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The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans.

Publication ,  Journal Article
Maybruck, BT; Lam, WC; Specht, CA; Ilagan, MXG; Donlin, MJ; Lodge, JK
Published in: mBio
December 17, 2019

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformansIMPORTANCECryptococcus neoformans is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents.

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Published In

mBio

DOI

EISSN

2150-7511

Publication Date

December 17, 2019

Volume

10

Issue

6

Location

United States

Related Subject Headings

  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • Morpholines
  • Molecular Structure
  • Models, Biological
  • Indomethacin
  • Drug Discovery
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclic AMP
  • Cryptococcus neoformans
 

Citation

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Maybruck, B. T., Lam, W. C., Specht, C. A., Ilagan, M. X. G., Donlin, M. J., & Lodge, J. K. (2019). The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans. MBio, 10(6). https://doi.org/10.1128/mBio.02264-19
Maybruck, Brian T., Woei C. Lam, Charles A. Specht, Ma Xenia G. Ilagan, Maureen J. Donlin, and Jennifer K. Lodge. “The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans.MBio 10, no. 6 (December 17, 2019). https://doi.org/10.1128/mBio.02264-19.
Maybruck, Brian T., et al. “The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans.MBio, vol. 10, no. 6, Dec. 2019. Pubmed, doi:10.1128/mBio.02264-19.

Published In

mBio

DOI

EISSN

2150-7511

Publication Date

December 17, 2019

Volume

10

Issue

6

Location

United States

Related Subject Headings

  • Signal Transduction
  • Receptors, G-Protein-Coupled
  • Morpholines
  • Molecular Structure
  • Models, Biological
  • Indomethacin
  • Drug Discovery
  • Cyclic AMP-Dependent Protein Kinases
  • Cyclic AMP
  • Cryptococcus neoformans