Andrej Perdih

Discovery of novel benzene 1,3-dicarboxylic acid inhibitors of bacterial MurD and MurE ligases by structure-based virtual screening approach

The peptidoglycan biosynthetic pathway provides an array of potential targets for antibacterial drug design, attractive especially with respect to selective toxicity. Within this pathway, the members of the Mur ligase family are considered as promising emerging targets for novel antibacterial drug design. Based on the available MurD crystal structures co-crystallised with N-sulfonyl glutamic acid inhibitors, a virtual screening campaign was performed, combining three-dimensional structure-based pharmacophores and molecular docking calculations. A novel class of glutamic acid surrogates-benzene 1,3-dicarboxylic acid derivatives-were identified and compounds 14 and 16 found to possess dual MurD and MurE inhibitory activity.

In silico discovery of 2-amino-4-(2,4-dihydroxyphenyl)thiazoles as novel inhibitors of DNA gyrase B

Cyclothialidines are a class of bacterial DNA gyrase B (GyrB) subunit inhibitors, targeting its ATP-binding site. Starting from the available structural information on cyclothialidine GR122222X (2), an in silico virtual screening campaign was designed combining molecular docking calculations with three-dimensional structure-based pharmacophore information. A novel class of 2-amino-4-(2,4-dihydroxyphenyl)thiazole based inhibitors (5-9) with low micromolar antigyrase activity was discovered.

Targeted molecular dynamics simulation studies of binding and conformational changes in E. coli MurD.

Enzymes involved in the biosynthesis of bacterial peptidoglycan, an essential cell wall polymer unique to prokaryotic cells, represent a highly interesting target for antibacterial drug design. Structural studies of E. coli MurD, a three‐domain ATP hydrolysis driven muramyl ligase revealed two inactive open conformations of the enzyme with a distinct C‐terminal domain position. It was hypothesized that the rigid body rotation of this domain brings the enzyme to its closed active conformation, a structure, which was also determined experimentally. Targeted molecular dynamics 1 ns‐length simulations were performed in order to examine the substrate binding process and gain insight into structural changes in the enzyme that occur during the conformational transitions into the active conformation. The key interactions essential for the conformational transitions and substrate binding were identified. The results of such studies provide an important step toward more powerful exploitation of experimental protein structures in structure‐based inhibitor design. Proteins 2007.

Binding free energy calculations of N-sulphonyl-glutamic acid inhibitors of MurD ligase.

The increasing incidence of bacterial resistance to most available antibiotics has underlined the urgent need for the discovery of novel efficacious antibacterial agents. The biosynthesis of bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. Structural studies of N-sulfonyl-glutamic acid inhibitors of MurD have made possible the examination of binding modes of this class of compounds, providing valuable information for the lead optimization phase of the drug discovery cycle. Binding free energies were calculated for a series of MurD N-sulphonyl-Glu inhibitors using the linear interaction energy (LIE) method. Analysis of interaction energy during the 20-ns MD trajectories revealed non-polar van der Waals interactions as the main driving force for the binding of these inhibitors, and excellent agreement with the experimental free energies was obtained. Calculations of binding free energies for selected moieties of compounds in this structural class substantiated even deeper insight into the source of inhibitory activity. These results constitute new valuable information to further assist the lead optimization process.

In silico discovery and biophysical evaluation of novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase B.

Bacterial DNA gyrase is an established and validated target for the development of novel antibacterials. In our previous work, we identified a novel series of bacterial gyrase inhibitors from the class of 4-(2,4-dihydroxyphenyl) thiazoles. Our ongoing effort was designated to search for synthetically more available compounds with possibility of hit to lead development. By using the virtual screening approach, new potential inhibitors were carefully selected from the focused chemical library and tested for biological activity. Herein we report on a novel class of 5-(2-hydroxybenzylidene) rhodanines as gyrase B inhibitors with activity in low micromolar range and moderate antibacterial activity. The binding of the two most active compounds to the enzyme target was further characterised using surface plasmon resonance (SPR) and differential scanning fluorimetry methods (DSF).

