Inhibitor discovery of full-length New Delhi metallo-β-lactamase-1 (NDM-1)

Abstract

New Delhi metallo-β-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (k cat/K m ratios between 0.03 to 1.28 µmol⁻¹.s⁻¹), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.

Figure 1. SDS-PAGE (5 to 12%) gels of NDM-1 purification.

Lane 1, Takara protein molecular weight marker; lane 2, boiled cell fraction of E. coli.BL21(DE3) containing pET30a-NDM-1 before induction; lane 3, boiled cell fraction of E. coli. BL21(DE3) containing pET30a-NDM-1 after a 14 h induction with 0.1 mM IPTG at 18°C; lane 4, crude protein after ultrasonication and centrifugation in supernatant solution; lane 5, crude protein after ultrasonication and centrifugation in precipitation; lane 6, His₆ tag-NDM-1 after Ni-NTA affinity chromatography; lane 7, full-length NDM-1 after removing the protease, uncut protein and affinity tag.

Figure 2. Molecular structures and IC₅₀ values of inhibitors against NDM-1.

Effects of inhibitors against full-length NDM-1 activity were mediated by meropenem hydrolysis. The precision of the IC₅₀ determinations was within the range of ±10%.

Figure 3. Structure of ticarcillin.

Figure 4. Binding model between compound 4 and NDM-1 in active site.

NDM-1 and compound 4 is shown as solid ribbon model and a stick model. Water-bridge and two zinc ions are shown as spheres, respectively. Key amino acids, His189, His120, His122, Asp124, Cys208 and His250, in active site are shown as line model.

Figure 5. The accurate partial charge values of two zinc ions, water-bridge and sulfur atom of compound 4.

Water-bridge and two zinc ions are shown as spheres. Compound 4 and key amino acids are shown as stick model and line model, respectively.