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Identification of Small-Molecule Inhibitors of Brucella Diaminopimelate Decarboxylase by Using a High-Throughput Screening Assay

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Identification of Small-Molecule Inhibitors of Brucella Diaminopimelate Decarboxylase by Using a High-Throughput Screening Assay

Pengfei Bie et al. Front Microbiol.

Abstract

Brucellosis, caused by intracellular gram-negative pathogens of the genus Brucella, continues to be one of the most pandemic zoonotic diseases in most countries. At present, the therapeutic treatment of brucellosis relies on a combination of multiple antibiotics that involves a long course of treatment, easy relapse, and high side effects from the use of certain antibiotics (such as streptomycin). Thus, the need to identify novel drugs or targets to control this disease is urgent. Diaminopimelate decarboxylase (DAPDC), a key enzyme involved in the bacterial diaminopimelate (DAP) biosynthetic pathway, was suggested to be a promising anti-Brucella target in our previous study. In this work, the biological activity of Brucella melitensis DAPDC was characterized, and a library of 1,591 compounds was screened for inhibitors of DAPDC. The results of a high-throughput screening (HTS) assay showed that 24 compounds inhibited DAPDC activity. In a further in vitro bacterial inhibition experiment, five compounds exhibited anti-Brucella activity (SID3, SID4, SID14, SID15, and SID20). These results suggested that the identified compounds can be used as potent molecules against brucellosis and that the application ranges of these approved drugs can be expanded in the future.

Keywords: diaminopimelate decarboxylase; enzyme inhibitors; high-throughput screening; lysine biosynthesis; saccharopine dehydrogenase.

Figures

FIGURE 1
FIGURE 1
Optimization of the diaminopimelate decarboxylase (DAPDC)-saccharopine dehydrogenase (SDH) coupled assay. (A) Standard curve of NADH concentrations. NADH (25–1,200 μM) was serially diluted in SDH assay buffer, and the absorbance at 340 nm was directly read in a PE EnVision multilabel plate reader. The data are depicted as the means ± SDs obtained from three independent experiments. A robust linear correlation was observed between the absorbance of NADH and its concentration. (B) Dependence of SDH activity on the concentration of NADH. The activity of SDH was tested at various concentrations of NADH (0–1,000 μM). The values represent the means ± SDs of SDH activity determined from three independent experiments. (C) Activity of SDH as a function of the concentration of L-lysine. The activity of SDH at various concentrations of L-lysine (0–1,200 μM) was measured. The values depicted are the means ± SDs of SDH activity determined from three independent experiments. (D) Activity of SDH as a function of the concentration of α-ketoglutarate. The activity of SDH at various concentrations of α-ketoglutarate ranging from 0 to 1,200 μM was measured. The values depicted are the means ± SDs of SDH activity determined from three independent experiments. (E) Time curves for the DAPDC reaction. The DAPDC activity was measured based on conditions optimized for SDH. Fifty nanograms of DAPDC was incubated with 20 mM DAP in DAPDC assay buffer containing 200 mM Tris–HCl (pH 8.0), 4 mM α-ketoglutarate, 600 μM NADH, 0.1 mM PLP, and an excess amount of SDH (17 ng/μl) in a total volume of 50 μl. Reactions were terminated by the addition of 25 μl of 8 M guanidine hydrochloride over a 3-min interval, and the absorbance at 340 nm was monitored. The data are depicted as the means ± SDs of Abs340 nm values obtained from three independent experiments. (F) Activity of DAPDC as a function of the concentration of DAP. The activity of DAPDC was tested at various concentrations of DAP (0–20 mM). The values depicted are the means ± SDs of DAPDC activity determined from three independent experiments.
FIGURE 2
FIGURE 2
Preliminary studies of diaminopimelate decarboxylase (DAPDC) enzyme activity inhibition using a US Food and Drug Administration (FDA)-approved drug library. Each compound was tested at a final concentration of 40 μM. Twenty-four compounds exhibited an inhibition rate greater than the cutoff value of 40%, representing a hit rate of 1.5%. All data were processed by using Microsoft Excel and GraphPad Prism 5.0 software.
FIGURE 3
FIGURE 3
Chemical structures of potential diaminopimelate decarboxylase (DAPDC) inhibitors identified from the primary screen.

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