Sinteza novih benzotiazolov kot zaviralcev bakterijske DNA-giraze in topoizomeraze IV s širokospektralnim protibakterijskim delovanjem

Žankar, Manca (author), Tomašič, Tihomir (mentor)

Hitro širjenje bakterijske odpornosti proti uveljavljenim protibakterijskim učinkovinam predstavlja eno izmed največjih groženj za javno zdravje. Bakterijski topoizomerazi tipa IIA, DNA-giraza in topoizomeraza IV, sta esencialna ter evolucijsko visoko ohranjena encima, ki katalizirata topološke spremembe in ohranjata integriteto DNA med podvojevanjem ter prepisovanjem. Fluorokinoloni delujejo na podenoto A DNA-giraze (GyrA) in podenoto C topoizomeraze IV (ParC), ki sta odgovorni za cepitev ter ponovno združitev verig bakterijske molekule DNA. Zaradi razvoja bakterijske odpornosti proti fluorokinolonom se raziskave vse bolj usmerjajo na podenoto B DNA-giraze (GyrB) in podenoto E topoizomeraze IV (ParE), ki s hidrolizo molekule ATP zagotavljata energijo za delovanje podenot GyrA in ParC ter predstavljata še neizkoriščeno tarčo protibakterijskih učinkovin. Pri eksperimentalnem delu magistrske naloge smo načrtovali in sintetizirali nove ATP-kompetitivne zaviralce DNA-giraze in topoizomeraze IV. Sintetiziranim spojinam je skupen osnovni skelet benzo[d]tiazol-6-karboksilna kislina, na katerega imajo na mesto 2 pripet 3-kloro-4-bromo-5-metil-1H-pirol-2-karboksamid ali 3,4-dikloro-5-metil-1H-pirol-2-karboksamid. Spojine z vezanim 3,4-dikloro-5-metil-1H-pirol-2-karboksamidom se med seboj razlikujejo po strukturi substituenta, ki je vezan na amidno skupino (N-etil, N-ciklopropil, N-izopropil in N-(2-metoksietil)). Spojine 7, 13, 23, 24 in 25 so bile testirane na rekombinantni DNA-girazi ter topoizomerazi IV iz bakterij Escherichia coli in Staphylococcus aureus. Kot najmočnejši zaviralec se je v encimskem testu izkazala spojina 13, ki ima na osnovni skelet vezan 3-kloro-4-bromo-5-metil-1H-pirol-2-karboksamid, na amidni skupini pa nima vezanega nobenega substituenta. Spojina 13 je izkazala dobro zaviralno delovanje na DNA-girazo iz bakterije E. coli (IC50, < 10 nM) in na DNA-girazo iz bakterije S. aureus (IC50, 15 nM), prav tako pa je delovala zaviralno tudi na topoizomerazo IV iz bakterije S. aureus, in sicer v nanomolarnem koncentracijskem območju (IC50, 90 nM). Spojinam 7, 13, 23, 24 in 25 smo določili tudi protibakterijsko delovanje na po Gramu pozitivnih bakterijskih sevih Staphylococcus aureus in Enterococcus faecalis ter na po Gramu negativnih sevih Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae in Escherichia coli. Spojine so izkazale močnejše delovanje proti po Gramu pozitivnim kot proti po Gramu negativnim bakterijam. Rezultati magistrske naloge predstavljajo pomemben doprinos k razumevanju strukturnih zahtev za protibakterijsko delovanje zaviralcev DNA-giraze in topoizomeraze IV.

• Language: Slovenian
• Keywords: bakterijska odpornost, benzotiazoli, DNA-giraza, protibakterijska učinkovina, topoizomeraza IV
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121728
• Publication date in RUL: 24.10.2020

Secondary language

• Language: English
• Title: Synthesis of novel benzothiazoles as inhibitors of bacterial DNA-gyrase and topoisomerase IV with broad-spectrum antibacterial activity

Abstract

Rapid development of resistance of the most prevalent bacterial pathogens to antibacterial drugs is one of the major threats to public health. Bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are essential and highly conserved enzymes that catalyze topological changes and maintain DNA integrity during replication and transcription. Fluoroquinolones act on subunit A of DNA gyrase (GyrA) and subunit C of topoisomerase IV (ParC), which are responsible for breakage and reunion of DNA double strand. Due to the development of bacterial resistance against fluoroquinolones, research is increasingly focused on the subunit B of DNA gyrase (GyrB) and subunit E of topoisomerase IV (ParE), which provide energy for GyrA and ParC activity by hydrolysis of the ATP molecule and represent an unexploited target of antibacterial drugs. In the experimental part of this master's thesis, we designed and synthesized new ATP-competitive inhibitors of DNA gyrase and topoisomerase IV. The synthesized compounds possessed benzo[d]thiazole-6-carboxylic acid scaffold, to which 3-chloro-4-bromo-5-methyl-1H-pyrrole-2-carboxamide or 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide is attached at position 2. Compounds with 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamide differ in the structure of the substituent attached to the amide group (N-ethyl, N-cyclopropyl, N-isopropyl and N-(2-methoxyethyl)). Compounds 7, 13, 23, 24 and 25 were tested for their inhibitory activities on recombinant DNA gyrase and topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most active compound in the enzyme assay was compound 13, which has the 3-chloro-4-bromo-5-methyl-1H-pyrrole-2-carboxamide bound to the central scaffold and unsubstituted amide group. Compound 13 displayed potent inhibitory activity on DNA gyrase from E. coli (IC50, < 10 nM) and on DNA gyrase from S. aureus (IC50, 15 nM), and it also exhibited inhibitory activity on topoisomerase IV from S. aureus in the nanomolar concentration range (IC50, 90 nM). Compounds 7, 13, 23, 24 and 25 were also tested for their antibacterial activity against Gram-positive bacterial strains Staphylococcus aureus and Enterococcus faecalis and against Gram-negative bacterial strains Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. The compounds exhibited stronger antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. The results of this master's thesis make an important contribution to understanding the structural requirements for the antibacterial activity of DNA gyrase and topoisomerase IV inhibitors.

• Keywords: antibacterial drug, bacterial resistance, benzothiazole, DNA gyrase, topoisomerase IV