Design, synthesis and evaluation of DNA gyrase B inhibitors based on the benzothiazole scaffold : doctoral dissertation

Gjorgjieva, Marina (author), Kikelj, Danijel (mentor), Peterlin-Mašič, Lucija (comentor)

Antibacterial resistance has become a huge problem lately. The big euphoria from the middle of the previous century with the discovery of the antibiotics did not last for long. Soon after the discovery of the first antibiotics, researchers realized that the fight with the microbes would not be so easy. The fast evolution of microorganisms and their ability to adapt to different external influences led to the development of resistance against most known and clinically used antibacterial drugs. Despite the increased need of new antibacterial compounds, not many new antibacterials reached the market in the last 20 years. DNA gyrase is a well-established and an attractive target in the search for new antibacterial compounds, with an important role in the process of DNA replication. DNA gyrase is an ATPase and the ATP binding site of the enzyme offers an opportunity for designing new antibacterial compounds with a mechanism of action that until now has not been enough exploited. Well-known structure of fragments of DNA gyrase offers great opportunity for structure-based design of new antibacterials, while many discovered inhibitors of DNA gyrase with different scaffolds and good inhibitory potential offer opportunity for ligand-based design of antibacterial compounds with an inhibitory activity on DNA gyrase. Dual targeting is an attractive approach in the design of new biologically active compounds, especially in the design of new antibacterial compounds, since the possibility for developing bacterial resistance is lower if two bacterial targets are inhibited simultaneously. Topoisomerase IV is a “twin” enzyme of DNA gyrase with a similar structure and function. Structural similarity between these two enzymes give an exceptional opportunity for designing dual targeting antibacterial compounds. Clinically used quinolones are a good example of successful dual inhibitors of DNA gyrase and topoisomerase IV, targeting their GyrA and ParC subunits. Lately, more attention has been paid to the less used part as antibacterial target of these enzymes, GyrB and ParE subunits, where the ATP-binding site is situated. Within the framework of the doctoral dissertation we designed, synthesized and biologically evaluated compounds with benzothiazole scaffold as potential inhibitors of DNA gyrase. Design of our compounds was based on modification of recently discovered inhibitors of DNA gyrase based on 4,5,6,7-tetrahydrobenzo-[d]thiazole scaffold in our research group. In this context we replaced the 4,5,6,7-tetrahydrobenzo-[d]thiazole scaffold with benzo[d]thiazole scaffold, performed similar structural modification as were done in the case of 4,5,6,7-tetrahydrobenzo-[d]thiazole compounds and studied the influence of the structural modification on the inhibitory activity. Our benzothiazole compounds were evaluated for inhibitory activities against DNA gyrase, isolated from Staphylococcus aureus and from Escherichia coli. Since topoisomerase IV is an enzyme that has similar structure, novel benzothiazole compounds were also evaluated for inhibitory activities against topoisomerase IV isolated also from S. aureus and E. coli. Most of our compounds exhibited potent in vitro inhibitory activities against E. coli DNA gyrase and some of the compounds also showed inhibitory activities against S. aureus DNA gyrase. The best inhibitor of E. coli gyrase