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

english
slovenian

doktorska dela

2020

Univerza v Ljubljani, Fakulteta za farmacijo
(Obvezni izvod spletne publikacije)

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 microM for bromo 4 derivative and IC50 = 0.011 microM 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.

12.05.2023