Razvoj novih N-fenilpirolamidov kot zaviralcev DNA-giraze in topoizomeraze IV : doctoral dissertation

Benedetto Tiz, Davide (avtor), Zidar, Nace (mentor), Kikelj, Danijel (komentor)

The greatest turning point in the moderrn treatment of infections is represented by the discovery of penicillin by Alexander Fleming in 1928. In the following decades, many other antibiotic classes have been discovered. With the high rate of use, sometimes even misuse of antibiotics, however, the degree of resistance to these agents increased, which complicates the treatment of infections that were previously manageable. Thus, the discovery of novel antibacterials is considered essential. We can fight antibacterial resistance by either searching for new ways of suppressing the bacterial growth or by preventing the emerging of resistance. This doctoral thesis focuses on the first mentioned strategy. The thesis describes the design, synthesis and evaluation of novel compounds endowed with inhibitory activities against bacterial topoisomerases - DNA gyrase and topoisomerase IV - enzymes critical for introducing topological changes to the DNA molecule and thus for the normal function of bacterial cells. DNA gyrase has important roles in the process of DNA replication and is a well-established and attractive target. DNA gyrase is an ATPase enzyme and its ATP binding site offers an opportunity for designing new antibacterial compounds with mechanism of action that until now has not been exploited enough. Well-known structure of DNA gyrase offers good opprotunities for structure-based design of new antibacterials, whereas many discovered small-molecule inhibitors of DNA gyrase with different scaffolds and good inhibitory properties can be used for ligand-based design of new antibacterials. The simultaneous inhibition of numerous bacterial targets to reduce target-based resistance has become an appealing strategy in medicinal chemistry. Topoisomerase IV possesses a similar structure and function as DNA gyrase. The similarity between these two enzymes provides the opportunity for designing dual targeting antibacterial compounds. Clinically used fluoroquinolones are well-known examples of dual inhibitors of topoisomerase IV and DNA gyrase, targeting their ParC and GyrA subunits. Lately, increased attention has been devoted to the GyrB and ParE subunits of these enzymes, where the ATP-binding site is located. In the present doctoral dissertation we designed, synthesized and biologically evaluated compounds with N-phenylpyrrolamide scaffold as potential inhibitors of GyrB and ParE. Beginning with the X-ray crystal structure of complexes of known inhibitors in the active site of GyrB, optimized N-phenyl-4,5-dibromopyrrolamides and N-phenyl-3,4-dichloro-5-methylpyrrolamides were prepared. We introduced structural modifications to investigate the optimal chemical space and the influence of such modifications on the inhibitory activity. Moreover, special focus was given on modifications that would increase the accumulation of inhibitors into the bacterial cells, such as the isosteric substitution of the terminal –COOH- group with less acidic groups or heterocycles. The prepared compounds were assayed for their inhibitory activities against DNA gyrase from Escherichia coli and Staphylococcus aureus. Since topoisomerase IV is an enzyme that has similar structure to DNA gyrase, novel N-phenylpyrrolamides were also evaluated against topoisomerase IV isolated also from S. aureus and E. coli. Selected compounds exhibited potent in vitro IC50 values against E. coli DNA gyrase and some of the compounds also showed good IC50 values against S. aureus DNA gyrase. The best inhibitors of E. coli DNA gyrase were zwitterionic compound 86 with excellent IC50 value of 6.3 nM and compound 9d with an IC50 value of 6.9 nM. Some compounds showed dual inhibition against DNA gyrase and topoisomerase IV. The best dual inhibitior was compound 9c, for which IC50 values on E. coli DNA gyrase, S. aureus DNA gyrase, E. coli topoisomerase IV and S. aureus topoisomerase IV were 9.9 nM, 99 nM, 9100 nM and 689 nM, respectively. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter aerogenes) were chosen for the evaluation of our prepared inhbitors. Additionally, we evaluated certain compounds against wild type E. coli strain and two E. coli mutant strains, JD17464 and JW5503. E. coli JD17464 is an lpxC deletion mutant with impaired outer membrane, while E. coli JW5503 is a tolC deletion mutant with defective efflux pump. Five derivatives (24, 25, 43b, 50 and 57) blocked the growth of E. faecalis by more than 85% at 50 μM concentration. In line with our expectations, the hydrazide derivative 24 displayed worse minimum inhibitory concentration (MIC = 12.5 μM) than the corresponding heterociclic compound 25 (MIC = 6.25 μM). The lower polarity of the heterocyclic ring in 25 compared to the more polar hydrazide of 24 is believed to facilitate the entry of this compound into the bacterial cells. The growth of E. faecium was inhibited by more than 65% by six compounds (7c, 8c, 25, 57, 76 and 87), and S. aureus was inhibited by more than 95% by five compounds (50, 57, 85, 86 and 87) at 50 μM concentration. Of notable interest was compound 57, with excellent MIC values of 1.56 μM against E. faecalis and E. faecium and of 0.78 μM against S. aureus. One compound (compound 87) showed significant inhibition of wild type E. coli (growth inhibition was 100%) and eight derivatives (7c, 10a, 10b, 18, 39, 43b, 50 and 61) showed good activities against E. coli tolC deletion mutant with defective efflux pump. These results suggest that active transporters are likely the main justification for the weaker activity of these compounds against Gram-negative bacteria. This work describing the discovery of new N-phenylpyrrolamides as potent inhibitors of DNA gyrase and topoisomerase IV with good antibacterial activities against Gram-positive bacteria (markedly compound 57) and Gram-negative bacteria (compound 87) represents a good starting point which, combined with a broader understanding of the requirements for the entry of small molecules into bacteria, will allow us to obtain brad-spectrum antibacterial agents in the future.

