A novel colistin adjuvant identified by virtual screening for ArnT inhibitors

Abstract

Background: Colistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa.

Objectives: Identification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants.

Methods: The available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A.

Results: A putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially.

Conclusions: This study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.

Figure 1.

Validation of BBN149 as a colistin resistance inhibitor. (a) Effect of the 18 putative ArnT inhibitors identified by in silico docking at 50 μM on the growth of the colistin-resistant isolate P. aeruginosa PA14 col^R 5 after 24 h at 37°C in MH supplemented or not with a sub-MIC concentration of colistin (8 mg/L). (b) Dose-dependent effect of BBN120, BBN149 and BBN153 on PA14 col^R 5 growth after 24 h at 37°C in MH supplemented with 8 mg/L colistin. In panels (a) and (b), growth values are expressed as the percentage relative to the cultures treated with equivalent concentrations of DMSO and represent the mean (±SD) of at least three independent experiments. (c) Effect of different concentrations of BBN149, and DMSO as control, on the MIC of colistin for PA14 col^R 5 as determined by chequerboard assays. The graph is representative of four independent experiments. *P < 0.05 (ANOVA).

Figure 2.

(a) Chemical structure of the diterpenoid ent-beyer-15-en-18-O-oxalate (BBN149). (b, c) Predicted binding mode of BBN149 to the catalytic site of the ArnT crystallographic structure. Two possible docking poses are shown in the two panels. The ligand is coloured cyan and shown as sticks, while the protein is coloured green. Residues within 5 Å from the ligand are shown as lines; those predicted to form H-bonds with BBN149 are shown as sticks and labelled. H-bond interactions are highlighted by black dashed lines. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.

Figure 3.

Time–kill curves of P. aeruginosa PA14 col^R 5, KK27 col^R 6 and ND76 exposed to 30 μM BBN149 in the presence or absence of colistin at 1× or 2× MIC (based on the values reported in Table 1). As a control, the strains were incubated in the presence of 0.3% DMSO and the same concentrations of colistin. The results are the mean (±SD) of two independent assays.

Figure 4.

Viability of 16HBE epithelial cells exposed to BBN149 at the indicated concentrations for 3 or 18 h. Viability was assessed through the MTT assay and expressed as a percentage relative to vehicle-only (DMSO) controls. Data are the mean (±SD) of three independent experiments.

Figure 5.

Effect of BBN149 on lipid A aminoarabinosylation. MALDI-TOF analysis of lipid A extracted from P. aeruginosa PA14 col^R 5 (a), KK27 col^R 6 (b) or TR1 col^R 6 (c) cultured in MH supplemented with 30 μM BBN149 or 0.3% DMSO as control. In (a) arrows indicate the addition of an aminoarabinose molecule (l-Ara4N; m/z + 131) or the removal of a phosphate group (m/z − 80) from the penta-acylated lipid A (m/z = 1446), the hexa-acylated lipid A (m/z = 1616) or a still unidentified lipid A form at m/z = 1348. Lipid A peaks differing by m/z ± 16 correspond to different hydroxylation states of the secondary C12 acyl chains. The m/z values of all peaks corresponding to aminoarabinosylated lipid A forms are in red, bold and underlined. Spectra were obtained in the negative-ion mode, thus m/z values correspond to (molecular mass – 1)/1 and are representative of three biological replicates. This figure appears in colour in the online version of JAC and in black and white in the printed version of JAC.