The Inhibitor Testing Core will assess inhibitors from the three Projects for antifungal activity. These assessments will follow an algorithm of highly standardized in vitro screens, followed by cell-based toxicity and murine maximum tolerated dose studies and a prioritized strategy of rigorous in vivo studies. In this way, the Core will help Project investigators identify those inhibitors with the highest translational potential.
- Test novel inhibitors for in vitro antifungal activity. Initially, we will perform standardized, highthroughput 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.
- Analyze mammalian toxicity profile of active novel inhibitors. 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.
- Test inhibitors for efficacy in animal models of the major invasive fungal pathogens. Inhibitors with in vitro activity, and acceptable toxicity, will be assessed in vivo using targeted animal models of the 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 and mucormycosis.
Synergies with Other Projects and Cores
The Inhibitor Testing Core will conduct detailed pre-clinical in vitro and in vivo evaluation of the novel inhibitors arising from Projects 1, 2, and 3. The Core will unify all three Projects by using standard methods, materials, and facilities for a complete evaluation of the activity of the inhibitors contributed by each Project. Importantly, the Core will promote synergy among the distinct Projects by analyzing for additive activity of combinations of active inhibitors generated from the three Projects. Results of inhibitor testing will be interpreted in regular meetings with each Project Leader, thereby interfacing each Project directly with Core leadership, and facilitating any possible additional chemical modifications needed.
The Core will provide exceptional value to the overall Program Project. First, it will efficiently test for in vitro and in vivo activity in one facility, allowing each Project team to focus on their particular biological pathway. Additionally, the combined Core personnel will provide practical expertise in each fungal pathogen. This will result in an independent assessment of each Project’s compounds for real translational potential, rather than theoretical antifungal effects. Outside of this comprehensive Core and the synergistic Program Project grant, limited resources and narrow focus would not allow the evaluation of combinations of novel inhibitors, nor the assessment of inhibitor efficacy/toxicity in relevant animal models of four separate invasive fungal diseases. Therefore, this Core provides essential support for true inter-Project synergy.