A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening

Getz, Rachel A.; Kwak, Grace; Cornell, Stacie; Mbugua, Samuel; Eberhard, Jeremy; Huang, Sheng Xiang; Abbasi, Zainab; Medeiros, Ana Santos de; Thomas, Richard K.; Bukowski, Brett; Dranchak, Patricia; Inglese, James; Hoffman, Charles S.

doi:10.1016/j.cellsig.2019.04.010

Cited by 7 publications

(3 citation statements)

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“…The E. coli lacZ gene encodes β -galactosidase, which can be readily measured using a colorimetric assay on protein extracts ( Hoffman and Winston, 1990 ). The Aequorea victoria green fluorescent protein (GFP) gene encodes a protein that can be assessed by fluorescence spectroscopy, fluorescence microscopy, or fluorescence scanning using live cells ( de Medeiros et al, 2013 ; Getz et al, 2019 ). The S. pombe ura4 gene encodes OMP decarboxylase, an enzyme acting in the uracil biosynthetic pathway.…”

Section: Yeast-based Phenotypes Associated With Camp Signalingmentioning

confidence: 99%

“…These include the length of cells at the time of forming a fission plate, which immediately precedes cytokinesis, the ability to grow in the presence of 1M KCl, mating competence, and the ability to successfully enter stationary phase ( Table 1 ). This wealth of phenotypes, together with the fact that PKA activity is not essential for S. pombe cell viability, has allowed us to construct a large collection of strains that express mammalian PDEs, adenylyl cyclases, and the human GNAS G α S that stimulates the nine mammalian transmembrane adenylyl cyclases ( Ivey et al, 2008 ; Alaamery et al, 2010 ; Demirbas et al, 2011a ; Demirbas et al, 2011b ; Ceyhan et al, 2012 ; Demirbas et al, 2013 ; de Medeiros and Hoffman, 2015 ; Getz et al, 2019 ).…”

Section: Yeast-based Phenotypes Associated With Camp Signalingmentioning

confidence: 99%

“…We have since expressed PDE11A4 from the more active adh1 promoter ( Russell and Hall, 1983 ). The increase in PDE activity allows us to conduct 5FOA assays in a strain that produces cAMP from a heterologously-expressed mammalian adenylyl cyclase ( Getz et al, 2019 ), thus avoiding the need for exogenous cyclic nucleotides in the 5FOA medium. One compound from the HTS, BC11-38, elevates cAMP levels, PKA activity and cortisol release by H295R adrenocortical cells.…”

Section: Fission Yeast-based Pde Inhibitor Screensmentioning

confidence: 99%

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Use of a Fission Yeast Platform to Identify and Characterize Small Molecule PDE Inhibitors

Hoffman

2022

Front. Pharmacol.

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Cyclic nucleotide phosphodiesterases (PDEs) have been proven to be targets for which highly selective and potent drugs can be developed. Mammalian genomes possess 21 genes whose products are pharmacologically grouped into 11 families; however related genes from pathogenic organisms display sufficient divergence from the mammalian homologs such that PDE inhibitors to these enzymes could be used to treat parasitic infections without acting on the related human PDEs. We have developed a platform for expressing cloned PDEs in the fission yeast Schizosaccharomyces pombe, allowing for inexpensive, but robust screening for small molecule inhibitors that are cell permeable. Such compounds typically display the expected biological activity when tested in cell culture, including anti-inflammatory properties for PDE4 and PDE7 inhibitors. The genetic pliability of S. pombe also allows for molecular genetic screens to identify mutations in target PDE genes that confer some resistance to these inhibitors as a way of investigating the PDE-inhibitor interaction. This screening method is readily accessible to academic laboratories as it does not require the purification of large quantities of a target protein. This allows for the discovery and profiling of PDE inhibitors to treat inflammation or of inhibitors of targets such as pathogen PDEs for which there may not be a sufficient financial motivation for pharmaceutical companies to identify selective PDE inhibitors using more traditional in vitro enzyme-based screening methods.

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Section: Yeast-based Phenotypes Associated With Camp Signalingmentioning

confidence: 99%