PROJECT SUMMARY. Infections caused by the filamentous fungus Aspergillus fumigatus and related speciesare associated with significant morbidity and mortality despite contemporary antifungal drug therapies. Manyfactors contribute to poor treatment outcomes including the physiological state of the fungus at the site ofinfection and the global emergence of triazole drug resistant strains. One major regulatory mechanism used bythe fungus to progress disease and resist triazole drug activity is proteolytic activation of the transcriptionalregulator, SrbA. Activation of SrbA in vivo is absolutely required for fungal virulence and intrinsic triazole drugresistance, as null mutants of SrbA regulatory factors such as the fungal specific activating serine proteaseRbdB and E3 ubiquitin ligases (DSCs) are avirulent in animal models of invasive aspergillosis (IA) and havesignificant increases in triazole susceptibility. The objective of this proposal in response to RFA-AI-17-036 is toidentify small molecules that inhibit SrbA activation and develop them into advanced therapeutic candidateswith broad-spectrum activity against triazole resistant filamentous fungi. Potent inhibitors of the SrbA-dependent signaling pathway will be developed for clinical use as an adjunctive therapy in combination with atriazole antifungal agent that is used to treat IA. The adjunctive therapy is expected to provide severaladvantages over triazole monotherapy, including growth inhibition in hypoxic conditions and increasedantifungal activity of the triazole drug in both drug susceptible and drug resistant infections. As the SrbApathway is conserved among most human fungal pathogens, some of which are inherently azole resistant, weanticipate broad spectrum clinical utility beyond infections caused by A. fumigatus. Our approach leverages theavailability of well characterized protease and ligase inhibitor chemical libraries, both known druggable targetsin many disease settings, with the expertise of Microbiotix Inc. and the Cramer Laboratory at Dartmouth. TheR21 phase of this application will utilize high-throughput cell based screens of defined targeted small moleculelibraries to identify and confirm SrbA regulatory protease and/or ligase inhibitors and validate their antifungalactivity, pathway specificity, and mammalian toxicity of early hits and leads. In the R33 phase, validated hitswill be chemically optimized, validated, defined pharmacologically, determine mechanism of action, and finallyproceed to in vivo pharmacologic and toxicology profiling and antifungal efficacy in established murine modelsof invasive aspergillosis.