Uporaba kemoinformatičnih in strukturno podprtih in silico metod za načrtovanje zaviralcev bakterijske DNA giraze ter modulatorjev kemokinskih receptorjev CXCR3 in CXCR4

doktorska disertacija

Anja Kolarič (Author), Nikola Minovski (Mentor), Andrej Perdih (Thesis defence commission member), Marko Anderluh (Co-mentor)

Abstract

V sklopu doktorske disertacije smo preučevali dve biološki tarči, DNA girazo in kemokinske receptorje CXCR3 in CXCR4, ki ju povezuje uporaba in silico pristopov pri načrtovanju zaviralcev bakterijske DNA giraze ter modulatorjev kemokinskih receptorjev CXCR3 in CXCR4. Pri preučevanju bakterijske DNA giraze smo s pomočjo in silico pristopov načrtovali nove potencialne protibakterijske učinkovine iz razreda novih zaviralcev bakterijske topoizomeraze (>>novel bacterial topoisomerase inhibtors<<; NBTI), ter jih nadalje pripravili z metodami sintezne organske kemije in njihovo protibakterijsko delovanje ovrednotiti z različnimi in vitro metodami. Z in silico študijami smo na podlagi znanih NBTI z in vitro eksperimentalno potrjenim zaviralnim učinkom na izoliran encim, izdelali in ovrednotili napovedni kvantitativni model odnosa med strukturo in delovanjem (QSAR model). Hkrati smo sestavili virtualno kombinatorno knjižnico novih NBTI strukturnih analogov, pri katerih je del ogrodja predstavljal fragment NBTI spojin vključenih v študijo, drugi, desni del spojine, ki se veže v girazo, pa je bil popolnoma inovativen. Novo nastalim virtualnim NBTI analogom smo nato napovedali biološko aktivnost z zgrajenim QSAR modelom. Z metodami molekulskega sidranja na podlagi poznane strukture tarče smo nato v naslednjih stopnjah identificirali nove virtualne zadetke z visoko napovedano afiniteto vezave ter potencialnim zaviranjem bakterijske DNA giraze iz seva Staphylococcus aureus. V naslednji stopnji smo zaradi zahtevne sinteze, strukture virtualnih NBTI zadetkov rahlo modificirali v skladu z bolj dostopno in izvedljivo sintezo, pri tem pa smo ohranili inovativni desni del molekule. Sintetiziranim analogom smo ovrednotili protibakterijski učinek, pri čemer je spojina z 1-fenilpirazolnim desnim fragmentom pokazala najbolj primeren protibakterijski profil proti S. aureusu, s selektivnostjo na bakterijski encim v primerjavi s podobnim človeškim in sprejemljivo stopnjo toksičnosti na človeških celicah. Ta spojina nam je nadalje služila kot izhodišče za izboljšanje protibakterijskega delovanja in razširitve spektra. S strukturno optimizacijo celotnega NBTI skeleta smo dosegli ustrezne fizikalno-kemijske lastnosti, pri čemer je bil glavni poudarek na izbiri monocikličnih desnih fragmentov substituiranih na para-mestu, ki bi zagotovili močno vezavo z aminokislinskimi ostanki v NBTI vezavnem žepu giraze. V novo sintetizirani seriji so NBTI s p-halogeno 2 fenilnim desnim fragmentom, izkazale izjemno zaviranje bakterijske DNA giraze S. aureus, z IC50 = 0,007 [mikro]M za bromo derivat in IC50 = 0,011 [mikro]M za jodo derivat. Tako izvrstna zaviralna jakost je posledica simetrične tvorbe halogene vezi med halogenom in karbonilnima kisikoma Ala68 iz obeh podenot GyrA, kar smo uspeli pokazati v kristalni strukturi kompleksa med p-kloro NBTI derivatom, DNA in DNA girazo iz S. aureusa. Prav tako pa se je to odrazilo predvsem na širokem spektru delovanja z visoko jakostjo na gramnegativne bakterije. Žal pa so naše NBTI spojine, kljub selektivnosti na bakterijski encim in minimalni toksičnosti na človeške celice, pokazale neželeno zaviranje hERG kalijevih kanalov. S kristalno strukturo kompleksa pa smo uspeli potrditi do sedaj predvidevan mehanizem stabilizacije enoverižne cepitve dvovijačne DNA. Namreč, kot prvim nam je uspelo kompleks NBTI-DNA-DNA giraza kristalizirati v eni sami orientaciji in s tem omogočiti povezavo orientacije spojine s konformacijo katalitičnega žepa, pri čemer smo uspeli pokazati, da do cepitve in stabilizacije samo ene verige DNA pride zaradi asimetričnosti fragmenta NBTI spojine, ki interkalira med bazne pare DNA. V nadaljnjem raziskovanju smo se posvetili potrjevanju alosteričnega modulatornega učinka že pripravljenih kemokinskih ligandov z in vitro metodami, ter uporabi in silico pristopov za odkrivanje njihovega vezavnega mesta ter razjasnitve povezave med strukturo in delovanjem na kemokinska receptorja CXCR3 in CXCR4. Predhodno sintetiziranim CXCR3 in CXCR4 kemokinskim ligandom, za katere je bilo ugotovljeno, da so sposobni negativno modulirati omenjena receptorja, smo z in vitro testom migracije celic negativni modulatorni učinek potrdili. Pri tem smo identificirali negativne modulatorje s selektivnim delovanjem na posamezni receptor, prav tako pa smo identificirali dualni alosterični ligand, ki je sposoben negativno modulirati oba tarčna receptorja. Z uporabo in silico metode molekulskega sidranja smo predvideli način vezave negativnih modulatorjev v preučevana CXCR3 in CXCR4 receptorja, ki odraža vezavo z aminokislinskimi ostanki, ki tvorijo alosterični vezavni žep in so bile že predhodno ugotovljene kot pomembne za modulacijo receptorjev. Prav tako smo ugotovili tudi vezavo z ostanki, ki so pomembni za vezavo kemokina in aktivacijo G proteina. Na podlagi teh rezultatov predvidevamo, da se naše spojine vežejo v alosterični žep, ki se delno prekriva z vezavnim mestom kemokina.