• Jezik: Angleški jezik
• Vrsta gradiva: Doktorska disertacija
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FFA - Fakulteta za farmacijo
• Kraj izida: Ljubljana
• Založnik: [D. Benedetto Tiz]
• Leto izida: 2019
• Št. strani: XVIII, 339 str.
• PID: 20.500.12556/RUL-137318
• UDK: 615.015.8(043.3)
• COBISS.SI-ID: 4726641
• Datum objave v RUL: 10.06.2022

Sekundarni jezik

• Jezik: Slovenski jezik
• Naslov: Discovery of novel N-phenylpyrrolamides as inhibitors of DNA gyrase and topoisomerase IV

Izvleček

Odkritje penicilina Alexandra Fleminga leta 1928 predstavlja enega izmed najpomembnejših dosežkov v sodobnem zdravljenju infekcijskih bolezni. V naslednjih desetletjih je temu odkritju sledilo odkritje številnih drugih razredov antibiotikov. Množična uporaba antibiotikov pa je privedla do tega, da se je povečal razvoj bakterij, ki izkazujejo odpornost na ta zdravila, kar otežuje zdravljenje tudi tistih okužb, ki so bile včasih obvladljive. Odkrivanje novih protibakterijskih učinkovin je zato nujno. Sodobna znanost se proti bakterijskim okužbam bori bodisi z iskanjem novih načinov zaviranja bakterijske rasti, bodisi s preprečevanjem razvoja bakterijske odpornosti. V okviru te doktorske naloge smo se osredotočili na prvo strategijo. V doktorski nalogi opisujemo načrtovanje, sintezo in vrednotenje novih zaviralcev bakterijskih topoizomeraz - DNA-giraze in topoizomeraze IV- encimov, ki imajo pomembno vlogo pri uravnavanju topološkega stanja molekule DNA in so zato ključni za normalno delovanje bakterijskih celic. DNA-giraza ima pomembno vlogo v procesu podvajanja DNA in je dobro uveljavljena tarča za razvoj protibakterijskih učinkovin. DNA-giraza je ATPazni encim, vezavno mesto za ATP pa ponuja priložnost za razvoj ATP-kompetitivnih protibakterijskih učinkovin, ki trenutno na trgu še niso prisotne. Struktura DNA-giraze je dobro raziskana, kar omogoča načrtovanje novih zaviralcev s pomočjo strukturno-podprtega načrtovanja. Pri načrtovanju novih inhibitorjev pa si lahko pomagamo tudi s številnimi v literature opisanimi zaviralci DNA-giraze iz različnih strukturnih razredov. Razvoj učinkovin, ki zavirajo več bakterijskih tarč je privlačna strategija za boj proti naraščajoči bakterijski odpornosti. Topoizomeraza IV ima podobno strukturo in funkcijo kot DNA-giraza. Podobnost med tema dvema encimoma zagotavlja možnost razvoja protibakterijskih učinkovin, ki hkrati delujejo na obe tarči. Skupina fluorokinolonov je dobro znan primer dvojnih zaviralcev DNA-giraze in topoizomeraze IV z delovanjem na podenoti GyrA iz DNA-giraze in ParC iz topoizomeraze IV. V zadnjem času so raziskovalci večjo pozornost namenili podenotama GyrB iz DNA-giraze in ParE iz topoizomeraze IV na katerih se nahaja ATP-vezavno mesto. V okviru doktorskega dela smo načrtovali, sintetizirali in biološko ovrednotili serije novih spojin z N-fenilpirolamidnim ogrodjem kot potencialnih zaviralcev DNA-giraze B (GyrB) in topoizomeraze IV. Na osnovi poznavanja vezave znanih zaviralcev v aktivno mesto GyrB smo pripravili več serij optimiziranih N-fenil-4,5-dibromopirolamidov in N-fenil-3,4-dikloro-5-metilpirolamidov. Da bi raziskali kemijski prostor smo v molekule uvajali strukturne spremembe in ugotavljali njihov vpliv na zaviralno delovanje. Poseben poudarek smo namenili strukturnim spremembam, ki bi povečale vstop zaviralcev v bakterijske celice, kot so uvedba izosternih zamenjav karboksilne skupine z manj kislimi skupinami ali heterocikli. Pripravljenim spojinam smo ovrednotili