T SS needle protein inhibitors for the treatment of P aeruginosa infection

DESCRIPTION provided by applicant The inability to treat many Gram negative bacterial infections effectively with existing antibiotics is a major medical crisis Pseudomonas aeruginosa is a prime example of clinical isolates from critically ill patients are resistant to three or
more drugs The overall goal of this project is to address the critical medical need by a novel approach of identifying specific inhibitors of the type three secretion system T SS targeting the extracellular T SS needle and developing them into novel therapeutic agents against P aeruginosa T SS is the major virulence factor contributing to the establishment and dissemination of P aeruginosa infections and is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes and weaken the hostandapos s innate immune response The presence of a functional T SS is significantly associated with poor clinical outcomes and death in patients and markedly reduces survival in animal infection models T SS inhibitors will be administered therapeutically and prophylactically in combination with anti pseudomonal agents to inhibit the T SS potentiate a robust host innate immune response and enhance the antibacterial activity of co administered antibiotics The strategy is to identify and optimize small molecules that interfere with the extracellular T SS needle polymerization or stability Such therapeutics will by pass P aeruginosa intrinsic resistance mechanisms caused by a poorly permeable outer membrane and efflux pumps Preliminary studies revealed a putative binding site for the phenoxyacetamide series of T SS inhibitors in the polymeric form of the needle protein PscF indicating that the needle is a target of the P aeruginosa T SS for small molecule inhibition The strategy of screening directly for compounds that alter the needle assembly or stability will capitalize on this newfound vulnerability and provide additional chemotypes of needle inhibitors for the drug development pipeline In other preliminary studies we developed methods for the purification of PscF and demonstrated a fluorescence based assay to monitor the polymerization of a purified T SS needle protein In Phase I a high throughput screen using purified PscF will be developed optimized and implemented to identify small molecules that inhibit needle polymerization or stability Diverse compound libraries will be
screened and resulting andapos hitsandapos will be confirmed in the screening assay in replicate prioritized b potency and selectivity by eliminating compounds that alter actin polymerization or stability or are promiscuous in multiple screens Confirmed potent selective andapos hitsandapos will be validated as T SS inhibitors by determining their ability to inhibit effector secretion and translocation from P aeruginosa and by ensuring that they are not cytotoxic do not disrupt mammalian cell membranes and do not affect bacterial growth or viability in vitro Preliminary SAR and in vitro ADME assays and will be used to prioritize analogs In Phase II the most promising of these T SS inhibitors will be optimized to develop lead compounds for efficacy and toxicity testing in animal models PUBLIC HEALTH RELEVANCE The increasing prevalence of antibiotic resistant strains of Pseudomonas aeruginosa is dire threat to the health of infected patients and represents an unmet medical need These bacteria are resistant to many drugs because of a thick outer membrane and efflux pumps and they weaken the patientandapos s innate immune cellular response by injecting toxins into the cells through a type three secretion system T SS Successful development of an inhibitor targeting the extracellular needle of this T SS virulence mechanism will provide a new weapon to combat acute infections such as pneumonia and bacteremia