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High-throughput screening for protein tyrosine phosphatase activity modulators.

ABSTRACT: Reversible phosphorylation of proteins, principally on serine, threonine, or tyrosine residues, is central to the regulation of most aspects of eukaryotic cell function. Dysregulation of protein kinases and protein phosphatases is linked to numerous human diseases. Consequently, many efforts have been made to target these enzymes with small molecules in order to develop new therapeutic agents. While protein kinase inhibitors have been successfully brought to the market, the development of specific protein phosphatase inhibitors is still in its infancy. The largest and most diverse protein phosphatase superfamily in humans is comprised by the protein tyrosine phosphatases, a group of over 100 enzymes. Here, we describe high-throughput screening methods to search for protein tyrosine phosphatase activity modulators. We illustrate the implementation of relatively simple phosphatase assays, using generic absorbance- or fluorescence-based substrates, in 384- or 1536-well microtiter plates. We discuss steps to optimize HTS assay quality and performance, and describe several PTP screening methods on the basis of previously performed successful HTS campaigns. Finally, we discuss how to confirm, follow up, and prioritize hit compounds, and point out a number of common pitfalls that are encountered in this process.

SUBMITTER: Tautz L

PROVIDER: S-EPMC8148610 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Previous studies from our laboratory and others have demonstrated that in addition to altering chromatin acetylation and conformation, histone deacetylase inhibitors (HDACi) disrupt the acetylation status of numerous transcription factors and other proteins. A whole genome yeast deletion library screen was used to identify components of the transcriptional apparatus that modulate the sensitivity to the hydroxamic acid-based HDACi, CG-1521. RESULTS: Screening 4852 haploid Saccharomyces cerevisiae deletion strains for sensitivity to CG-1521 identifies 407 sensitive and 80 resistant strains. Gene ontology (GO) enrichment analysis shows that strains sensitive to CG-1521 are highly enriched in processes regulating chromatin remodeling and transcription as well as other ontologies, including vacuolar acidification and vesicle-mediated transport. CG-1521-resistant strains include those deficient in the regulation of transcription and tRNA modification. Components of the SAGA histone acetyltransferase (HAT) complex are overrepresented in the sensitive strains, including the catalytic subunit, Gcn5. Cell cycle analysis indicates that both the wild-type and gcn5? strains show a G1 delay after CG-1521 treatment, however the gcn5? strain displays increased sensitivity to CG-1521-induced cell death compared to the wild-type strain. To test whether the enzymatic activity of Gcn5 is necessary in the response to CG-1521, growth assays with a yeast strain expressing a catalytically inactive variant of the Gcn5 protein were performed and the results show that this strain is less sensitive to CG-1521 than the gcn5? strain. CONCLUSION: Genome-wide deletion mutant screening identifies biological processes that affect the sensitivity to the HDAC inhibitor CG-1521, including transcription and chromatin remodeling. This study illuminates the pathways involved in the response to CG-1521 in yeast and provides incentives to understand the mechanisms of HDAC inhibitors in cancer cells. The data presented here demonstrate that components of the SAGA complex are involved in mediating the response to CG-1521. Additional experiments suggest that functions other than the acetyltransferase activity of Gcn5 may be sufficient to attenuate the effects of CG-1521 on cell growth.

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High-throughput screening for protein tyrosine phosphatase activity modulators.

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