1. Development of the dynamic model of the MurD ligase and design of novel inhibitors of the Mur ligase (MurC-MurF) family
An essential component of the bacterial cells – the bacterial peptidoglycan – is traditionally a target of choice with respect to selective toxicity in antibacterial drug design. Peptidoglycan provides rigidity and strength that are necessary for the bacterial cells to withstand high internal osmotic pressure. Four members of the ADP-forming bacterial Mur ligase family – MurC, MurD, MurE and MurF – are involved in the intracellular steps of the peptidoglycan assembly, catalyzing the synthesis of the peptide moiety and represent novel promising targets for antibacterial drug design.
Figure 1. Three dimensional structure of the bacterial MurD ligase (PDB: 2UAG) comprised of three protein domains
In the scope of our research activities we are deriving a dynamical model of the E. coli MurD enzyme by combining available experimental data with results from our molecular simulation studies to get a full atomistic picture of the MurD enzyme. We are examining the substrates binding process as well movement of its C-terminal domain. Hybrid QM/MM approach is utilized, to evaluate the proposed MurD reaction pathways. We are also performing binding free energies calculations using Linear Interaction energy (LIE) method to identify driving forces responsible for binding of several classes of MurD inhibitors.
PERDIH, Andrej, KOTNIK, Miha, HODOŠČEK, Milan, ŠOLMAJER, Tomaž. Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD. Proteins, 2007, 68, 243-254. Link
PERDIH, Andrej, ŠOLMAJER, Tomaž. MurD ligase from Escherichia coli: C-terminal domain closing motion. Computational and theoretical chemistry, 2012, 979, 73-81.Link
PERDIH, Andrej, HODOŠČEK, Milan, ŠOLMAJER, Tomaž. MurD ligase from E. coli : tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method. Proteins, 2009, 74, 3, 744-759. Link
PERDIH, Andrej, WOLBER, Gerhard, ŠOLMAJER, Tomaž. Molecular dynamics simulation and linear interaction energy study of D-Glu-based inhibitors of the MurD ligase. Journal of computer-aided molecular design, 2013, 27, 723-738. Link
Based on the available structural data we are designing virtual screening campaigns to identify novel classes of Mur ligase inhibitors (MurC-MurF). We are combining classical drug design methods such as molecular docking calculations and pharmacophore modeling approaches with a our integrated dynamical approach to drug design which include molecular dynamics simulation studies and virtual screening experiments using multiple protein structures. In collaboration with experimental partners in Slovenia and abroad our designed compounds are assayed and characterised in inhibition assays, steady state kinetics experiments, antibacterial activity determination and structural studies of binding.
Figure 2. Example of drug design study of resulting in a discovery of rigid multiple Mur ligase inhibitors using three dimensional structure-based pharmacophore models.
PERDIH, Andrej, HRAST, Martina, BARRETEAU, Hélène, GOBEC, Stanislav, WOLBER, Gerhard, ŠOLMAJER, Tomaž. Inhibitor design strategy based on an enzyme structural flexibility : a case of bacterial MurD ligase. J. Chem. Inf. Model, 2014, 54, 1451-1466. Link
PERDIH, Andrej, HRAST, Martina, BARRETEAU, Hélène, GOBEC, Stanislav, WOLBER, Gerhard, ŠOLMAJER, Tomaž. Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF). Bioorg. Med. Chem., 2014, 22, 4124-4134. Link
ENIYAN, Kandasamy, KUMAR, Anuradha, RAYASAM, Geetha Vani, PERDIH, Andrej, BAJPAI, Urmi. Development of a one-pot assay for screening and identification of Mur pathway inhibitors in Mycobacterium tuberculosis. Scientific reports, 2016, 6, 35134-1-35134-12. Link
2. Design of novel inhibitors of the bacterial DNA gyrase B
Bacterial DNA gyrase, a type II topoisomerase, remains one of the most investigated and validated targets for the development of novel antibacterials. Its absence in the mammalian organism and the crucial role it plays in the bacterial DNA replication cycle make this enzyme a very suitable target for the development of potential drugs from the perspective of selective toxicity. DNA gyrase is comprised of two subunits; gyrase A and B which both form a heterodimeric structure A2B2. Currently, the 6-fluoroquinolones are the only class of DNA gyrase inhibitors used in clinical practice and they act by stabilizing the complex between the DNA molecule and DNA gyrase thus preventing bacterial DNA from unwinding and duplicating.
Figure 3. Three-dimensional structure of the 43kDa dimmer of the DNA gyrase B subunit (PDB: 1EI1)
OBLAK, Marko, KOTNIK, Miha, ŠOLMAJER, Tomaž. Discovery and development of ATPase inhibitors of DNA gyrase as antibacterial agents. Cur. Med. Chem., 2007, 14, 2033-2047. Link
We are designing inhibitors of the DNA gyrase B subunit targeting its ATP binding site. The design starting points represent available structural information about the binding of several natural products which act as ATP competitive inhibitors (e.g cyclothialidines and coumarins). In the design process we are using structure-based drug design methods such as molecular docking calculations and pharmacophore modelling approaches. Our designed compounds are first assayed in standard inhibition assays, and further characterized by differential scanning fluorimetry (DSF) surface plasmon resonance measurements (SPR) and microscale thermophoresis (MST). In collaboration with external experimental partners antibacterial activity is determined and structural X-ray studies of binding are conducted.
Figure 4. Example of the drug design study where bithiazole inhibitors of DNA gyrase B were discovered using structure-based drug design approach and determination of the X-ray structure of the complex between DNA gyrase B and the inhibitor (PDB: 4DUH).
BRVAR, Matjaž, PERDIH, Andrej, RENKO, Miha, ANDERLUH, Gregor, TURK, Dušan, ŠOLMAJER, Tomaž. Structure-based discovery of substituted 4, 5`-bithiazoles as novel DNA gyrase inhibitors. Journal of Medicinal Chemistry, 2012, 55, 6413-6426. Link
BRVAR, Matjaž, PERDIH, Andrej, HODNIK, Vesna, RENKO, Miha, ANDERLUH, Gregor, JERALA, Roman, ŠOLMAJER, Tomaž. In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B. Bioorganic & Medicinal Chemistry, 2012, 20, 2572-2580. Link
BRVAR, Matjaž, PERDIH, Andrej, OBLAK, Marko, PETERLIN-MAŠIČ, Lucija, ŠOLMAJER, Tomaž. In silico discovery of 2-amino-4-(2, 4-dihydroxyphenyl)thiazoles as novel inhibitors of DNA gyrase B. Bioorganic & Medicinal Chemistry Letters, 2010, 20, 958-962. Link