Inhibitor Testing Core

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ABSTRACT: Inhibitor Testing Core Rigorous testing of novel inhibitors to define their efficacy against invasive fungal infections is the crux of this translational research. The Inhibitor Testing Core will conduct detailed in vitro and in vivo evaluations of the novel inhibitors arising from all three Projects for antifungal activity. This will unify the Projects by utilizing standard methods, materials, and facilities to depict the full scope and activity of each inhibitor. Fungal species present several inherent challenges to complete and accurate inhibitor assessment, including distinct disease subcategories and emergence of antifungal resistance in some species. Comprehensive evaluation therefore requires a prioritized approach by investigators knowledgeable in the nuances of fungal biology, clinical disease states, and currently available antifungals, coupled with a background in the discovery and development of antifungal compounds and strategies. The Inhibitor Testing Core provides robust expertise to support inhibitor validation, as its leadership has evaluated hundreds of compounds in vitro and in vivo and helped bring several to clinical trials or market. We will initially test novel inhibitors for in vitro antifungal activity by performing standardized, high-throughput evaluations of antifungal susceptibility for inhibitors from each of the three Projects. Subsequently, inhibitors of particular interest will be tested in a prioritized manner against a broader array of clinically-relevant fungi, as well as in combination with other antifungal agents to assess for synergistic activity. These synergy tests will be performed with FDA-approved antifungal classes (polyenes, azoles, echinocandins, flucytosine), with immunosuppressants (calcineurin inhibitors, Tor inhibitors, purine biosynthesis inhibitors), and with selected pathway inhibitors from other Projects in this proposal. We will then analyze mammalian toxicity profile of active novel inhibitors. To accomplish this, we will define the in vitro toxicity of active inhibitors as measured by mammalian cell cytotoxicity using human red blood cell hemolysis, followed by human respiratory cells (A549), and human kidney cells (HEK293). Secondary evaluation will define animal toxicity, including a murine maximum tolerated dose and evaluation of acute and chronic murine toxicities. Lastly, we will test inhibitors for efficacy in animal models of the major invasive fungal pathogens. Inhibitors showing activity in vitro, and acceptable toxicity, will be assessed in vivo using targeted animal models of the three major invasive fungal infections. The choice of animal models will be guided by in vitro antifungal activity against the various fungal species. Testing will be also prioritized to begin with invasive candidiasis, followed by invasive aspergillosis, and then proceed to cryptococcal meningitis/pulmonary cryptococcosis. Invertebrate models of mucormycosis will be utilized for Project 1 inhibitors when applicable. Evaluating novel inhibitors via broad testing and in multiple animal models would not be possible outside of the Inhibitor Testing Core or between unconnected grants.