benzothiazole