Herboxidiene Features That Mediate Conformation-Dependent SF3B1 Interactions to Inhibit Splicing

Area of Science:

• Molecular Biology
• Biochemistry
• Cancer Research

Background:

• Small molecules targeting the spliceosome SF3B complex are effective cancer cell growth inhibitors.
• These compounds interfere with early spliceosome assembly, specifically the U2 snRNP's interaction with the intron branch point sequence.

Purpose of the Study:

• Investigate factors influencing inhibitor interactions with SF3B to block pre-mRNA splicing.
• Conduct structure-activity relationship analysis of the splicing inhibitor herboxidiene.

Main Methods:

• Utilized an inactive herboxidiene analog (iHB) as a competitor in in vitro experiments.
• Performed order-of-addition experiments to assess inhibitor-SF3B interactions.
• Analyzed structure-activity relationships of herboxidiene, focusing on the tetrahydropyran ring.

Main Results:

• Inhibitor interactions with SF3B are long-lasting and sensitive to temperature and ATP presence.
• SF3B conformations influence inhibitor efficacy; not all observed conformations are productive for inhibition.
• Herboxidiene's tetrahydropyran ring features are critical for SF3B interaction and splicing inhibition.
• A carboxylic acid substituent on herboxidiene interacts with SF3B1, maintaining occupancy and counteracting SF3B transition.

Conclusions:

• Extended model for early spliceosome assembly and inhibitor mechanisms.
• SF3B inhibitors do not disrupt ATP-dependent U2 snRNP rearrangements essential for branch site recognition.
• Specific interactions between herboxidiene and SF3B1 are crucial for sustained inhibitor binding and splicing disruption.