Prochelators with Metal-Dependent Antifungal Activity against Candida albicans

Abstract

Candidemia is second-most frequent hospital associated bloodstream infection in the United States. Current clinical antifungals are limited to a handful of classes. Concerned with increasing resistance, the World Health Organization (WHO) published its first fungal pathogen priority list, within which Candida albicans is listed as one of four species where there is urgent need for novel antifungal research.1 The most common causal agent of candidemia, C. albicans is a commensal fungus which colonizes the oral, intestinal, and genitals tracts that upon opportunity may cause infection. Azoles, a class of antifungals used to treat these infections, exert fungistatic activity by targeting cell membrane biosynthesis, specifically through the inhibition of an iron-dependent cytochrome p450. C. albicans respond to azole pressure by altering its metal homeostasis, particularly of iron and copper. Evidence indicates that azole antifungal activity can be potentiated using iron chelators. Iron is an essential trace metal required for DNA synthesis and cellular respiration. Similar to synthetic-iron chelation, the immune system sequesters iron from invading pathogens, an approach termed nutritional immunity. Employing synthetic chelators to mimic nutritional immunity could be a compelling strategy to improve azole efficacy. However, current chelators indiscriminately chelate iron from the host and pathogen. A solution to this could include using prochelators that cannot bind iron until activation by the infectious microenvironment.This work investigates the use of reductively-activated prochelators to inhibit C. albicans growth and improve azole efficacy. These prochelators, previously studied for anticancer activity, contain aroylhydrazone and thiosemicarbazone chemical moieties with (S,N,O) and (S,N,S) donor atoms. Linking two together via a disulfide bond blocks one of the donor atoms and prevents the prochelator from binding iron. Using cellular growth assays combined with spectrometric and spectroscopic analyses, I identify two prochelators, (AH1-S)2 and (IT1-S)2, that transiently inhibit C. albicans growth in both a standard laboratory strain and in a clinical isolate. The prochelators modulate azole activity against C. albicans in a non-linear concentration-dependent manner but have mixed outcomes in a macrophage co-culture setting. Investigation into the stability of the compounds under cellular assay conditions revealed the (IT1-S)2 is stable whereas (AH1-S)2 is susceptible to media-induced degradation in a light-sensitive manner. Spectroscopic and spectrometric analyses revealed that (AH1-S)2 prevents bulk iron accumulation under fluconazole stress through formation of an iron-chelator complex. (AH1-S)2 also demonstrates copper-dependent activity where non-toxic copper levels transform the fungistatic prochelator into a fungicide, likely through a mechanism of copper accumulation. Indeed, (AH1-S)2 and (IT1-S)2 complex with copper in buffer and cellular medium. A [Cu(IT1-S)2]PF6 complex was synthesized with an apparent logarithmic binding constant of 18.1  0.1. Collectively, the data collected herein employs a prochelation strategy against a fungal pathogen that targets metal-dependent vulnerabilities to modulate azole efficacy.