njihovo zaviralno delovanje na DNA-girazo iz bakterij Escherichia coli in Staphylococcus aureus. Zaradi podobnosti v strukturah obeh encimov, smo zaviralno delovanje spojin ovrednotili tudi na encimu topoizomeraza IV iz E. coli in S. aureus. Izbrane spojine so izkazale močno zaviralno delovanje na DNA-girazo iz E. coli, nekatere spojine pa so izkazale tudi dobro zaviralno delovanje na DNA-girazo iz S. aureus. Najmočnejša zaviralca DNA-giraze iz E. coli sta bila amfoterna spojina (l-(5-karboksi-2-(3,4-dikloro-5-metil-lH-pirol-2-karboksamido)fenil)piperidin-3-il) metanamonijev klorid (86) z odlično vrednostjo IC50 6,3 nM in spojina (R)-4-(5-((1-karboksietil)karbamoil)-2-(3,4-dikloro-5-metil-1H-pirol-2-karboksamido)fenoksi)piperidin-1-ijev klorid (9d) z vrednostjo IC50 6,9 nM. Nekatere spojine so izkazale zaviralno delovanje tako na DNA-girazo, kot na topoizomerazo IV. Najboljši dualni zaviralec je bila spojina (R)-4-(5-((1-karboksietil)karbamoil)-2-(3,4-dikloro-5-metil-1H-pirol-2-karboksamido)fenoksi)piperidin-1-ijev klorid (9c) z vrednostmi IC50 na DNA-girazo iz E. coli 9,9 nM, na DNA-girazo iz S. aureus 99 nM, na topoizomerazo IV iz E. coli 9100 nM in na topoizomerazo IV iz S. aureus 689 nM. Protibakterijsko delovanje pripravljenih zaviralcev smo ovrednotili na bakterijah iz družine ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa in Enterobacter aerogenes). Nekatere izmed spojin smo dodatno testirali na divji sev bakterije E. coli in dva mutirana seva E. coli, JD17464 in JW5503. E. coli JD17464 je sev z mutiranim genom za lpxC s povečano prepustnostjo zunanje membrane, medtem ko je E. coli JW5503 sev z mutiranim genom za tolC z oslabljeno izlivno črpalko. Pet spojin (24, 25, 43b, 50 in 57) je pri koncentraciji 50 microM zaviralo rast bakterije E. faecalis za več kot 85%. V skladu z našimi pričakovanji je spojina 25 izkazala boljšo protibakterijsko delovanje (vrednost MIK = 6,25 microM) kot njen hidrazidni analog 24 (MIK = 12,5 microM). Sklepamo, da nižja polarnost 1,3,4-oksadiazol-2-tionskega obroča, ki je prisoten v spojini 25, v primerjavi s polarnim hidrazidom 24 omogoča boljše prodiranje te spojine v bakterijske celice. Šest spojin (7c, 8c, 25, 57, 76 in 87) je zaviralo rast E. faecium za več kot 65%, pet spojin (50, 57, 85, 86 in 87) pa je zavrlo S. aureus za več kot 95% pri 50 microM koncentraciji. Zlasti zanimiva je bila spojina 57 z odličnimi vrednostmi MIK 0,78 microM proti S. aureus in 1,56 microM proti E. faecalis in E. faecium. Spojina 87 je izkazala dobro zaviranje rasti divjega seva E. coli (zaviranje je bilo 100%), osem derivatov (7c, 10a, 10b, 18, 39, 43b, 50 in 61) pa je izkazalo dobro delovanje na sev E. coli z mutirano izlivno črpalko. Ti rezultati kažejo, da so nekatere spojine podvržene aktivnemu črpanju iz bakterijske celice, kar je verjetno glavni razlog za njihovo šibkejše delovanje proti po Gramu negativnim bakterijam. Odkritje novih N-fenilpirolamidov kot močnih zaviralcev DNA-giraze in topoizomeraze IV z dobrimi protibakterijskim delovanjem proti po Gramu pozitivnim bakterijam (zlasti spojina 57) in po Gramu negativnim bakterijam (zlasti spojina 87). Rezultati predstavljajo dobro izhodišče, ki nam lahko skupaj s širšim razumevanjem zahtev potrebnih za vstop majhnih molekul v bakterije v prihodnosti omogoči odkritje protibakterijskih učinkovin s širokim spektrom delovanja.

• Ključne besede: antibakteijske spojine, DNA giraze, topoizomeraze IV, zaviralci DNA giraz, protibakterijske učinkovine, N-fenilpirolamidi