A Cephalosporin-Tripodalamine Conjugate Inhibits Metallo-β-Lactamase with High Efficacy and Low Toxicity

Abstract

The wide spread of metallo-β-lactamase (MBL)-expressing bacteria has greatly threatened human health, and there is an urgent need for inhibitors against MBLs. Herein, we present a cephalosporin-tripodalamine conjugate (DPASC) as a potent MBL inhibitor with a block-release design. The cephalosporin tag blocks the ligand binding site to reduce toxicity and is cleaved by MBLs to release active ligands to inhibit MBLs in situ. The screening of MBL-expressing pathogenic strains with 16 μg/mL DPASC showed a decrease of the minimum inhibitory concentration of meropenem (MEM) by 16 to 512-fold, and its toxicity was minimal to human HepG2 cells, with an IC₅₀ exceeding 512 μg/mL. An in vivo infection model with Galleria mellonella larvae showed an increased 3-day survival rate of 87% with the coadministration of DPASC and MEM, compared to 50% with MEM alone and no toxicity at a dose of 256 mg/kg of DPASC. Our findings with DPASC demonstrate that it is an effective MBL inhibitor and that the block-release strategy could be useful for the development of new MBL inhibitors.

Keywords: bacterial infection; cephalosporin conjugate; inhibitor; metallo-beta-lactamase; self-immolative linkage.

FIG 1

Structure and the inhibiting mechanism of DPASC.

FIG 2

Synthesis and enzymatic decomposition of DPASC. (A) Synthesis of DPASC. (B) Proposed cleavage of DPASC and formation of DPAS⁻ in the presence of metallo-β-lactamases.

FIG 3

¹H NMR spectra of DPASC before (black) and after treatment with Ca²⁺(red), Mg²⁺(blue), or Zn²⁺(green). Inset: enlarged view of spectra between 7.3 to 8.8 ppm.

FIG 4

Inhibitory and stability properties of DPASC. (A) Inhibition of NDM-1 by DPASC (black) or DPASH (red). Experimental data are shown as solid dots, and the fitted curves are shown as solid lines. (B) Inhibition of ADH by DPASC (black) or DPASH (red). Experimental data are shown as solid dots, and the fitted curves are shown as solid lines. (C) High-performance liquid chromatography (HPLC) of DPASC after cleavage by NDM-1 with excess ZnSO₄ with a m/z of 471.81 corresponding to DPASC, a m/z of 260.06 corresponding to DPASH, and a m/z of 321.74 corresponding to the DPAS-zinc(II) complex. (D) HPLC chromatography of DPASC incubated with fetal bovine serum (FBS).

FIG 5

Molecular modeling of the DPAS-NDM-1 complex. (A) Docked structure of DPAS⁻ in the active site of NDM-1. (B) Time-dependent root mean square deviation (RMSD) plots of the DPAS-NDM-1 complex; protein backbone (red) and DPAS⁻ (black). (C) The distance between the zinc ions and the sulfur atom of Cys208; Zn1 (black) and Zn2 (red). (D) The distance between the zinc ions and the sulfur atom of DPAS⁻; Zn1 (black) and Zn2 (red).

FIG 6

(A) Time-killing curve of strain 42 in MH (black), 32 μg/mL DPASC (red), 1 μg/mL MEM (blue), and a combination of 1 μg/mL MEM and 32 μg/mL DPASC (green). The dotted line indicates the limit of detection. (B) Survival rates of G. mellonella larvae. Larvae (10 per group) were infected with E. coli 42 (5.0 × 10⁵ CFU), followed by the injection of water (black), MEM (4 mg/kg, red), or a combination of MEM (4 mg/kg) and DPASC (64 mg/kg) (blue). The survival of the larvae was recorded for 3 days. Results were obtained from three independent experiments and are shown as the mean ± the standard error of the mean (SEM). P values were determined via a one-way ANOVA, using the Bonferroni post hoc correction. The overall P value, F statistic, and total number of degrees of freedom were 0.0001, 58.88, and 8.