Stability and binding effects of silver(I) complexes at lipoxygenase-1.

An anti-inflammatory complex of Ag(I), namely [Ag(tpp)3(asp)](dmf) [tpp = triphenylphosphine, aspH = aspirin, dmf = N,N-dimethylformamide], was synthesized in an attempt to develop novel metallotherapeutic molecules. STD (1)H NMR experiments were used to examine if this complex binds to LOX-1. The (1)H NMR spectra in buffer Tris/D2O betrayed the existence of two complexes: the complex of aspirin and the complex of salicylic acid produced after deacetylation of aspirin. Nevertheless, the STD spectra showed that only the complex of salicylic acid is bound to the enzyme. Molecular docking and dynamics were used to complement our study. The complexes were stabilized inside a large LOX-1 cavity by establishing a network of hydrogen bonds and steric interactions. The complex formation with salicylic acid was more favorable. The in silico results provide a plausible explanation of the experimental results, which showed that only the complex with salicylic acid enters the binding cavity.

A novel 2-oxoindolinylidene inhibitor of bacterial MurD ligase: Enzyme kinetics, protein-inhibitor binding by NMR and a molecular dynamics study.

N-(5-(5-nitro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)4-oxo-2-thioxo-1,3-thiazolidin-3-yl)nicotinamide, a 2-oxoindolinylidene derivative with novel structure scaffold, was evaluated for inhibition potency against the MurD enzyme from Escherichia coli using an enzyme steady-state kinetics study. The compound exerted competitive inhibition with respect to UMA, a MurD substrate, and affected bacterial growth. Furthermore, we isolated and purified (13)C selectively labeled MurD enzyme from E. coli and evaluated the binding interactions of the new compound using the (1)H/(13)C-HSQC 2D NMR method. Molecular dynamics calculations showed stable structure for the MurD-inhibitor complex. The binding mode of novel inhibitor was determined and compared to naphthalene-N-sulfonamide-d-Glu derivatives, transition state mimicking inhibitors, UMA and AMP-PCP, an ATP analog. It binds to the UDP/MurNAc binding region. In contrast to transition state mimicking inhibitors, it does not interact with the enzyme's C-terminal domain, which can be beneficial for ligand binding. A pharmacophore pattern was established for the design of novel drugs having a propensity to inhibit a broad spectrum of Mur enzymes.

The binding mode of second-generation sulfonamide inhibitors of MurD: clues for rational design of potent MurD inhibitors.

A series of optimized sulfonamide derivatives was recently reported as novel inhibitors of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD). These are based on naphthalene-N-sulfonyl-D-glutamic acid and have the D-glutamic acid replaced with rigidified mimetics. Here we have defined the binding site of these novel ligands to MurD using (1)H/(13)C heteronuclear single quantum correlation. The MurD protein was selectively (13)C-labeled on the methyl groups of Ile (δ1 only), Leu and Val, and was isolated and purified. Crucial Ile, Leu and Val methyl groups in the vicinity of the ligand binding site were identified by comparison of chemical shift perturbation patterns among the ligands with various structural elements and known binding modes. The conformational and dynamic properties of the bound ligands and their binding interactions were examined using the transferred nuclear Overhauser effect and saturation transfer difference. In addition, the binding mode of these novel inhibitors was thoroughly examined using unrestrained molecular dynamics simulations. Our results reveal the complex dynamic behavior of ligand-MurD complexes and its influence on ligand-enzyme contacts. We further present important findings for the rational design of potent Mur ligase inhibitors.

Novel 2-thioxothiazolidin-4-one inhibitors of bacterial MurD ligase targeting D-Glu- and diphosphate-binding sites.

Mur ligases are involved in cytoplasmic steps of bacterial peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have designed and synthesized a focused chemical library of compounds combining the glutamic acid moiety and the 2-thioxothiazolidin-4-one, thiazolidine-2,4-dione, 2-iminothiazolidin-4-one or imidazolidine-2,4-dione ring connected by a benzylidene group. These compounds were designed to target the d-Glu- and the diphosphate-binding pockets of the MurD active site and were evaluated for inhibition of MurD ligase from Escherichia coli. The most potent compounds (R)-9 and (S)-9 inhibited MurD with IC(50) values of 45 µM and 10 µM, respectively. The specific binding mode of (R)-9 in MurD active site was established by high-resolution NMR spectroscopy.

Second-generation sulfonamide inhibitors of D-glutamic acid-adding enzyme: activity optimisation with conformationally rigid analogues of D-glutamic acid.

D-Glutamic acid-adding enzyme (MurD) catalyses the essential addition of d-glutamic acid to the cytoplasmic peptidoglycan precursor UDP-N-acetylmuramoyl-l-alanine, and as such it represents an important antibacterial drug-discovery target enzyme. Based on a series of naphthalene-N-sulfonyl-d-Glu derivatives synthesised recently, we synthesised two series of new, optimised sulfonamide inhibitors of MurD that incorporate rigidified mimetics of d-Glu. The compounds that contained either constrained d-Glu or related rigid d-Glu mimetics showed significantly better inhibitory activities than the parent compounds, thereby confirming the advantage of molecular rigidisation in the design of MurD inhibitors. The binding modes of the best inhibitors were examined with high-resolution NMR spectroscopy and X-ray crystallography. We have solved a new crystal structure of the complex of MurD with an inhibitor bearing a 4-aminocyclohexane-1,3-dicarboxyl moiety. These data provide an additional step towards the development of sulfonamide inhibitors with potential antibacterial activities.

5-Benzylidenethiazolidin-4-ones as multitarget inhibitors of bacterial Mur ligases.

Mur ligases participate in the intracellular path of bacterial peptidoglycan biosynthesis and constitute attractive, although so far underexploited, targets for antibacterial drug discovery. A series of hydroxy-substituted 5-benzylidenethiazolidin-4-ones were synthesized and tested as inhibitors of Mur ligases. The most potent compound 5 a was active against MurD-F with IC(50) values between 2 and 6 microm, making it a promising multitarget inhibitor of Mur ligases. Antibacterial activity against different strains, inhibitory activity against protein kinases, mutagenicity and genotoxicity of 5 a were also investigated, and kinetic and NMR studies were conducted.

NMR and molecular dynamics study of the binding mode of naphthalene-N-sulfonyl-D-glutamic acid derivatives: novel MurD ligase inhibitors.

The presented series of naphthalene-N-sulfonyl-D-glutamic acid derivatives are novel MurD ligase inhibitors with moderate affinity that occupy the D-Glu binding site. We performed an NMR study including transfer NOE to determine the ligand bound conformation, as well as saturation transfer difference experiments to obtain ligand epitope maps. The difference in overall appearance of the epitope maps highlights the importance of hydrophobic interactions and shows the segments of molecular structure that are responsible for them. Transfer NOE experiments indicate the conformational flexibility of bound ligands, which were then further examined by unrestrained molecular dynamics calculations. The results revealed the differing degrees of ligand flexibility and their effect on particular ligand-enzyme contacts. Conformational flexibility not evident in the crystal structures may have an effect on ligand-binding site adaptability, and this is probably one of the important reasons for the only moderate activity of novel derivatives.