Invasive fungal infections are a leading cause of death in immunocompromised patients. Translating molecular understanding into clinical benefit is difficult because fungal pathogens and their hosts have similar eukaryotic physiology. As a result, current antifungals have limited clinical efficacy, are poorly fungicidal in the host, are in some cases toxic, and are increasingly ineffective due to emerging drug resistance. Over the last two decades, through genetic and pharmacologic approaches we established that calcineurin (CN) phosphatase is a key determinant for invasive fungal disease and a potential target for antifungal drug development. The CN inhibitor FK506 significantly inhibits fungal CN but is also immunosuppressive in the host and not fungal-selective. Our overall objective is to leverage our pathogenic fungal CN-FK506-FKBP12 complex X-ray structures for structure- guided design of broad-spectrum non-immunosuppressive FK506 analogs using complementary medicinal chemistry and combinatorial biosynthesis to develop a novel paradigm of fungal-specific antifungals. Our central hypothesis is that a structure-based approach to design antifungals using molecular modeling, NMR dynamics, and molecular dynamic (MD) simulations for specific targeting of fungal CN will lead to improved therapeutic intervention. For a broader treatment perspective, we will focus on the major and newly recalcitrant clinical fungal pathogens: Aspergillus fumigatus, Candida albicans, Candida auris, Cryptococcus neoformans, and Rhizopus oryzae. In Aim 1, we will synthesize FK506/FK520 analogs with chemical modifications at the C21 and C22 positions, and at other structurally relevant positions (C9, C31) by combinatorial biosynthetic and synthetic strategies. The C21 and C22 residues will be modified using different starter molecules through single-step reactions. In parallel, we will employ combinatorial biosynthetic approach to produce modified FK506 analogs through genetic manipulation of Streptomyces species, the natural producer of FK506. Analogs with high affinity to form fungal CN-FKBP12 complexes will be screened for antifungal activity. In Aim 2, we will define selective determinants of fungal CN inhibition with our pathogenic fungal CN ternary complex X-ray structures, coupled with NMR-based inhibitor binding dynamics and MD simulations to selectively define the inhibitor interactions that differentiate fungal and human CN-FK506-FKBP12 complex formation. Quantitative mapping of protein ligand interactions, together with genetic mutational analyses, will enable design of optimized and more selective analogs that minimize mammalian immunosuppression and enhance antifungal activity. In Aim 3, we will test analogs for additional in vitro antifungal activity and define activity on mammalian CN through primary murine T cell activation assays. Analogs that exhibit promising antifungal activity and reduced immunosuppression will be tested for efficacy in murine models of invasive candidiasis, aspergillosis, and cryptococcosis. This work capitalizes on our structural biology and NMR dynamics experience to design and synthesize novel fungal- specific calcineurin inhibitors as a unique antifungal approach that are also active against drug-resistant isolates.

R56AI112595

2014-08-04

2021-08-31