Starting from the available structural information about the binding of the natural product inhibitor, clorobiocin, we identified a novel series of 4,5`bithiazoles inhibitors of the DNA gyrase B with a low micromolar inhibitory activity, by implementing a two-step structure-based design procedure. This novel class of DNA gyrase inhibitors was extensively investigated by various techniques: Differential Scanning Fluorimetry (DSF), Surface Plasmon Resonance (SPR) and microscale thermophoresis (MST). The binding mode of the most potent inhibitor was revealed by Xray crystallography, confirming our initial in silico binding model. The project leader is a corresponding author of the paper together with prof. T. Solmajer.
COBISS.SI-ID: 4999450
In this study we presented a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data (Perdih et al. Proteins 2007 in Perdih et al. CTC 2012) as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays and one compound from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with the aminothiazole compound were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to a hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities how computational tools can be proficiently used at all stages of the drug discovery process. The project leader is the first and corresponding author of the paper.
COBISS.SI-ID: 5462810
In this study as part of our investigation of the MurD bacterial target two recently discovered classes of the MurD ligase inhibitors were investigated resulting from the lead optimization phases of the N-sulfonamide D-Glu MurD inhibitors. Molecular dynamics simulations, based on novel structural data, in conjunction with the Linear Interaction Energy (LIE) method suggested the transferability of our previously obtained LIE coefficients (Perdih et al. J. Mol. Model 2009) to further D-Glu based classes of MurD inhibitors. Analysis of the observed dynamical behavior of these compounds in the MurD active site was supported by static drug design techniques. These results complement the current knowledge of the MurD inhibitory mechanism and provide valuable support for the D-Glu paradigm of the inhibitor design. The project leader is the first and corresponding author of the paper.
COBISS.SI-ID: 5303066
Off-path simulation (OPS) technique, an extension of the well established Replica Path Method (RPATh), was applied to compare the relative energy of the two by C-terminal domain closing motions of the MurD ligase generated by TMD simulations (Perdih et al. Proteins 2007). The first C-terminal domain closing process commenced from the experimental open structure in which this domain is located out of plane to the N-terminal and central domains (open structure pdb:1EEH). In the second trajectory the conformational movement is confined to this plane only (open structure pdb:1E0D). The obtained results indicated that the C-terminal domain movement occurring from the "out of plane" starting structure is coupled with much higher energy changes compared to the motion of this domain within this plane which is more freely available to the enzyme. The project leader is the first author of the paper.
COBISS.SI-ID: 4840730
The Daspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrugresistant E. faecium. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. In the second design stage, a validated ligandbased pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Asl-fm inhibitors with 7-amino-9H-purine and 7-amino-1H-pyrazolo[3,4d] pyrimidine scaffolds in the low micromolar range. Investigation of the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds represent the first inhibitors of Asl-fm reported to date. The project leader is a corresponding author of the paper together with prof. A. Zega.
COBISS.SI-ID: 5274906