neoformans electron transport chain and suggest that the effect of microplusin on the growth of the fungi may be related to the damage of the classical respiratory chain, probably at the copper-containing complex IV. Although we cannot entirely discard the effects of Fe2+ on microplusin, our assumption that microplusin is preferentially a copper chelator is based on the fact that the four respiratory complexes that have iron as prosthetic groups or bound to the heme Protein Tyrosine Kinase inhibitor group remained functional, whereas complex IV, the only complex that has copper as a prosthetic group, was affected by microplusin. We also show that microplusin stimulated the alternative respiratory

pathway in C. neoformans, likely buy Trichostatin A to compensate for the damaged classical electron transport chain. The alternative pathway is not coupled to oxidative phosphorylation and ATP synthesis, and hence, energy production in microplusin-treated yeasts is likely to be deficient. However, uncoupled respiration helps the cells to manage reactive oxygen species production under stress conditions. Similar to complex IV, the assembly and functioning of other copper proteins, such as the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1), might also be compromised in microplusin-treated C. neoformans. Microplusin

at concentrations ≥3.12 μM clearly inhibited C. neoformans melanization as well as reduced laccase activity. This further suggests that the copper-chelating ability of microplusin may affect the loading of copper ions to laccase apoenzyme. In addition, we observed that copper supplementation of the medium prevented the inhibition of melanization by microplusin, according to 1 : 1 binding ratio

(Silva et al., 2009). A correct laccase metallation is reportedly crucial for its biological activity, as shown for the laccase produced by the avirulent Δvph1 mutant of C. neoformans. mafosfamide Defective vesicular acidification disrupts the insertion of copper cofactors into proteins, resulting in the inability of Δvph1 laccase to catalyze phenolic compounds to melanin (Erickson et al., 2001). As expected, addition of 1 mM of the copper chelator BCS to the medium abolished laccase activity not only in the Δvph1 mutant but also in the wild-type strain and copper supplementation, restored laccase activity as well as induced its transcription (Zhu et al., 2003). Therefore, these data support the hypothesis that microplusin sequesters copper and may affect the availability of this metal to copper-dependent enzymes, such as laccase. The microplusin concentrations that inhibited melanization (≥3.12 μM) also increased the autopolymerization of l-dopa. l-dopa autopolymerization is a process that occurs spontaneously by exposure to light (Mason, 1955). Microplusin probably stimulates the spontaneous autopolymerization of the products derived from l-dopa oxidation; however, this possible action did not interfere with its inhibitory effect on melanization of C.