was exhibited by compound 2-((6-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzo[d]thiazol-2-yl)amino)-2-oxoacetic acid (13) with an IC50 of 33 nM. Certain compounds showed dual inhibition against gyrase and topoisomerase IV isolated from both bacterial types. The best dual inhibition was observed by compound 2-((2-(4,5-dichloro-1H-pyrrole-2-carboxamido)benzo[d]thiazol-6-yl)amino)-2-oxoacetic acid (27) (IC50 values for E. coli DNA gyrase, S. aureus DNA gyrase, E. coli topoisomerase IV and S. aureus topoisomerase IV are 0.087, 0.51, 1.8 and 1.7 μM, respectively). Molecular docking studies showed that benzothiazole compounds bind to the ATP-binding site of the gyrase, which was further confirmed with a crystal structure of compound 2-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido)benzothiazol-6-yl)amino)-2-oxoacetic acid (24) bound to the ATP binding site of the gyrase. Our benzothiazole-based inhibitors of DNA gyrase and topoisomerase IV were biologically evaluated for antibacterial activity against Gram-positive (S. aureus ATCC 25923, Enterococcus faecalis ATCC 29212) and Gram-negative (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853). In general, most of the compounds did not show significant antibacterial activity against all tested bacterial strains. Notable activities showed only compounds ethyl 2-((6-(4,5-dichloro-1H-pyrrole-2-carboxamido)benzo[d]thiazol-2-yl)amino)-2-oxoacetate (11) and 3-((2-(4,5-dibromo-1H-pyrrole-2-carboxamido) benzo[d]thiazol-6-yl)amino)-3-oxopropanoic acid (25) against Gram-positive E. faecalis with 47 % and 59 % inhibition of the bacterial growth of E. faecalis at a 50 μM concentration after 24 h incubation, respectively. Compound 25 was found as potential ligand of AcrAB-tolC efflux pump, which could explain its weak antibacterial activity against Gram-negative bacterial strains (12 % inhibition of the growth of E. coli after a 24 h incubation and 11 % inhibition of the growth of P. aeruginosa after 24 h incubation). Furthermore, in a larger screening project, benzothiazole inhibitors of DNA gyrase, synthesized within the framework of this doctoral dissertation, were found to possess potent antiviral activity against influenza viruses (Influenza A/H1N1, Influenza A/H3N2 and Influenza B). Considering the importance of Hsp90 chaperon in the replication of virus influenza and also the similarities in the structure of the ATP binding sites of Hsp90 and Gyrase B (members of the GHKL group of enzymes with resembles in the ATP binding sites) we hypothesized that the antiviral activity of benzothiazole compounds is probably due to the inhibition of Hsp90. In a microscale thermophoresis assay, eleven of our compounds showed good binding to Hsp90 with Kd in the range of 0.26-51 μM. Compound 27 has the best Kd value (0.26 μM) which is highly comparable with the already known Hsp90 inhibitor, 17-desmethoxy-17-N,N-dimethylaminoethylamino geldanamycin (17-DMAG) (Kd = 0.27 μM). This study provided an excellent series of compounds with dual inhibition of both DNA gyrase and topoisomerase IV from E. coli and S. aureus. The crystal structure of compound 24 in the ATP-binding place of DNA gyrase B gives also opportunities for further optimization of these compounds in order to improve their inhibitory activities. The finding that compounds have antiviral activity against virus influenza with inhibition of Hsp90 host chaperone, gives a good starting point for designing and developing compounds with dual action that will combine both antibacterial and antiviral activity in the same molecule.

