Sinteza in vrednotenje 4-aminopiperidin-1-il-etoksi substituiranih N-fenilpirolamidnih zaviralcev DNA-giraze

Keber, Lara (author), Zidar, Nace (mentor), Peršolja, Peter (comentor)

Protibakterijske učinkovine danes sodijo med najpogosteje uporabljene zdravilne učinkovine, pogosta pa je tudi njihova nepravilna uporaba. Neizogibna posledica njihove široke uporabe je razvoj proti antibiotikom odpornih patogenov, kar spodbuja vse večjo potrebo po novih učinkovinah. Dobro uveljavljene tarče protibakterijskih učinkovin predstavljajo bakterijske topoizomeraze, med katere prištevamo DNA-girazo in topoizomerazo IV. Omenjena encima uvrščamo med topoizomeraze tipa II, ki katalizirajo cepitev obeh verig DNA hkrati. DNA-giraza in topoizomeraza IV imata podobno heterotetramerno strukturo, zato je možno načrtovati zaviralce, ki hkrati zavirajo oba encima. DNA-giraza je zgrajena iz dveh podenot GyrA in dveh podenot GyrB. Znano je, da ima podenota GyrA katalitično vlogo pri cepitvi in ponovni združitvi molekule DNA, medtem ko je podetnota GyrB odgovorna za vezavo ATP, katerega hidroliza sprošča energijo, ki je potrebna za katalizo reakcije. Z analizo kristalnih struktur kompleksov DNA-giraze B z že znanimi zaviralci smo načrtovali in sintetizirali osem novih potencialnih ATP-kompetitivnih zaviralcev. Zasnovali smo dva razreda spojin in njihovo potencialno učinkovitost ovrednotili z izvedbo biološkega testiranja na encimih DNA-giraza in topoizomeraza IV iz Escherichia coli ter človeški topoizomerazi IIα. Vsem sintetiziranim spojinam sta skupna N-fenilpirolamidni in 4-aminopiperidinski del, razlikujejo pa se predvsem v trifluorometilnem (razred A) oziroma ciklopropilnem delu (razred B). Spojinam smo ovrednotili protibakterijsko delovanje na izbranih grampozitivnih in gramnegativnih bakterijskih sevih. Na encimskih testih sta se kot najaktivnejši izkazali spojina 20 s srednjo zaviralno koncentracijo (IC50) 80 nM na DNA-girazo iz E. coli, ki ima v svoji strukturi trifluorometilni fragment in karboksilno kislino, ter spojina 26 s ciklopropilnim fragmentom ter 1,3,4-oksadiazol-2-onskim heterociklom z vrednostjo IC50 79 nM. Pri protibakterijskih testih se je kot najbolj obetavna izkazala spojina 28 z minimalno zaviralno koncentracijo (MIK90) 4 µg/mL proti Staphylococcus aureus ATCC 29213 in vrednostjo MIK90 8 µg/mL proti S. aureus ATCC 43300. Perspektivna je tudi spojina 18 z MIK90 vrednostjo 16 µg/mL proti Klebsiella pneumoniae in MIK90 vrednostjo 32 µg/mL proti Acinetobacter baumannii.

• Language: Slovenian
• Keywords: protibakterijska učinkovina, DNA-giraza, N-fenilpirolamid, topoizomeraza IV, zaviralec
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158219
• Publication date in RUL: 30.05.2024

Secondary language

• Language: English
• Title: Synthesis and evaluation of 4-aminopiperidin-1-yl-ethoxy-substituted N-phenylpyrrolamide inhibitors of DNA gyrase

Abstract

Antibacterial drugs are among the most commonly used and often missused drugs. The widespread use has led to the inevitable development of antibiotic-resistant pathogens, increasing the need for new antibacterial agents. Bacterial topoisomerases, including DNA gyrase and topoisomerase IV, are well-established drug targets. Both enzymes are classified as type II topoisomerases, which catalyze the cleavage of both DNA strands simultaneously. Due to similar heterotetrameric structure of DNA gyrase and topoisomerase IV, there is a possibility of designing antibacterial substances that simultaneously inhibit both enzymes. DNA gyrase consists of two GyrA and two GyrB subunits. It is known that the GyrA subunit plays a catalytic role in the cleavage and recombination of the DNA molecule, while the GyrB subunit is responsible for the binding of ATP, whose hydrolysis releases the energy needed to catalyze the reaction. Through studies of co-crystal structures of DNA-gyrase B with its known inhibitors, we designed and synthesized eight new potential ATP-competitive inhibitors. We designed two classes of compounds and evaluated their potential efficacy by biological testing on DNA gyrase and topoisomerase IV from Escherichia coli and human topoisomerase IIα. All synthesized compounds share the N-phenylpyrrolamide and 4-aminopiperidine moiety, but differ in the trifluoromethyl (class A) and cyclopropyl fragments (class B). The compounds were tested for antibacterial activity against gram-positive and gram-negative bacterial strains. According to the enzyme tests, 20 and 26 were shown to be the most active. Compound 20 with trifluoromethyl fragment and a carboxyl group in its structure displays a half-maximal inhibitory concentration (IC50) of 80 nM on DNA gyrase from E. coli. Compound 26 with a cyclopropyl fragment and a 1,3,4-oxadiazol-2-one heterocycle shows an IC50 value of 79 nM. According to antibacterial tests, compound 28 was shown to be the most promising agent against gram-positive bacterial strains with minimum inhibitory concentration (MIC90) of 4 µg/mL against Staphylococcus aureus ATCC 29213 and a MIC90 value of 8 µg/mL against S. aureus ATCC 43300. Compound 18 also demonstrated a very promising antibacterial activity with MIC90 value of 16 µg/mL against Klebsiella pneumoniae and MIC90 value of 32 µg/mL against Acinetobacter baumannii.

• Keywords: antibacterial agent, DNA gyrase, inhibitor, N-phenylpyrrolamide, topoisomerase IV