Colistin is a last resort treatment option for many multidrug resistant Gram-negative bacteria. However, colistin resistance immediately appeared as colistin was introduced in clinical setting. 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.
Pseudomonas aeruginosa, is among the bacteria for which antibiotics are urgently needed. In Cystic Fibrosis (CF), colistin has been used for decades suggesting that resistance to this antibiotic might become soon critical for CF patients. Indeed, an increase of colistin-resistant P. aeruginosa isolates in CF has been recently reported.
The antibacterial activity of colistin relies on its interaction with LPS. Aminoarabinosylation of the lipid A moiety of LPS is strictly required for the development of colistin resistance in P. aeruginosa. Therefore, the main objective of this project is to develop small molecules that revert colistin resistance by targeting lipid A aminoarabinosylation.
Taking advantage of the resolved structure of the aminoarabinose transferase, which catalyzes the last step of lipid A aminoarabinosylation, and using a structure-guided in silico approach, we have identified a short list of candidate hits from the screening of a library of natural compounds. Among them we have identified one compound, named BBN149, which is active at relatively low concentration in reversing colistin resistance to colistin sensitivity of a P. aeruginosa tester strain.
Growth-inhibition assays and computational analysis strongly suggest that BBN149 is an inhibitor of ArnT activity.
The present project comprises the following aims: modification of BBN149 by medicinal chemistry to produce derivatives with optimized potency and specificity; analysis of the efficacy of BBN149 and its derivatives on different model systems.
Global resistance to antimicrobial medicines is recognized as one of the greatest threat to human health. In 2017 the world health organization (WHO) included Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacteriaceae, in the list of bacteria for which antibiotics are urgently needed as first priority (https://www.who.int/en/news-room/detail/27-02-2017-who-publishes-list-of...). Indeed, our ability to treat common infection diseases is heavily compromised by the appearance of multi-resistant pathogens, which restrict the therapeutic options for the treatment of common infectious diseases.
In this scenario, colistin is considered the last therapeutic options for multidrug resistant Gram-negative pathogens, including P. aeruginosa and K. pneumoniae.
In P. aeruginosa it was recently found that ArnT-mediated lipid A aminoarabinosylation is absolutely required to acquire colistin resistance (7). Indeed, Ara4N, encoding the ArnT enzyme, defective mutants are unable to develop colistin resistance (7). Moreover, it was demonstrated that other lipid A modifications, such as those mediated by either endogenous (EptA) or plasmid-mediated PEtN transferases, has marginal effects on colistin resistance in P. aeruginosa. These findings strongly support the notion that pharmacological inhibition of Ara4N biosynthetic enzymes inactivate the colistin-resistance in P. aeruginosa and in other Gram-negative bacteria such as K. peneumoniae and B. cenocepacia. Based on this it is reasonably to surmise that Arn-mediated lipid A aminoarabinosylation can be inhibited by drugs blocking ArnT activity with the consequent recovering of sensitivity to colistin.
Thus, starting from the crystal structure of ArnT and the identification of its active site (8) and using a docking-based virtual screening of an in-house chemical library within the catalytic site of ArnT we have identified BBN149 as a lead compounds to inhibit colistin resistance in gram-neg bacteria.
In the present project, by combining computational and medicinal approaches, a library of semi-synthetic analogs of BBN149 will be synthesized at CTD. The resulting compounds will be tested either with microbiology studies, as reported above, and with computational approaches. By these approaches we expect to confirm thatt compounds able to interact with the catalytic site of ArnT are a privileged tool for an efficient reversal of colistin-resistant strains to its susceptibility.
The results of this project is expected to enlarge the therapeutic options for infectious diseases and positively impact the outcome of gram-negative infections.
The proposed project takes advantage of our team which combines broad expertise in molecular genetics of antibiotic resistance and infectious disease, in particular cystic fibrosis lung infections, medicinal and computational chemistry, host-pathogen interaction in cystic fibrosis, and CF clinicians.