Alteration of RNA Splicing by Small-Molecule Inhibitors of the Interaction between NHP2L1 and U4

Diouf B, et al., doi: 10.1177/2472555217735035, SLAS Discov., 2017

Described herein is the development of time-resolved FRET (TR-FRET) assay to assess the interaction between proteins NHP2L1 and U4 (RNA), the two key components of spliceosome. Furthermore, a high-throughput TR-FRET screen was used to identify small molecules that disrupt the interaction between NHP2L1 and U4(RNA) and verified the results by using orthogonal methods. Topotecan and related camptothecin derivatives were identified as the most effective small molecules that interfere with NHP2L1 and U4 interaction. Topotecan binding to NHP2L1 protein or to U4 5' SL was studied by surface plasmon resonance (SPR) using a Pall ForteBio Pioneer instrument equipped with a COOH5 sensor chip. The sensor chip surface was activated using NHS and EDC. Neutravidin was immobilized onto COOH5 sensor chip by using a routine amine coupling chemistry. Any remaining active sites on the sensor chip surface were blocked with ethanolamine. Biotin-NHP2L1 and U4 5?SL-biotin were injected on separate flow channels. One flow channel on the chip was used as a reference. Topotecan was prepared in running buffer and injected using the OneStep injection feature, which exploits Taylor dispersion to generate a continuous analyte titration in a single injection. SPR data obtained were globally fitted to a 1:1 binding model and equilibrium dissociation constants (KD) were determined. Overall results of this study provide new insights into how small molecules can interfere with the interaction between NHP2L1 and U4. The findings may provide important implications for the development of therapeutics that alter RNA splicing and thereby gene function.

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