• Language: English
• Keywords: protibakterijske učinkovine, bakterijska rezistenca, DNA-giraza, inhibitorji, načrtovanje, sinteza, vrednotenje, disertacije
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [M. Gjorgjieva]
• Year: 2016
• Number of pages: XXII, 219 str.
• PID: 20.500.12556/RUL-143774
• UDC: 615.281.9(043.3)
• COBISS.SI-ID: 286920960
• Publication date in RUL: 12.01.2023

Secondary language

• Language: Slovenian
• Title: Načrtovanje, sinteza in vrednotenje zaviralcev DNA giraze B z benzotiazolskim skeletom

Abstract

Bakterijska odpornost proti večini protibakterijskih učinkovin postaja vse večji problem. Odkritje antibiotikov v sredini prejšnjega stoletja je vzbudilo velika pričakovanja, ki pa so se žal kmalu izkazala kot nerealna. Kmalu po tem, ko so znanstveniki odkrili prvi antibiotik, so ugotovili, da borba z mikroogranizmi ne bo tako lahka. Sposobnost mikroogranizmov, da se prilagodijo različnim zunanjim vplivom je bila vzrok za pojav odpornosti proti protibakterijskim učinkovinam. Zaradi tega se potreba po novih protibakterijskih učinkovinah vedno bolj povečuje, med tem ko je vse bolj zaskrbljujoče dejstvo, da je na trg v zadnjih dvajsetih letih prišlo zelo malo novih protibakterijskih učinkovin. DNA giraza je encim, ki ima pomembno vlogo pri replikaciji DNA molekule v bakterijski celici in predstavlja dobro uveljavljeno in privlačno tarčo v iskanju novih protibakterijskih učinkovin. DNA giraza je ATPaza in njeno ATP vezavno mesto ponuja dobro priložnost za načrtovanje novih protibakterijskih učinkovin z mehanizmom delovanja, ki do sedaj ni dovolj izkoriščen v boju proti bakterijam. Pri načrtovanju novih inhibitorjev DNA giraze se lahko izkoristijo znane strukturne informacije o posameznih delih giraze (kot je struktura ATP vezavnega mesta), ki ponujajo priložnost za strukturno podprto načrtovanje novih inhibitorjev ter že znane inhibitorje DNA giraze (tudi inhibitorji, ki se vežejo v ATP vezavno mesto), ki ponujajo priložnost za načrtovanje novih inhibitorjev na osnovi ligandov. Načrtovanje inhibitorjev, ki bi delovali na dve ali več tarč je zelo privlačen pristop pri iskanju novih učinkovin in posebej pri iskanju novih protibakterijskih učinkovin, zaradi tega ker se z modulacijo dveh ali več tarč v bakterijski celici zmanjšuje možnost za razvoj bakterijske rezistence. Topoizomeraza IV je encim, ki je po svoji strukturi in funkciji zelo podoben DNA girazi. Podobnost v strukturi med tema dvema encimoma ponuja dobro priložnost za načrtovanje protibakterijskih učinkovin, ki bi imeli dve tarči v bakterijski celici. Dober primer za protibakterijske učinkovine, ki inhibirajo tako DNA girazo kot topoizomerazo IV so dobro znani in v klinični praksi veliko uporabljani kinoloni. Kinoloni so inhibitorji gyrA in parC podenot DNA giraze in topoizomeraze IV. V zadnjem času je vedno večja pozornost namenjena gyrB podenoti giraze in parE podenoti topoizomeraze IV, ki sta bili doslej manj izkoriščeni. V okviru doktorskega dela smo načrtovali, sintetizirali in biološko ovrednotili potencijalne inhibitorje DNA giraze, ki vsebujejo benzotiazolski skelet. Načrtovanje je izhajalo iz že znanih inhibitorjev DNA giraze s 4,5,6,7-tetrahidrobenzo[d]tiazolnim ogrodjem, ki so bili pred kratkim odkriti v naši raziskovalni skupini. Pri tem smo 4,5,6,7-tetrahidrobenzo-[d]tiazolni obroč zamenjali z benzo[d]tiazolnim obročem ter naredili podobne strukturne modifikacije kot v 2,6- diamino-4,5,6,7-tetrahidrobenzo-[d]tiazolski seriji in študirali vpliv strukturnih sprememb na inhibitorno aktivnost. Benzotiazolne spojine, ki so bile sintetizirane v sklopu doktorskega dela so bile najprej testirane na inhibitorno aktivnost na DNA girazo iz Escherichia coli in Staphylococcus aures. Glede na zgoraj omenjeno podobnost v strukturi med DNA girazo in topoizomerazo IV, so bile pripravljene spojine testirane tudi na topoizomerazo IV, prav tako iz E. coli