Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

• Published in: The Journal of biological chemistry Vol. 288; no. 8; pp. 5840 - 5848
• Main Authors: Huang, Weigang, Barrett, Matthew, Hajicek, Nicole, Hicks, Stephanie, Harden, T. Kendall, Sondek, John, Zhang, Qisheng
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22-02-2013; American Society for Biochemistry and Molecular Biology
• Subjects: Biological Assay - methods; Chemistry, Pharmaceutical - methods; Cyclic AMP - metabolism; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical - methods; Enzyme Inhibitors - pharmacology; HEK293 Cells; High-throughput Screening (HTS); Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Models, Biological; Models, Chemical; Phosphatidylinositol; Phospholipase C; Phospholipases - chemistry; Receptors, G-Protein-Coupled - metabolism; Signal Transduction; Small Molecule Libraries; Small Molecules; Substrate Specificity; Type C Phospholipases - antagonists & inhibitors; Type C Phospholipases - metabolism; Phosphatidylinositol; Small Molecules; High-throughput Screening (HTS); Inositol 1,4,5-Trisphosphate; Phospholipase C
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.422501

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.
Background: Phospholipase C (PLC) isozymes are increasingly attractive therapeutic targets; however, pharmacological modulators are lacking.
Results: A facile fluorescent high-throughput screen was developed and used to identify small molecule inhibitors of PLC activity.
Conclusion: The new assay is robust and suitable for the rapid discovery of novel PLC modulators.
Significance: This new methodology eliminates the major roadblock hampering the discovery of small molecule PLC inhibitors.