SF3B1 thermostability as an assay for splicing inhibitor interactions

Area of Science:

• Molecular Biology
• Cancer Biology
• Drug Discovery

Background:

• The spliceosome protein SF3B1 is crucial for pre-mRNA splicing and is frequently mutated in cancer.
• SF3B1 is the target of natural product drugs with anti-tumor properties, though its exact mechanism of action is debated.
• Understanding SF3B1's role and inhibitor interactions is vital for cancer therapy.

Purpose of the Study:

• To establish a thermostability assay for SF3B1 to investigate inhibitor interactions in a functional context.
• To determine if SF3B1 binding by inhibitors correlates with changes in its thermostability.
• To explore the potential of SF3B1 thermostability as a biomarker for inhibitor binding and conformational status.

Main Methods:

• Developed an assay to measure SF3B1 thermostability in nuclear extracts.
• Tested the effect of natural product inhibitors and analogs on SF3B1 thermostability.
• Investigated a synthetic SF3B1 ligand (WX-02-23) for its effects on thermostability and in vitro splicing.

Main Results:

• Both active and antagonistic inhibitors altered SF3B1 thermostability, suggesting binding alone doesn't impair function.
• SF3B1 thermostability varied between nuclear extracts, indicating conformational differences.
• The synthetic ligand WX-02-23 increased SF3B1 thermostability and inhibited splicing, mimicking natural product inhibitors.

Conclusions:

• Altered SF3B1 thermostability serves as a valuable indicator of inhibitor binding, complementing functional splicing assays.
• SF3B1 thermostability can be used to study factors influencing its conformation.
• This research provides insights into cancer drug mechanisms targeting SF3B1.