Seeinstructions): The widespread occurrence of antibiotic resistance among bacteria is causing increasing concern as a major threat to public health. Current antibiotics cause death or growth arrest in the target bacteria. As a result, antibiotic use exerts strong selective pressure to favor antibiotic resistant strains. Novel antimicrobial reagents that suppress pathogen virulence without selecting for antibiotic resistance provide a promising alternative approach for treatment of infectious diseases. Group A Streptococcus (GAS) is an important human pathogen affecting millions of people globally each year. The streptokinase (SK) is a major GAS virulence factor that activates human plasminogen. In our previous studies, we have established the streptokinase/plasminogen interaction as a critical factor in GAS pathogenesis. We propose to take advantage of this observation and design novel antimicrobial reagents for the treatment of GAS infection. In the preliminary study, we has screened 55,000 small compounds for inhibitors of SK expression and 20 candidate hit compounds have been identified. In specific aim I, the candidate compounds will befurther characterized and optimized to generate proper SK expression inhibitors for studying the roles of SK in GAS virulence. An additional high throughput screen of up to 500,000 more small compounds will be performed by taking advantage of the NIH Molecular Libraries and Imaging roadmap initiative. In specific aim II, Global effects of candidate compounds on GAS gene expressionwill be studied to provide clues for identification of the targets. GAS two-component system FasBCAX that has been demonstrated to regulate SK expression in published reports will be tested as prime candidate targets. In specific aim III,a number of murine GAS infection models established in our preliminary studies will be used to elucidate the effects of candidate compounds on GAS virulence in vivo. Data collected from the above studies will further our understanding of the contribution of SK to GAS infection and identify small compounds that can inhibit GAS virulence. As a result, alternative approach to treat bacterial infection by interfering with GAS virulence without unduly introducing selection pressure for resistance can be exploredto supplement antibiotic treatment. RELEVANCE (Seeinstructions): Group A Streptococcus (GAS) is an important human pathogen infecting millions of people. Streptokinase (SK) is one of the major GAS virulence factors. The overall objective of this proposal is to screen for small compounds to inhibit SK expression and explore these compounds both as tools to study GAS pathogenesis and as novel therapeutic reagents for treating GAS infections to supplement antibiotic treatment.