Type 3 secretion system inhibitors to treat Pseudomonas aeruginosa keratitis

Contact lens-associated bacterial keratitis is a serious infection of the eye that can result in blindness if treatment is not effective. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is the most common pathogen associated with contact lens use, and multi-drug resistant strains are increasing in prevalence. P. aeruginosa forms biofilms on the corneal epithelium, resulting in increased resistance to antibiotics and killing by neutrophils, further complicating the treatment of these infections. Most clinical isolates from these infections are cytotoxic strains that express ExoU, a potent cytotoxin that is injected into host cells by the type 3 secretion system (T3SS), which is associated with increased virulence, antibiotic resistance, and greater morbidity. Clearly, novel approaches to treating these infections are needed. The T3SS of P. aeruginosa is a virulence factor that is essential for this organism to establish infection in the eye. The overall goal of this proposal is to develop a novel topical therapy for the treatment of contact lens-associated keratitis that targets the T3SS of P. aeruginosa. In an ongoing program at Microbiotix (MBX), we discovered and chemically optimized a novel phenoxyacetamide (PhA) chemical series of potent inhibitors of the T3SS of P. aeruginosa, resulting in the zwitterionic (ZW) analog sub-series of PhAs with excellent in vitro potency and ADM E properties. Several ZW analogs were efficacious in a murine non-neutropenic pneumonia model with P. aeruginosa infection consistent with their efficacy in in vitro assays and in murine models of infection. Our strategy for developing novel treatments for P. aeruginosa keratitis is to evaluate a select set of these ZW PhA T3SS inhibitors using in vitro and in vivo assays designed to identify a lead candidate suitable for further development as a topical adjunctive therapy to anti-pseudomonal antibiotics for P. aeruginosa keratitis. In this Phase I proposal, we will prioritize members of a library of ZW analogs for T3SS inhibitory activity and cytotoxicity using a panel of in vitro cellbased assays and keratitis clinical isolates with human corneal epithelial cell lines. Based on the results of these studies, we will identify up to 6 analogs for evaluation of tolerability and efficacy in a murine P. aeruginosa keratitis model as an adjunctive therapy with an ophthalmic antibiotic, with the goal of selecting a lead candidate that is suitable for further development in a follow-on Phase II proposal. The Phase 11 research will comprise lead optimization for an ophthalmic indication, evaluation of ophthalmic antibiotic combinations, formulation, and pre-clinical studies that are required for an IND application to the FDA. We will accomplish the goals of Phase I by completing the following Specific Aims: Aim 1. Prioritize select PhA T3SS inhibitors using in vitro assays relevant to ophthalmic infection. Aim 2. Identify a lead candidate based on in vivo efficacy in a murine P. aeruginosa keratitis model.