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

• A MurD inhibitor with novel structure scaffold is discovered.
• New inhibitor exerts competitive inhibition with respect to UMA, a MurD substrate.
• Its binding mode is determined by NMR and molecular dynamics simulations.
• Binding of the novel inhibitor is not affected by MurD domain's movements.
• A pharmacophore pattern for the design of potent Mur inhibitors is provided.

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 13C selectively labeled MurD enzyme from E. coli and evaluated the binding interactions of the new compound using the 1H/13C-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.

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