Mur ligases have essential roles in the biosynthesis of peptidoglycan, and they represent attractive targets for the design of novel antibacterials. MurD (UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase) is the second enzyme in the series of Mur ligases, and it catalyzes the addition of D-glutamic acid (D-Glu) to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA). Because of the high binding affinity of D-Glu toward MurD, we synthesized and biochemically evaluated a series of N-substituted D-Glu derivatives as potential inhibitors of MurD from E. coli, which allowed us to explore the structure-activity relationships.The substituted naphthalene-N-sulfonyl-D-Glu inhibitors, which were synthesized as potential transition state analogues, displayed IC50 values ranging from 80 to 600 microM. In addition, the high-resolution crystal structures of MurD in complex with four novel inhibitors revealed details of the binding mode of the inhibitors within the active site of MurD. Structure-activity relationships and cocrystal structures constitute an excellent starting point for further development of novel MurD inhibitors of this structural class.
BackgroundPenicillin-binding proteins (PBPs) are well known and validated targets for antibacterial therapy. The most important clinically used inhibitors of PBPs β-lactams inhibit transpeptidase activity of PBPs by forming a covalent penicilloyl-enzyme complex that blocks the normal transpeptidation reaction; this finally results in bacterial death. In some resistant bacteria the resistance is acquired by active-site distortion of PBPs, which lowers their acylation efficiency for β-lactams. To address this problem we focused our attention to discovery of novel noncovalent inhibitors of PBPs.Methodology/Principal FindingsOur in-house bank of compounds was screened for inhibition of three PBPs from resistant bacteria: PBP2a from Methicillin-resistant Staphylococcus aureus (MRSA), PBP2x from Streptococcus pneumoniae strain 5204, and PBP5fm from Enterococcus faecium strain D63r. Initial hit inhibitor obtained by screening was then used as a starting point for computational similarity searching for structurally related compounds and several new noncovalent inhibitors were discovered. Two compounds had promising inhibitory activities of both PBP2a and PBP2x 5204, and good in-vitro antibacterial activities against a panel of Gram-positive bacterial strains.ConclusionsWe found new noncovalent inhibitors of PBPs which represent important starting points for development of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.
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.
Quantitative structure-activity relationship (QSAR) studies on a set of Streptococcus pneumoniae MurD transitionstate inhibitors were performed, using a comprehensive set of molecular descriptors calculated by CODESSA software. Multiple and best multiple linear regressions were applied to generate models for predicting their inhibitory activity. The results (the best model had r 2 0.8818, s 2 0.0749, F 87.04 and r 2 cv 0.8488) demonstrate the importance of hydrogen bonding and that a matching conformation of ligands for interaction with the enzyme active site is required.
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