in S. aureus. Benzotiazolne spojine so pokazale močno in vitro inhibitorno aktivnost na E. coli DNA girazo, in nekatere spojine pa so izkazale tudi zaviralno aktivnost na S. aureus DNA girazo. Najboljši inhibitor na E. coli DNA girazo je bila spojina 2-((6-(4,5-dibromo-1H-pirol-2-karboksamido)benzo[d]tiazol-6-il)amino)-2-oksoacetna kislina (13), z nanomolarno IC50 vrednostjo 33 nM. Določene spojine so pokazale dvojno inhibicijo, tako na DNA girazo iz E. coli in S. aureus kot tudi na topoizomerazo IV iz E. coli in S. aureus. Najboljši inhibitor obeh encimov je bila spojina 2-((2-(4,5-dikloro-1H-pirol-2-karboksamido)benzo[d]tiazol-6-il)amino)-2-oksoocetna kislina (27) s 87 nM in 510 nM IC50 vrednostmi za inhibicijo DNA giraze iz E. coli in S. aureus ter 1.8 in 1.7 μM IC50 vrednostmi za inhibicijo topoizomeraze IV in iz E. coli in S. aureus. Študije sidranja so pokazale, da se benzotiazolni inhibitorji vežejo v ATP vezavno mesto DNA giraze, kar smo potrdili tudi s kristalno strukturo kompleksa giraze B iz E. coli in 2-((2-(4,5-dibromo-1H-pirol-2-karboksamido)benzotiazol-6-il)amino)-2-oksoocetne kisline (24) vezane v ATP vezavnem mestu. Benzotiazolni inhibitorji DNA giraze in topoizomeraze IV so bili testirani tudi za protibakterijsko aktivnost na Gram-pozitivne (S. aureus ATCC 25923, Enterococcus faecalis ATCC 29212) in Gram-negativne bakterijske seve (Escherichia coli ATCC 25922 in Pseudomonas aeruginosa ATCC 27853). Na splošno spojine niso pokazale znatne protibakterijske aktivnosti na vse testirane bakterijske seve. Edine opazne aktivnosti sta pokazali spojini etil 2-((6-(4,5-dikloro-1H-pirol-2-karboksamido)benzo[d]tiazol-2-il)amino)-2-oksoacetat (11) in 3-((2-(4,5-dibromo-1H-pirol-2-karboksamido)benzo[d]tiazol-6-il)amino)-3-oksopropanojska kislina (25) (47 % in 59 % inhibicija rasti bakterije E. faecalis pri 50 μM koncentraciji po 24 h inkubaciji). Za spojino 25 je bilo ugotovljeno, da je potencialni ligand AcrAB-tolC efluksne črpalke in s tem tudi lahko razložimo njeno slabo protibakterijsko aktivnost na testirane Gram-negativne bakterijske seve (12 % inhibicija rasti E. coli po 24 h inkubacije in 11 % inhibicija P. aeruginosa po 24 h inkubacije). Nadalje smo naše spojine vključili v projekt rešetanja spojin, ki je bil namenjen odkrivanje novih spojin s protivirusnim delovanjem. Pri tem so nekateri benzotiazolni inhibitorji giraze izkazali protivirusno aktivnost na viruse influence A/H1N1, A/H3N2 in influence B. Hsp90 šaperon je pomemben pri replikaciji virusa influence in pri tem, ker je struktura ATP vezavnega mesta DNA giraze zelo podobna strukturi ATP vezavnega mesta šaperona Hsp90 (oba sta člana GHKL grupe encimov, ki imajo podobnosti v strukturi ATP vezavnega mesta), ki je udeležen pri procesiranju virusnih proteinov, smo domnevali, da je protivirusna aktivnost benzotiazolnih inhibitorjev giraze B morda posledica inhibicije Hsp90. Pri mikroforeznem testu, s katerim smo ovrednotili vezavo benzotiazolnih inhibitorjev giraze na Hsp90, je enajst benzotiazolnih spojin izkazalo vrednosti za konstanto disocijacije Kd v območju od 0.26 do 51 μM. Najboljšo aktivnost je pokazala spojina 27, s Kd vrednostjo 0.26 μM, ki je primerljiva Kd vrednosti znanega inhibitorja Hsp90 17-desmetoksi-17-N,N-dimetilaminoetilaminogeldanamicina (17-DMAG) (Kd = 0.27 μM). Z raziskavami v okviru doktorskega dela smo uspeli pripraviti spojine, ki imajo tako inhibitorno delovanje na DNA girazo kot tudi na topoizomerazo IV iz E. coli in S. aureus. Kristalna struktura spojine 24 v kompleksu z DNA girazo B iz E. coli daje priložnost za nadaljnjo optimizacijo teh spojin za izboljšanje njihovih inhibitornih aktivnosti. Ugotovitev, da imajo naši benzotiazolni inhibitorji giraze tudi delovanje proti virusu influence in se vežejo na Hsp90 šaperon, daje dobro izhodišče za nadaljnje načrtovanje in razvoj učinkovin, ki bi imele protibakterijsko in protivirusno delovanje.