Keywords

encimi DNA topoizomeraze;zaviralci bakterijske giraze;kemokinski receptorji;alosteričen učinek;sinteza CXCR3;sinteza CXCR4;

Data

Language:	Slovenian
Year of publishing:	2020
Typology:	2.08 - Doctoral Dissertation
Organization:	UL FFA - Faculty of Pharmacy
Publisher:	[A. Kolarič]
UDC:	615.015.8:615.4:54(043.3)
COBISS:	32373251
Views:	169
Downloads:	0
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Application of cheminformatics and structure-based in silico methods for design of inhibitors of bacterial DNA gyrase and modulators of chemokine receptors CXCR3 and CXCR4
Secondary abstract:	Two biological targets were studied within the dissertation, the DNA gyrase and CXCR3 and CXCR4 chemokine receptors, joined by the use of in silico approaches in the design of bacterial DNA gyrase inhibitors and the modulators of CXCR3 and CXCR4 chemokine receptors. In our quest for DNA gyrase inhibitors, novel potential antibacterial compounds from the class of novel bacterial topoisomerase inhibitors (NBTIs) were designed using in silico tools, synthesized and their antibacterial effect was evaluated with various in vitro methods. Using in silico studies, we have developed and validated a predictive quantitative structure-activity relationship (QSAR) model based on known NBTIs with in vitro experimentally confirmed enzyme inhibition data. At the same time, we assembled a virtual combinatorial library of new NBTI structural analogues, in which one part of the NBTI scaffold represented fragments of NBTIs included in the study and the other, right hand side part was completely innovative. The biological activity of thus newly created virtual NBTI analogs was then predicted by the QSAR model. Structure-based molecular docking calculations were used for identification of novel virtual NBTI hits with highly predicted binding affinity, and potential inhibitory effect on bacterial DNA gyrase from Staphylococcus aureus. Due to demanding synthesis, the structures of the virtual NBTI hits were slightly modified according to a more accessible and feasible synthesis, while maintaining the innovative right hand side of the molecule. The antibacterial effect of the synthesized analogs was then evaluated, with compound comprising innovative 1-phenylpyrazole right hand side fragment showing the most appropriate antibacterial profile against S. aureus, with selectivity for the bacterial enzyme over orthologous human enzyme and acceptable levels of toxicity on human cells. This compound further served as a starting point for improving antibacterial activity and widening the spectrum. Structural optimization of the entire NBTI scaffold resulted in suitable physicochemical properties, with the main emphasis on the selection of monocyclic right hand side fragments substituted at the para-position that would provide strong binding with amino acid residues delineating the NBTI gyrase binding pocket. In the newly synthesized series, NBTI compounds with a p-halogen substituted phenyl right hand side fragment expressed remarkable inhibition of bacterial DNA gyrase, with S. aureus IC50 = 0.007 [micro]M for bromo 4 derivative and IC50 = 0.011 [micro]M for iodine derivative. Such excellent enzyme inhibitory potency is due to the formation of a symmetrical halogen bond between the halogen atom and backbone carbonyl oxygen of Ala68 from both GyrA subunits, which we have shown in the crystal structure of the complex between p-chloro NBTI derivative, DNA, and DNA gyrase from S. aureus. This was also reflected in the broad spectrum and strong potency against Gram-negative bacteria. Unfortunately, despite the selectivity for bacterial enzyme and minimal toxicity to human cells, NBTI compounds have shown undesirable inhibition of hERG potassium channels. With the crystal structure of the complex, we were also able to confirm the until now predicted mechanism of DNA single-strand stabilization by NBTIs. Namely, as very first we succeeded to crystallize the NBTI-DNA-DNA gyrase complex in a single orientation, thereby allowing the connection of the compound orientation to the conformation of the catalytic pocket. We have been able to show that the cleavage and stabilization of only one DNA strand occurs due to the asymmetry of the NBTI fragment that intercalates between DNA base pairs. In the second part of our work, we have focused on confirming the in vitro allosteric modulatory effect of already synthesized chemokine ligands, and on the use of in silico approaches for prediction of their binding mode along with clarification of the relationship between their structure and activity on chemokine receptors CXCR3 and CXCR4. For pre-synthesized CXCR3 and CXCR4 chemokine ligands with established ability of negative modulation, their effect on the modulation was additionally confirmed by using in vitro transwell migration assay. We have identified negative modulators acting selectively on a single receptor, and also confirmed a dual allosteric ligand capable to negatively modulate both receptors. Using in silico molecular docking calculations, we have predicted the binding mode of these negative modulators into the studied CXCR3 and CXCR4 receptors. The binding with amino acid residues that form an allosteric binding pocket and have been previously identified as important for receptor modulation was revealed as well as binding with residues, which are important for chemokine binding and activation of G protein. Therefore, we predict that our compounds bind into an allosteric pocket that partially overlaps with the chemokine binding site.
Secondary keywords:	Bakterijska rezistenca;Farmacevtska kemija;
Type (COBISS):	Dissertation
Thesis comment:	Univ. v Ljubljani, Fak. za farmacijo
Pages:	255 str.
ID:	15504124