This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features!
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log in
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 May 2;85(10):e02923-18.
doi: 10.1128/AEM.02923-18. Print 2019 May 15.

Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi

Affiliations

Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi

Asumi Sugahara et al. Appl Environ Microbiol. .

Abstract

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 three-dimensional (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.IMPORTANCE A candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified by in silico screening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed in Saccharomyces cerevisiae Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison with S. cerevisiae PKC. Analysis of the AGS1 mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungus Magnaporthe grisea revealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization in M. grisea, and this melanization, which is required for pathogenicity of M. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of 4HNR, 3HNR, and SCD1, which are essential for melanization in M. grisea, were suppressed by both PKC inhibitors.

Keywords: PKC inhibitor; cell wall integrity signaling; filamentous fungi; in silico screening; protein kinase C.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Chemical structure of Z-705.
FIG 2
FIG 2
In silico screening of fungal PKC inhibitors. (A) Alignment of the amino acid sequences of MgPkc1p and human PKCθ. Asterisk, identical residue; colon, highly similar residue; period, weakly similar residue; hyphen, deletion. (B) Three-dimensional model with ligands docked to MgPkc1p. Red, α-helix; cyan, β-sheet; white, random structure. Magenta sticks indicate seven ligands. Ligands bind along the right antiparallel β-sheet.
FIG 3
FIG 3
Sensitivity of the S. cerevisiae wild-type and yeast-filamentous fungal chimeric PKC (YF-PKC) strains to Z-705. (A) Construction of YF-PKC cassettes. KanMX, Geneticin resistance marker. (B) Sensitivity to Z-705 of the S. cerevisiae wild-type (WT; strain BY4741) and YF-PKC strains (SC, MG, and AN) on plate cultures. Serial 10-fold dilutions of cell suspensions were spotted onto synthetic galactose minimal medium (SG; Gal 1%) plates containing or not containing dimethyl sulfoxide (DMSO) or Z-705, and the plates were incubated at 30°C for 60 h. (C) Sensitivity to Z-705 of the S. cerevisiae wild-type and YF-PKC strains in liquid cultures. Cells were inoculated into SG liquid medium (Gal 1%) containing DMSO or Z-705 and incubated at 30°C for 96 h. In panel C, the optical density at 660 nm (OD660) was measured every 6 h (24 h to 96 h).
FIG 4
FIG 4
Inhibition of MLP1 mRNA level by Z-705. Induction of the MLP1 transcript by cell wall stress is inhibited by Z-705. The wild-type (WT; BY4741) and YF-PKC (SC, MG, and AN) strains were incubated in SG (Gal 1%) containing 5% DMSO or 15.5 μM Z-705 for 30 min. The mRNA levels were determined by quantitative reverse transcription-PCR (RT-PCR) using specific primers (Table 2). Each value represents the ratio of MLP1 expression relative to that of RPL28S in each strain. *, P < 0.05; **, P < 0.01 (Student's t test).
FIG 5
FIG 5
Inhibition of Mpk1p phosphorylation by Z-705 and STS. (A) Mpk1p phosphorylation is inhibited by Z-705. The wild-type (WT; BY4741) and YF-PKC (SC, MG, and AN) strains were incubated in yeast extract-peptone-dextrose (YPD) medium containing 1% DMSO (solvent for Z-705) or 7.77 μM Z-705 for 30 min and then transferred for 60 min to YPD medium containing 100 μg/ml Congo red (CR). Mpk1p phosphorylation was detected by immunoblotting with anti-phospho-p44/42 MAPK antibody; anti-Mpk1p antibody was used to detect Mpk1p regardless of phosphorylation (loading control); H2A, histone H2A. Ctrl indicates the standard sample, which was obtained from the WT treated with CR and used throughout this study. (B) Mpk1p phosphorylation is inhibited by STS. Cells were incubated in YPD medium containing 1% DMSO (solvent for STS) or 6.70 μM STS at 30°C for 30 min and then transferred for 60 min to YPD medium containing 100 μg/ml Congo red (CR). Mpk1p phosphorylation was detected as described above.
FIG 6
FIG 6
Inhibitory effects of Z-705 and STS on AGS1 gene expression and hyphal melanization in M. grisea, induced by cell wall stress. (A) Mycelia of the wild-type strain (Guy11) were incubated in complete medium (CM) containing 0 (−) or 0.01 (+) μg/ml MCFG (MCFG), with or without 1% DMSO or with or without the indicated concentration of Z-705 or STS for 24 h. AGS1 mRNA levels were determined by quantitative RT-PCR using specific primers (Table 2). (B) Melanization in the wild-type M. grisea Guy11 strain in liquid culture. Cells were cultured in CM in the absence or presence of 0.01 μg/ml MCFG, with or without 1% DMSO, or with 1% DMSO and the indicated concentrations of Z-705 or STS, for 24 h.
FIG 7
FIG 7
Expression of the melanin biosynthesis genes SCD1, 3HNR, and 4HNR in M. grisea. Mycelia of the wild-type strain (Guy11) were incubated in CM containing 0.01 μg/ml MCFG and 1% DMSO, without or with the indicated concentrations of Z-705 or STS for 24 h. The mRNA levels were determined by quantitative RT-PCR using specific primers (Table 2). Expression is shown relative to that of the ACTIN gene under each condition. *, P < 0.05; **, P < 0.01 (Tukey’s multiple-comparison test).
FIG 8
FIG 8
Model of cell wall integrity signaling in M. grisea. Pkc1 activates the Mps1 MAPK cascade, and the expression of the 4HNR, 3HNR, and SCD1 genes is regulated by the transcription factor Pig1 via Mps1 (31). 4HNR, 3HNR, and SCD1 are involved in melanin biosynthesis and are essential for infection (37). The expression of the AGS1 gene is predicted to be regulated by the transcription factor Rlm1 via Mps1. Ags1p synthesizes α-1,3-glucan, which acts as a stealth factor in M. grisea by blocking host recognition of fungal invasion and is required for fungal infection of live rice cells (22, 38). The fact that expression of both melanin and α-1,3-glucan biosynthetic genes is regulated by PKC in M. grisea suggests that PKC is directly or indirectly associated with the pathogenicity of this fungus (see details in the Discussion).

References

    1. Klis FM, Boorsma A, De Groot P. 2006. Cell wall construction in Saccharomyces cerevisiae. Yeast 23:185–202. doi:10.1002/yea.1349. - DOI - PubMed
    1. Latgé JP. 2010. Tasting the fungal cell wall. Cell Microbiol 12:863–872. doi:10.1111/j.1462-5822.2010.01474.x. - DOI - PubMed
    1. Yoshimi A, Miyazawa K, Abe K. 2016. Cell wall structure and biogenesis in Aspergillus species. Biosci Biotechnol Biochem 80:1700–1711. doi:10.1080/09168451.2016.1177446. - DOI - PubMed
    1. Mazu TK, Bricker BA, Flores-Rozas H, Ablordeppey SY. 2016. The mechanistic targets of antifungal agents: an overview. Mini Rev Med Chem 16:555–578. doi:10.2174/1389557516666160118112103. - DOI - PMC - PubMed
    1. Georgopapadakou NH, Tkacz JS. 1995. The fungal cell wall as a drug target. Trends Microbiol 3:98–104. doi:10.1016/S0966-842X(00)88890-3. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite

AltStyle によって変換されたページ (->オリジナル) /