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

Autor: Funato Y; Laboratory of Biorecognition Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan. funato.yosuke.3i@kyoto-u.ac.jp., Mimura M; Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, 565-0871, Osaka, Japan., Nunomura K; Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 565-0871, Osaka, Japan., Lin B; Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 565-0871, Osaka, Japan., Fujii S; Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 565-0871, Osaka, Japan., Haruta J; Center for Supporting Drug Discovery and Life Science Research, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, 565-0871, Osaka, Japan., Miki H; Laboratory of Biorecognition Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, 615-8510, Japan. miki.hiroaki.4e@kyoto-u.ac.jp.
Jazyk: angličtina
Zdroj: Scientific reports [Sci Rep] 2024 Oct 25; Vol. 14 (1), pp. 25432. Date of Electronic Publication: 2024 Oct 25.
DOI: 10.1038/s41598-024-76269-1
Abstrakt: 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.
(© 2024. The Author(s).)
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje