Identification and Characterization of Chemical Compounds that Inhibit Leucyl-tRNA Synthetase from Pseudomonas aeruginosa

Abstract

Background: Pseudomonas aeruginosa is an opportunistic multi-drug resistance pathogen implicated as the causative agent in a high-percentage of nosocomial and community acquired bacterial infections. The gene encoding leucyl-tRNA synthetase (LeuRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized.

Methods: LeuRS was kinetically evaluated and the KM values for interactions with leucine, ATP and tRNA were 6.5, 330, and 3.0 μM, respectively. LeuRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen over 2000 synthetic and natural chemical compounds.

Results: The initial screen resulted in the identification of two inhibitory compounds, BT03C09 and BT03E07. IC50s against LeuRS observed for BT03C09 and BT03E07 were 23 and 15 μM, respectively. The minimum inhibitory concentrations (MIC) were determined against nine clinically relevant bacterial strains. In time-kill kinetic analysis, BT03C09 was observed to inhibit bacterial growth in a bacteriostatic manner, while BT03E07 acted as a bactericidal agent. Neither compound competed with leucine or ATP for binding LeuRS. Limited inhibition was observed in aminoacylation assays with the human mitochondrial form of LeuRS, however when tested in cultures of human cell line, BT03C09 was toxic at all concentration whereas BT03E07 only showed toxic effects at elevated concentrations.

Conclusion: Two compounds were identified as inhibitors of LeuRS in a screen of over 2000 natural and synthetic compounds. After characterization one compound (BT03E07) exhibited broad spectrum antibacterial activity while maintaining low toxicity against human mitochondrial LeuRS as well as against human cell cultures.

Keywords: E. coli; Pseudomonas aeruginosa; antibiotics; drug discovery; leucyl-tRNA synthetase; protein synthesis..

Fig. (1).. An alignment of the amino acid sequence of LeuRS from P. aeruginosa, E. coli, and T. thermophilus.

The protein sequences were downloaded from the National Center for Biotechnology Information (NCBI). Accession numbers for LeuRS from E. coli (Ec), P. aeruginosa (Pa), and T. thermophilus (Tth) sequences are NP_252676, AMK98521 and BAD69984, respectively. Sequence alignments were performed using Vector NTI Advance ^(R) 11.5 (Invitrogen). The structural motifs, HIGH and KMSKS are enclosed by solid line boxes. The editing hydrolytic active site composed of two regions are enclosed by dashed line boxes. Specific amino acids that interact with Leu (•), ATP (♦) and tRNA (★) are indicated. Cysteine and histidine residues forming zinc-binding knuckles are indicated (▲).

Fig. (2).. Determination of the activity of P. aeruginosa LeuRS and the kinetic parameters governing interactions with its three substrates: leucine, ATP, and tRNA.

P. aeruginosa LeuRS was titrated into the aminoacylation assay (A) as described in “Methods and Materials” at concentrations between 0.1 and 0.8 μM. Background activity was minimal and was subtracted from values at all concentrations of LeuRS. Initial velocities for the interaction of LeuRS with ATP (B) and leucine (C) were both determined using the ATP:PP_i exchange reaction. Initial velocity for the interaction of LeuRS with tRNA were determined using the aminoacylation reaction (D). The concentration of LeuRS in the aminoacylation reactions and the exchange reactions was 0.5 μM and 0.035 μM, respectively. Initial velocities were determined and the data were fit to a Michaelis-Menten steady-state model using XLfit 5.3 (IDBS) to determine.K_M and V_max. The kinetic parameters were determined for interaction of LeuRS with the three substrates (E).

Fig. (3).

The chemical structure of the hit compounds. The structure of BT03C09 (A) and BT03E07 (B).

Fig. (4).. Characterization of enzymatic and bacterial inhibition of BT03C09 and BT03E07.

IC₅₀ values for the inhibitory activity of BT03C09 (A) and BT03E07 (B) against P. aeruginosa LeuRS were 23 μM and 15 μM, respectively. IC₅₀s were determined with the test compounds serially diluted from 200 μM to 0.4 μM into aminoacylation assays containing P. aeruginosa LeuRS at 0.1 μM. The “% Positive” indicates the percent of activity observed relative to activity in assays where only DMSO was added to the assay in the absence of compound. The curve fits and IC₅₀ values were determined using the Sigmoidal Dose-Response Model in XLfit 5.3 (IDBS). The activity of the hit compounds against the growth of cultures containing (C) E. faecalis, and (D) E. coli tolC mutant bacteria were determined using broth microdilution susceptibility testing. Compounds were added to bacterial cultures at 4×MIC. Samples were analyzed by plating and determination of colony forming units (CFU) at 0, 2, 4, 6 and 24 h. Filled squares (■) represents cultures containing BT03E07 and filled triangles (▲) represents cultures containing BT03C09. Filled circles (•) represent growth of control cultures in the absence of compound.

Fig. (5).

Determination of the compound inhibition of human mitochondrial LeuRS and the toxicity relative to the control staurosporine in human cell cultures. Inhibitory activity of BT03E07 (A) and BT03C09 (B) against the activity of human mitochondrial LeuRS (hmLeuRS). The concentration of hmLeuRS was 0.075 μM. MTT assays were performed after 24 hours exposure to the indicated dose of drug under standard tissue culture conditions as described in “Materials and Methods”. To determine the CC₅₀ of BT03E07, the compound concentration ranged from 25 to 300 μg/ml (C). The control staurosporine was serially diluted in assays from 1 to 0.001 μg/ml to determine CC₅₀ (D). The data points represents an average value for assays carried out in triplicate. The “% Positive” indicates the percent of activity observed relative to activity in assays where only DMSO was added to the assay in the absence of compound. The curve fits and CC₅₀ values were determined using the Sigmoidal Dose-Response Model in XLfit 5.3 (IDBS).