Pseudomonas aeruginosa infections result in high mortality rates and constitute a significant health care burden. Antimicrobials are the mainstay of anti-pseudomonal therapy;however, increasing antibiotic resistance mandates development of new approaches to combat infections. The P. aeruginosa quorum sensing homoserine lactone C12 produces deleterious host effects including apoptosis and repression of NF- ?B transcription. Inhibition of C12-mediated host effects is predicted to attenuate P. aeruginosa virulence. By small molecule screening, we have identified triazolo[4,3-a]quinolines as C12 inhibitors that both rescue NF-?B activity and inhibit apoptosis. Our lead compound (TQ416) is potent (EC50 ~400 nM) and has demonstrated activity in a model of C12-induced cutaneous inflammation. Studies proposed in this application will employ TQ416 in models of P. aeruginosa lung infection to investigate the efficacy of inhibiting C12-mediated host effects as a novel anti-pseudomonal approach. The objective of Specific Aim 1 is to establish activity of TQ416 as a C12 inhibitor in the mouse lung using LPS and C12 to mimic infection. Initial studies will employ reporter mice that express luciferase in response to LPS-stimulated NF-?B activation to rapidly establish TQ416 dosing regimens that inhibit C12. This approach will be extended to confirm that TQ416 inhibits C12 to restore physiologically relevant NF?B-dependent processes (e.g. cytokine secretion, leukocyte infiltration), and that TQ416 prevents C12-induced apoptosis in the lung. These studies will accomplish MILESTONE 1: Verification that TQ416 inhibits C12 activity to restore NF?B transcription and prevent apoptosis in vivo. To investigate the role of C12-induced host effects in P. aeruginosa virulence, Specific Aim 2 will test TQ416 efficacy in resolvable, lethal and chronic models of P. aeruginosa lung infection. TQ416-mediated reduction of P. aeruginosa virulence will indicate that C12-mediated host effects contribute to P. aeruginosa infectivity and will accomplish MILESTONE 2: Validation of the hypothesis that inhibition of C12-mediated host effects is a useful therapeutic approach for P. aeruginosa infections. R33 phase studies aim to develop C12 inhibitor drug candidates with high potency and optimized pharmacological properties. Extended analysis of the triazolo[4,3- a]quinoline scaffold (>1000 analogs) will be conducted in addition to extensive screening (~300,000 compounds) to identify new C12 inhibitor scaffolds. Metabolism of potent lead compounds will then be assessed in microsomal stability assays. Pharmacokinetics and toxicity of potent and stable C12 inhibitors will be established in vivo. Subsequently, efficacy of validated, drug-like compounds will be assessed in models of P. aeruginosa lung infection. We anticipate these studies will identify 2 or more drug-like leads from distinct chemical classes to achieve MILESTONE 3: Delivery of drug-like validated C12 inhibitors for clinical development.
Pseudomonas aeruginosa secrete a small signaling molecule that initiates deleterious host effects;this application will test a novel anti pseudomonal approach that involves inhibiting these host responses.