Atividade antivirulência do composto de coordenação CTP, [cobre(teofilina)2(1,10-fenantrolina)(H2O).5H2O], sobre espécies de Candida não-albicans: foco na inibição de aspártico peptidases secretadas

Abstract

The rise of infections caused by Candida non-albicans species resistant to conventional antifungals, particularly in immunocompromised patients, highlights the urgent need for novel therapeutic strategies. Secreted aspartic peptidases (Saps) play key roles in fungal pathogenesis by facilitating adhesion, tissue damage, and immune evasion, making them promising drug targets. Coordination compounds involving transition metals and biologically active organic molecules, such as CTP [copper(theophylline)₂(1,10-phenanthroline)(H₂O).5H₂O], have shown potential against Candida spp. This study investigated the antivirulence activity of CTP against Saps from six Candida non-albicans species using in silico, in vitro, and in vivo approaches. Clinical isolates were cultured in YCB medium supplemented with bovine serum albumin (BSA) to induce Sap production. Significant BSA consumption was dose-dependently inhibited by CTP (IC₅₀: 40.5-70.7 µM) without fungicidal effects, suggesting direct interference with Sap activity. Agar-albumin plate assays demonstrated that pre-treatment of yeast cells with CTP (100-12.5 µM) significantly reduced Sap secretion, as evidenced by lower enzymatic activity compared to untreated cells. Flow cytometry experiments confirmed that CTP significantly decreased the expression of Sap homologs, reducing both the percentage of labeled cells and fluorescence intensity. Sap enzymatic activity was measured using a fluorogenic peptide substrate specific for aspartic peptidases in cell-free, Sap-rich supernatants. Results showed that Saps degraded the substrate with values ranging from 267.17 to 476.75 arbitrary fluorescence units (AFU). In the presence of CTP, proteolytic activity was dose-dependently inhibited (IC₅₀: 6.82-8.5 µM), similar to pepstatin A inhibition (IC₅₀: 4.62-6.85 µM). Molecular docking studies indicated that CTP effectively binds to the catalytic site of Candida Saps, forming an enzymeinhibitor complex with binding energy values ranging from -7.9 to -9.1 kcal/mol. In in vitro and in vivo assays, concentrated fungal supernatants exhibited high enzymatic activity (779.3-1041.7 AFU), which was completely inhibited by CTP and pepstatin A. CTP showed no cytotoxicity up to 20 µM (CC₅₀: 53.66 µM) and reversed the cytotoxic effects induced by C. auris, C. albicans, and C. parapsilosis supernatants. In Galleria mellonella models, fungal supernatants significantly increased larval mortality (30-70%), an effect eliminated by preheating or treatment with CTP and pepstatin A (10 µM), ensuring total larval survival. In infection assays, larval mortality (50-90%) was reduced by CTP, with greater efficacy at higher concentrations (2-5×MIC). These findings highlight CTP as a promising candidate for antivirulence therapies against Candida species resistant to conventional antifungals.