Characterizing the Physiochemical Properties of Copper Chelating Agents: an Effort Towards Understanding their Antifungal Activity

Abstract

<p>An increase in drug-resistant infections and the paucity of new antibiotics present a major world health problem. Cryptococcus neoformans (C. neoformans) is an opportunistic fungal pathogen responsible for life-threatening infections in immunocompromised individuals and occasionally in those with no known immune impairment. The Franz lab recently identified several copper (Cu) chelators containing O, S and O, N donor atoms that exhibit Cu-dependent antifungal activity against C. neoformans. Interestingly, the O, O analogs of these chelators do not exhibit anti-fungal activity. Here, using UV-visible spectroscopy the Cu(I) binding properties of these ligands are determined. The lipophilic properties of these ligands and their bis-Cu(II) complexes were also determined using the traditional shake flask method. Lipophilicity and binding studies indicate that ligands exhibiting Cu-dependent antifungal activity are able to bind Cu(II) with a binding affinity, Log K_(〖CuL〗_2)^', greater than 19 and they form hydrophobic bis-Cu(II) complexes. Further, inductively coupled plasma-mass spectrometry (ICP-MS) was used to analyze total metal content of C. neoformans fungal cells treated with these ligands and Cu(II). This analysis revealed that the ligands displaying antifungal activity increased Cu, zinc (Zn), and iron (Fe) levels in the fungal cells dramatically compared to the ligand or Cu only treatment. Lastly, a new group of linear and cyclic thiohydroxamic acids (O,S donor atoms) was screened for their effect on C. neoformans’ growth, in the presence and absence of Cu(II). These studies indicate that cyclic thiohydroxamic acids are able to elicit Cu-dependent antifungal activity opening the possibility of a new class of metallo-antifungals. Further initial attempts were made to understand the Cu(II) binding properties of these thiohydroxamic acids using calcein fluorescence competition assays. The results from this work suggest that small molecules, capable of binding Cu(II) to form hydrophobic complexes, can deliver Cu to fungal cells altering not only their Cu but also intracellular Zn and Fe levels. This hypothesis about Cu delivery agents sets the stage for future work in genome-wide approaches to probe how alteration in metal levels affects different biochemical pathways to induce Cu-dependent antifungal activity.</p>