Chemical probes for imaging and treatment of otitis media

Project Summary Otitis media (OM), or infection of the middle ear, is the second most common illness diagnosed in children in the United States. It results in greater than $2 billion in costs per year and is responsible for 24% of all pediatric antibiotic prescriptions. Acute OM is most commonly caused by the bacteria Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus, with Pseudomonas aeruginosa being the most common cause of chronic OM. Diagnosis of OM currently relies on otoscopy, which has low sensitivity and specificity and is unable to differentiate bacterial infection from viral infection or other causes of inflammation. These limitations result in over-diagnosis and over-prescription of antibiotics which contributes to growing antimicrobial resistance (AMR) and subsequent treatment failures. Therefore, there is a significant unmet need for improved diagnostic tools for OM that can enable faster and more effective treatment decisions while also reducing the potential for AMR development. We propose to address these current shortcomings in OM diagnosis and treatment by developing chemical probes that will enable initial imaging to confirm an infection and then immediate treatment using phototherapy, thus avoiding the use of antibiotics. We will focus on the bacterial D,D-carboxypeptidases (DD-CPases), a class of proteases involved in processing of glycopeptides in the cell membrane to facilitate formation of the bacterial cell wall. These proteases are ideal imaging targets as they are active and present within the outer cell membrane, can be targeted with small molecule inhibitors that form covalent bonds to the active site and exist only in bacteria. Our central hypothesis is that a probe carrying a fluorescent phototherapy dye that covalently labels the bacterial DD-CPases of the five major OM-causing bacteria will enable specific detection of OM by fluorescence imaging and then effective treatment through simple and non-toxic light exposure. In support of this hypothesis, all five common OM- causing bacteria have a DD-CPase that lack human homologs and have conserved actives sites suitable for covalent inhibition. Moreover, recent evidence suggest that PDT can be used as an effective antibacterial treatment strategy. Therefore, we will accomplish our goal of identifying a novel diagnostic and treatment strategy for OM through completion of the following primary aims: Aim 1: Develop a covalent active site probe that targets the DD-CPases of the five primary OM-causing bacteria. Aim 2: Demonstrate that a single covalent probe can be used to image and kill the five most common OM-causing bacteria in vitro. Aim 3: Use validated probes from in vitro studies to confirm imaging and clearance of each of the five common OM-causing bacteria in a mouse model of OM.