Unveiling the Antifungal Mechanisms of CTP, a New Copper(II)- Theophylline/1,10-Phenanthroline Complex, on Drug-Resistant Non-albicans Candida Species

Candida species are the commonest opportunistic human fungal pathogens worldwide, with Candida albicans serving as the primary representative. However, non- albicans Candida species (NACs) have been on the rise in terms of incidence, and they are associated with outbreaks and instances of antifungal resistance. The search for new strategies to combat antifungal-resistant Candida strains is of paramount importance. Recently, our group reported the anti- Candida activity of a coordination compound containing copper(II) complexed with theophylline (theo) and 1,10-phenanthroline (phen), known as “CTP” – Cu(theo) ₂ phen(H ₂ O).5H ₂ O. In the present work, we investigated the mechanisms of action of CTP against six medically relevant, antifungal-resistant NACs, including C. auris , C. glabrata , C. haemulonii , C. krusei , C. parapsilosis and C. tropicalis . CTP effectively inhibited the activity of mitochondrial dehydrogenases, leading to an increase in intracellular reactive oxygen species production. CTP treatment resulted in significant damage to the plasma membrane, as evidenced by the passive incorporation of propidium iodide, and DNA fragmentation as evidenced by the TUNEL assay. Scanning electron microscopy images confirmed that the fungal surface underwent substantial alterations due to CTP treatment, including the formation of invaginations that led to cavitations and lysis. These surface modifications significantly impacted the ability of Candida cells to adhere to the polystyrene surface and form a robust biofilm structure. Moreover, CTP was effective in disassembling mature biofilms formed by these NACs. CTP represents a promising avenue for the development of novel antifungals with innovative mechanisms of action against clinically relevant NACs that are resistant to antifungals commonly used in clinical settings.