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

Highlights

• Screening of inhibitors against intracellular survival of Cryptococcus neoformans is presented.

• Ca²⁺ channel blocker fendiline hydrochloride is identified as a potential candidate.

• Fendiline triggers phagosomal acidification and intracellular fungal killing.

• Mechanistic studies reveal intracellular calcium rise upon drug treatment.

• Fendiline may be a promising drug scaffold for anticryptococcal therapy.

Abstract

The fungal pathogen Cryptococcus neoformans poses a major threat to immunocompromised patients and is a leading killer of human immunodeficiency virus (HIV)-infected patients worldwide. Cryptococci are known to manipulate host macrophages and can either remain latent or proliferate intracellularly within the host phagocyte, a favourable niche that 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 C. neoformans. The Prestwick Chemical Library^® of FDA-approved small molecules was screened for compounds that limit the intracellular replication of a fluorescently-tagged C. neoformans reference strain (H99-GFP) in macrophages. Preliminary screening revealed 19 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, this study highlights a novel approach for identifying new lead compounds and unravels a pharmacologically promising scaffold towards the development of novel antifungal therapies for this neglected disease.