Novel antifungal compound Z-705 specifically inhibits protein kinase C of filamentous fungi

The cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungus Aspergillus nidulans and in MgPkc1p in the rice blast fungus Magnaporthe grisea are lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried out in silico screening by threedimensional (3D) structural modeling and performed growth inhibition tests for M. grisea on agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed in Saccharomyces cerevisiae cells in which the kinase domain of native S. cerevisiae PKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis of MLP1, which encodes a downstream factor of PKC in S. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress in M. grisea. We discuss the relationships between PKC and melanin biosynthesis.