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.

ent-Beyerane Diterpenes as a Key Platform for the Development of ArnT-Mediated Colistin Resistance Inhibitors

“…To assess the colistin adjuvant activity of the newly synthesized compounds ( 2–15 ), we first performed the same screening assay that allowed the identification of compound 1 as a colistin potentiator. 22 Briefly, a reference P. aeruginosa strain evolved in vitro toward colistin resistance (PA14 col R 5), characterized by a colistin MIC of 64 μg/mL and that was demonstrated to depend on lipid A aminoarabinosylation for colistin resistance, 45 , 46 was cultured in the presence of a fixed, subinhibitory concentration of colistin (8 μg/mL) and different concentrations of each compound of interest (4–64 μM). As control, the effect of the compounds on PA14 col R 5 growth in the absence of colistin was also assessed.…”

“…Recently, a unique in-house library of natural products available in our group was screened in silico against the catalytic site of the ArnT enzyme to identify putative inhibitors of the Ara4N-dependent colistin resistance mechanism. 22 This led to the selection of the ent -beyerene diterpene 1 (formerly known as BBN149), isolated from the leaves of Fabiana densa var. ramulosa , with a colistin adjuvant activity versus colistin-resistant P. aeruginosa strains, without any significant effect on colistin-susceptible strains.…”

Biofilm Production by Carbapenem-Resistant Klebsiella pneumoniae Significantly Increases the Risk of Death in Oncological Patients

“…Colistin is considered as an antibiotic of last resort for treating severe CRKP infections, because of increasing microbial resistance and associated toxicity ( Elnahriry et al., 2016 ; Schwarz and Johnson, 2016 ; Rojas et al., 2017 ; Wang et al., 2018 ; Ghirga et al., 2020 ). In this study, we found a 10.5% of CRKP resistant to colistin.…”

A New Surface Charge Neutralizing Nano-Adjuvant to Potentiate Polymyxins in Killing Mcr-1 Mediated Drug-Resistant Escherichia coli

“…A novel colistin adjuvant that inhibits ArnT, which catalyzes the last committed step of lipid A aminoarabinosylation and is associated with a positive charge increase in cells [ 7 ], was identified using in silico screening [ 11 ]. Therefore, the strategy appears to be a promising candidate for lead optimization in the development of colistin resistance inhibitors.…”