Exploiting the vulnerable active site of a copper-only superoxide dismutase to disrupt fungal pathogenesis

Natalie G Robinett,Edward M Culbertson,Ryan L Peterson,Hiram Sanchez,David R Andes,Jeniel E Nett,Valeria C Culotta

doi:10.1074/jbc.ra118.007095

Natalie G Robinett, Edward M Culbertson + Show 5 more

Open Access

https://doi.org/10.1074/jbc.ra118.007095

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Abstract

Copper-only superoxide dismutases (SODs) represent a new class of SOD enzymes that are exclusively extracellular and unique to fungi and oomycetes. These SODs are essential for virulence of fungal pathogens in pulmonary and disseminated infections, and we show here an additional role for copper-only SODs in promoting survival of fungal biofilms. The opportunistic fungal pathogen Candida albicans expresses three copper-only SODs, and deletion of one of them, SOD5, eradicated candidal biofilms on venous catheters in a rodent model. Fungal copper-only SODs harbor an irregular active site that, unlike their Cu,Zn-SOD counterparts, contains a copper co-factor unusually open to solvent and lacks zinc for stabilizing copper binding, making fungal copper-only SODs highly vulnerable to metal chelators. We found that unlike mammalian Cu,Zn-SOD1, C. albicans SOD5 indeed rapidly loses its copper to metal chelators such as EDTA, and binding constants for Cu(II) predict that copper-only SOD5 has a much lower affinity for copper than does Cu,Zn-SOD1. We screened compounds with a variety of indications and identified several metal-binding compounds, including the ionophore pyrithione zinc (PZ), that effectively inhibit C. albicans SOD5 but not mammalian Cu,Zn-SOD1. We observed that PZ both acts as an ionophore that promotes uptake of toxic metals and inhibits copper-only SODs. The pros and cons of a vulnerable active site for copper-only SODs and the possible exploitation of this vulnerability in antifungal drug design are discussed.

Highlights

Copper-only superoxide dismutases (SODs) represent a new class of SOD enzymes that are exclusively extracellular and unique to fungi and oomycetes
We find that SOD5 lost over 50% of its copper to pyrithione zinc (PZ) following only 2 h dialysis, whereas Cu,Zn-SOD1 was refractory to copper loss in the presence of PZ (Fig. 6C)
We describe a potential new drug target worthy of consideration: the extracellular copper-only superoxide dismutases that are important for fungal pathogenesis, unique to the fungal kingdom, and selectively targetable by small molecules, leaving their highly conserved mammalian Cu,Zn-SOD counterparts unperturbed

Summary

Results

Life-threatening Candida bloodstream infections are often seeded by fungus-colonized implanted devices such as intravenous catheters, which account for between 25 and 40% of hospital-acquired candidemia infections (36 –40). Our studies with Cu(II) binding to copper-only SOD5 suggested that this enzyme with its unusual open active site might be hypersensitive to inhibition by metal chelators or other selective small molecules. To this end, we optimized a highthroughput assay for SOD activity based on the water-soluble tetrazolium salt WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)5-(2,4-disulfo-phenyl)-2H-tetrazolium, monosodium salt) [46, 47]. In addition to CQ, pyrithione zinc (PZ), ciclopirox (CP), and chloroxine (CX) were identified as selective inhibitors against copper-only SOD5 versus Cu,Zn-SOD1 (Fig. 5, A–E) These compounds, together with chelators from our targeted approach, were further validated and analyzed to determine IC50 values (Table 2). Our studies implicate a third property of such agents: enzyme inactivation through metal chelation

Discussion

Experimental procedures

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