Pharmacological inhibition of colistin resistance in Gram-negative cystic fibrosis pathogens

Background and rationale Resistance to colistin, a last-resort antibiotic for the Gram-negative bacteria, is mediated by modifications of the lipidA of the bacterial outer membrane. In Pseudomonas aeruginosa the ArnT transferase is a key enzyme for colistin resistance (1). Thus, inhibition of ArnT activity is expected to reduce colistin resistance. Hypothesis and objectives We have previously identified a potential inhibitor of ArnT, named BBN149, from which the ent-beyerane scaffold was identified as privileged platform for further development of ArnT inhibitors (2, 3). Based on this, we aim at the simplification of the ent-beyerane complex structure into drug-like synthetic molecules with improved biological activity. Essential methods A model of ArnT from P. aeruginosa was generated and used to predict the binding mode of small molecules within the catalytic site and to produce a library of ent-beyerane diterpens. A small focused library of abietane-type diterpenoid derivatives was produced by combining the abietane scaffold with the functional groups identified by in silico modeling. Compounds activity was determined by conventional microbiological methods. Design and assembly of liposomal formulations by thin lipid film hydration technique. Results A library of 21284 compounds was generated in silico, from which the top-ranking 1000 compounds were selected for further analysis of binding modes. From this, 41 compounds with a higher score than the reference BBN149, were suggested for organic synthesis. Among them, the abietane-type diterpenoids were selected for the synthesis of a small focused library. The colistin adjuvant activity of ten selected compounds was determined and the structure-activity relationship are under evaluation for additional cycles of design-synthesis-bioassay. Liposomes were assembled using the isostevic acid as a prototype of ent-beyerane derivatives. Liposomes loaded with colistin in the aqueous core and isostevic acid in the lipid bilayer were produced and their biologic activity is under investigation. Additionally, inhalable dosage forms are under investigation. Conclusions The entire procedure for identification and iterative modification of small molecules with potential inhibitory activity of ArnT has been set up and applied for the identification of optimized ArnT inhibitors, Additionally, nanovehicles for delivery of the ArnT inhibitors has been produced and advanced formulations are under investigation.