Pseudomonas aeruginosa is a common cause of serious infections in patients with immune deficiency (e.g., HIV and cancer), cystic fibrosis (CF), on mechanical ventilation or with burn wounds. While P. aeruginosa exhibits an alarming variety of intrinsic drug resistances, aminoglycosides (AMGs) such as tobramycin and amikacin, are still effective in treating diseases caused by this pathogen, including CF and ventilator- associated pneumonia. Nevertheless, the long-term utility of even these agents is in jeopardy due to the increasing prevalence of strains with intrinsic or acquired resistances. Recently, we discovered two members of a membrane stress reduction network, a two-component system (AmgRS) and a membrane protease (FtsH), that protect P. aeruginosa from AMGs in vitro and in vivo. Deletion of these genes yielded P. aeruginosa strains that are up to 16-fold more sensitive to inhibition by AMGs, even if they carry the rmtD 16S rRNA methylase AMG-resistance allele or are grown as biofilms. In addition, the AMG tobramycin was more effective in rescuing mice infected with these P. aeruginosa mutants from lethality. The goal of this project is to discover and develop new drugs that increase the efficacy of AMGs against P. aeruginosa. Our strategy is to identify small molecules that inhibit FtsH and/or AmgRS activity and to develop them into innovative adjunctive therapies that potentiate AMGs. Such therapies will significantly improve clinical treatment outcomes because they are expected to (a) increase the AMG-mediated killing of P. aeruginosa biofilms and planktonic cells, (b) sensitize clinical isolates exhibiting AMG resistance, (c) decrease the rate of development of AMG resistance, (d) reduce AMG-associated oto- and nephrotoxicities by enabling the use of lower doses, and (e) extend the spectrum of more AMGs to include P. aeruginosa. In preliminary studies, we constructed and optimized a cell- based reporter strain to identify FtsH or AmgRS inhibitors. This strain carries a transcriptional fusion of the amgRS- and ftsH-responsive P. aeruginosa gene PA2549 promoter to a luciferase operon, and exhibits increased signal in response to deletion of ftsH and decreased signal in response to deletion of amgRS. This novel reporter strain was validated in a pilot screen of known bioactive compounds, exhibiting a Z'-Factor >0.5 and successfully identifying compounds that increased or decreased RLU values. In Phase I, we will apply the PA2549-lux reporter strain to screen libraries of >300,000 discrete chemical compounds and natural products, confirm the hits, and validate them as potent, selective potentiators of AMG antibiotics vs. P. aeruginosa. The strongest potentiators acting on multiple AMGs and clinical P. aeruginosa isolates will be profiled to exclude those exhibiting cytotoxicity or membrane effects and will be re-synthesized to confirm structure. Analogs will be synthesized to demonstrate responsive SAR and favorable predicted ADME properties. In Phase II, we will develop the most promising validated hits into lead compounds by optimizing their activity and specificity and evaluate them for efficacy and toxicity in animal models of infection.
This research is aimed at discovering new drugs that increase the effectiveness of existing aminoglycoside antibiotics against pathogenic Pseudomonas aeruginosa strains. New drugs that inhibit the bacterial membrane stress response network will reduce the pathogen's ability to survive antibiotic therapy. These new drugs will prolong the duration of effective concentrations of existing antibiotics, thus reducing selection for resistanc and improving therapy.
