Development of a high-throughput screening system targeting the protein-protein interactions between PRL and CNNM

• Published in: Scientific reports Vol. 14; no. 1; pp. 25432 - 13
• Main Authors: Funato, Yosuke, Mimura, Mai, Nunomura, Kazuto, Lin, Bangzhong, Fujii, Shintarou, Haruta, Junichi, Miki, Hiroaki
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 25.10.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 631/154; 631/45; Animals; Antineoplastic drugs; Compound screening; Cyclin M (CNNM); Drug development; Drug interaction; Drug metabolism; Drug screening; Energy transfer; Fluorescence resonance energy transfer; Fluorescence Resonance Energy Transfer - methods; HEK293 Cells; High-throughput screening; High-Throughput Screening Assays - methods; Homogenous time-resolved fluorescence resonance energy transfer (HTR-FRET; HTRF; Humanities and Social Sciences; Humans; Magnesium; Magnesium - metabolism; Membrane proteins; multidisciplinary; Neoplasm Proteins - antagonists & inhibitors; Neoplasm Proteins - metabolism; Pharmacokinetics; Phosphatase of regenerating liver (PRL); Protein Binding; Protein interaction; Protein Tyrosine Phosphatases - antagonists & inhibitors; Protein Tyrosine Phosphatases - metabolism; Protein-protein interaction (PPI); Proteins; Science; Science (multidisciplinary); Protein-protein interaction (PPI); Homogenous time-resolved fluorescence resonance energy transfer (HTR-FRET; Compound screening; HTRF; Cyclin M (CNNM); Phosphatase of regenerating liver (PRL)
• DOI: 10.1038/s41598-024-76269-1
• ISSN: 2045-2322

Phosphatase of regenerating liver (PRL) is an oncogenic protein that promotes tumor progression by directly binding to cyclin M (CNNM) membrane proteins and inhibiting their Mg 2+ efflux activity. In this study, we have developed a high-throughput screening system to detect the interactions between PRL and CNNM proteins based on homogenous time-resolved fluorescence resonance energy transfer (HTR-FRET, HTRF). We optimized the tag sequences attached to the recombinant proteins of the CNNM4 CBS domains and PRL3 lacking the carboxyl terminal CAAX motif, and successfully detected the interaction by observing the FRET signal in the mixture of the tagged proteins and fluorophore-conjugated antibodies. Moreover, we performed compound library screening using this system and discovered several compounds that could efficiently inhibit the PRL-CNNM interaction. Characterization of one candidate compound revealed that it was relatively stable compared with thienopyridone, a known inhibitor of the PRL-CNNM interaction. The candidate compound can also inhibit PRL function in cells: suppression of CNNM-dependent Mg 2+ efflux, and has sufficient in vitro drug metabolism and pharmacokinetic properties. Overall, these results demonstrate the effectiveness of this screening system for identifying novel inhibitors of the PRL-CNNM interaction, which could contribute to the development of novel anti-cancer drugs.