Recent Developments of DNA Poisons - Human DNA Topoisomerase IIα Inhibitors - as Anticancer Agents

DNA topoisomerases are an important family of enzymes that catalyze the induction of topological changes in the DNA molecule. Their ability to modulate the topology of the DNA makes DNA topoisomerases a key player in several vital cell processes such as replication, transcription, chromosome separation and segregation. Consequently, they already represent an important collection of macromolecular targets for some of the established anticancer drugs on the market as well as serve as templates in the development of novel anticancer drugs especially supported by recent structural advances in the field. The aim of this review is to provide an overview of the recent developments in the field of DNA poisons - a major class of human topoisomerase IIα inhibitors - of which several are already in clinical use. Due to frequently experienced occurrence of serious side effects of these molecules during therapy, especially cardiotoxicity issues, further drug design efforts were initiated already yielding novel promising compounds that have overcome this issue and already entered into clinical studies. Some of the presented and discussed chemical classes include intercalators, non-intercalators and redox-dependent poisons of human topoisomerase IIα. In particular, this review focuses on the currently available structure-based standpoint of molecular design and on the medicinal chemists perspective of this field of anticancer drug design.

Combinatorially-generated library of 6-fluoroquinolone analogs as potential novel antitubercular agents: a chemometric and molecular modeling assessment

The virtual combinatorial chemistry approach as a methodology for generating chemical libraries of structurally-similar analogs in a virtual environment was employed for building a general mixed virtual combinatorial library with a total of 53.871 6-FQ structural analogs, introducing the real synthetic pathways of three well known 6-FQ inhibitors. The druggability properties of the generated combinatorial 6-FQs were assessed using an in-house developed drug-likeness filter integrating the Lipinski/Veber rule-sets. The compounds recognized as drug-like were used as an external set for prediction of the biological activity values using a neural-networks (NN) model based on an experimentally-determined set of active 6-FQs. Furthermore, a subset of compounds was extracted from the pool of drug-like 6-FQs, with predicted biological activity, and subsequently used in virtual screening (VS) campaign combining pharmacophore modeling and molecular docking studies. This complex scheme, a powerful combination of chemometric and molecular modeling approaches provided novel QSAR guidelines that could aid in the further lead development of 6-FQs agents.

Molecular dynamics simulation and linear interaction energy study of d-Glu-based inhibitors of the MurD ligase

The biosynthetic pathway of the bacterial peptidoglycan, where MurD is an enzyme involved at the intracellular stage of its construction, represents a collection of highly selective macromolecular targets for novel antibacterial drug design. 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 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.

MurD ligase from E. coli: Tetrahedral intermediate formation study by hybrid quantum mechanical/molecular mechanical replica path method

MurD (UDP‐N‐acetylmuramoyl‐L‐alanine:D‐glutamate ligase), a three‐domain bacterial protein, catalyses a highly specific incorporation of D‐glutamate to the cytoplasmic intermediate UDP‐N‐acetyl‐muramoyl‐L‐alanine (UMA) utilizing ATP hydrolysis to ADP and Pi. This reaction is part of a biosynthetic path yielding bacterial peptidoglycan. On the basis of structural studies of MurD complexes, a stepwise catalytic mechanism was proposed that commences with a formation of the acyl‐phosphate intermediate, followed by a nucleophilic attack of D‐glutamate that, through the formation of a tetrahedral reaction intermediate and subsequent phosphate dissociation, affords the final product, UDP‐N‐acetyl‐muramoyl‐L‐alanine‐D‐glutamate (UMAG). A hybrid quantum mechanical/molecular mechanical (QM/MM) molecular modeling approach was utilized, combining the B3LYP QM level of theory with empirical force field simulations to evaluate three possible reaction pathways leading to tetrahedral intermediate formation. Geometries of the starting structures based on crystallographic experimental data and tetrahedral intermediates were carefully examined together with a role of crucial amino acids and water molecules. The replica path method was used to generate the reaction pathways between the starting structures and the corresponding tetrahedral reaction intermediates, offering direct comparisons with a sequential kinetic mechanism and the available structural data for this enzyme. The acquired knowledge represents new and valuable information to assist in the ongoing efforts leading toward novel inhibitors of MurD as potential antibacterial drugs. Proteins 2009.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Parks nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents.