1,10-Phenanthroline-5,6-Dione-Based Compounds Are Effective in Disturbing Crucial Physiological Events of Phialophora verrucosa.

Phialophora verrucosa is a dematiaceous fungus able to cause chromoblastomycosis, phaeohyphomycosis and mycetoma. All these fungal diseases are extremely difficult to treat and often refractory to the current therapeutic approaches. Therefore, there is an urgent necessity to develop new antifungal agents to combat these mycoses. In this context, the aim of the present work was to investigate the effect of 1,10-phenanthroline-5,6-dione (phendione) and its metal-based derivatives [Ag(phendione)2]ClO4 = ([Ag(phendione)2]+) and [Cu(phendione)3](ClO4)2.4H2O = ([Cu(phendione)3]2+) on crucial physiological events of P. verrucosa conidial cells. Using the CLSI protocol, we have shown that phendione, [Ag(phendione)2]+ and [Cu(phendione)3]2+ were able to inhibit fungal proliferation, presenting MIC/IC50 values of 12.0/7.0, 4.0/2.4, and 5.0/1.8 µM, respectively. [Cu(phendione)3]2+ had fungicidal action and when combined with amphotericin B, both at sub-MIC (½ × MIC) concentrations, significantly reduced (~40%) the fungal growth. Cell morphology changes inflicted by phendione and its metal-based derivatives was corroborated by scanning electron microscopy, which revealed irreversible ultrastructural changes like surface invaginations, cell disruption and shrinkages. Furthermore, [Cu(phendione)3]2+ and [Ag(phendione)2]+ were able to inhibit metallopeptidase activity secreted by P. verrucosa conidia by approximately 85 and 40%, respectively. Ergosterol content was reduced (~50%) after the treatment of P. verrucosa conidial cells with both phendione and [Ag(phendione)2]+. To different degrees, all of the test compounds were able to disturb the P. verrucosa conidia-into-mycelia transformation. Phendione and its Ag+ and Cu2+ complexes may represent a promising new group of antimicrobial agents effective at inhibiting P. verrucosa growth and morphogenesis.

1,10-phenanthroline inhibits the metallopeptidase secreted by Phialophora verrucosa and modulates its growth, morphology and differentiation.

Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis.

Estudos de peptidases secretadas por Phialophora verrucosa: efeito de inibidores proteolíticos/agentes quelantes na biologia celular / Studies peptidases secreted by Phialophora verrucosa: effects of proteolytic inhibitors / chelating agents in cell biology

Phialophora verrucosa é um dos agentes etiológicos da cromoblastomicose, uma micose que afeta os tecidos cutâneos e subcutâneos. Esta doença é crônica, recidivante e de difícil tratamento. Vários estudos têm demonstrado que peptidases secretadas estão associadas a importantes processos biológicos. Neste trabalho, foi relatada a presença de atividade peptidásica extracelular em P. verrucosa. A peptidase secretada por este fungo apresentou ótima atividade em condições acídicas e à 37ºC. A atividade proteolítica foi completamente inibida por 1,10-Fenantrolina (1,10-Fen) e EGTA, sugerindo a presença de metalopeptidase em P. verrucosa. Zinco (Zn2+) foi o único metal capaz de estimular a atividade peptidásica, de forma dose dependente. Esta atividade enzimática gerou a hidrólise de proteínas do soro e de matriz extracelular como soro albumina humana (HSA), imunoglobulina G (IgG), fibrinogênio, colágeno (tipo I e IV), fibronectina e laminina. Queratina também foi eficientemente hidrolisada pela atividade peptidásica de P. verrucosa. Esses dados sugerem o envolvimento dessa enzima na disseminação fúngica e evasão do sistema imune do hospedeiro. O crescimento de P. verrucosa na presença de 1,10-Fen e seus derivados complexados a metais, como [Cu(fendio)3](ClO4)2•4H2O e [Ag(fendio)2]ClO4 (Fen-Cu2+ e Fen-Ag+, respectivamente) e a forma livre de metal (Fendio) foi também testado. 1,10-Fen foi o composto mais efetivo na inibição da proliferação fúngica. Fen-Cu2+, Fen-Ag+ e Fendio também apresentaram intensa atividade antimicrobiano. O complexo Cu2+ e Fendio foram fungicidas, enquanto Fen-Ag+ demonstrou atividade fungistática. Conídios tratados com 1,10-Fen e seus derivados apresentaram importantes alterações morfológicas, indicativas de morte celular, como determinado por microscopia eletrônica de transmissão. Além disto, Fen-Cu2+ e Fen-Ag+ foram capazes de inibir a atividade proteolítica, enquanto Fendio não afetou a hidrólise do substrato proteico. 1,10-Fen e Fen-Cu2+ inibiram a filamentação de P. verrucosa. No entanto, Fen-Ag+ foi capaz de inibir a produção de esterol por este fungo. Os resultados também sugerem um efeito antifúngico sinérgico entre Fen-Cu2+ e anfotericina B (AMB). Nossos dados suportam a hipótese de que inibidores de metalopeptidases/agentes quelantes apresentam potencial para serem utilizados em modelos futuros para o tratamento da cromoblastomicose.