Novel cell-based in vitro screen to identify small molecule inhibitors against intracellular replication of Cryptococcus neoformans in macrophages

The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of HIV patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche which also renders them relatively insensitive to antifungal agents. Here we report an attempt to address this limitation by using a fluorescence-based drug screening method to identify potential inhibitors of intracellular proliferation of Cryptococcus neoformans. We screened the Prestwick Chemical Library® of FDA-approved small molecules for compounds that limit the intracellular replication of a fluorescently-tagged Cryptococcus neoformans reference strain, H99-GFP in macrophages. Preliminary screening revealed 19 out of 1200 compounds that could significantly reduce intracellular growth of the pathogen. Secondary screening and host cell cytotoxicity assays highlighted fendiline hydrochloride as a potential drug candidate for the development of future anticryptococcal therapies. Live cell imaging demonstrated that this Ca2+ channel blocker strongly enhanced phagosome maturation in macrophages leading to improved fungal killing and reduced intracellular replication. Whilst the relatively high dose of fendiline hydrochloride required renders it unfit for clinical deployment against cryptococcosis, our study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards development of novel antifungal therapy for this neglected disease.