Identification and characterization of novel small molecule compounds with acinetobacter-specific inhibitory activities : a chemical genetics approach

Abstract

Acinetobacter species have emerged rapidly as multidrug resistant nosocomial pathogen, particularly Acinetobacter baumanii, in which pan-drug resistant clones were reported, capable of resisting all commercially available drugs. Therapeutic options are severely scarce due to the antibiotics void in the past decades; with situation further worsen by the new acquisitions of resistant genes from other bacteria via horizontal gene transfer. New drugs are desperately needed to meet the demand, but whether broad-spectrum antibiotics remain as the best way to combat horizontal gene transfer related multidrug resistance are in doubt. A narrow-spectrum antibacterial may provide a better solution to resolve the current situation. An Acinetobacter specific small molecule compound, G11, was identified by high throughput screening of a structural diverse chemical library. The bacteriostatic compound demonstrated broad antibacterial activites against clinical isolates of multidrug-resistant A. baumannii and non-baumannii Acinetobacter species tested, but did not have antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium, Enterococcus faecalis, Staphylococcus aureus, Serratia marcescens and Providencia stuartii. The minimal inhibitory concentration of the compound against A. baumannii is at low micromolar range, making it an ideal lead compound for future drug-like development. Resistant mutants of G11 were selected and whole genome sequenced. Comparison between the G11 sensitive and resistant clones revealed that mutations in the outer membrane protein A (OmpA) were responsible for the G11 resistances. Absence of OmpA in the bacteria were not lethal, but allows the bacteria to resist the antibacterial effect of G11. However, this loss of OmpA adaptation was penalized with the loss of biofilm formation function, consequently leading to the loss of virulence in in-vivo infections models. Less frequent missense mutations of the OmpA were found; those clones would preserve the virulence and biofilm formation ability. However, as observed in all mutants with OmpA, all clones demonstrated less resistance towards β-lactam class antibiotics. Those observations were ideal traits of a potential drug, for that the bacteria must either be susceptible to the drug, or resistant to the drug at a physiologic cost; both of which will assist the host to clear the pathogen. This was something we anticipated, without broad-spectrum antibiotics affecting other species, resistance mechanisms must be self originated; this likely will disrupt the delicate homeostatic cellular environments leading to sub-optimal growth. Finally through a preliminary analogue screening, we were able to obtain enhanced small molecule analogues of G11, compound 13 and 18, which exhibited lower toxicity than G11 and retained the Acinetobacter specific antibacterial effect. Those compounds will lay the foundation for better development of narrow-spectrum antibiotics against the problematic multidrug resistant A. baumannii. (425 words)