Mechanism of anti-tumor compounds that inhibit the spliceosome
The spliceosome is a dynamic ribonucleo-protein complex that is in charge of removing introns from pre-mRNA. Five small nuclear ribonucleic proteins (snRNPs) (U1, U2, U4, U5, U6) build the spliceosome, and each contain their own special RNA. In particular, U2 snRNP is unique, in that it guides the spliceosome to the correct branch point sequence. SF3B1 is the largest and keystone protein of U2 snRNP. Mutations in SF3B1 have been associated with misregulation in splicing and disease such as cancer. Spliceostatin A, Pladienolide B and herboxidiene are small drug molecules that target SF3B1. They are used as tools to help study the importance of SF3B1 and initiation of splicing assembly. However, the mechanism of action of these molecules is not known. More studies need to research how these drugs bind and inhibit. I used in vitro order of addition splicing assays, and studied SF3B1 in different conditions: temperature, time, and competition assay. My findings clearly demonstrate that temperature affects competition between active and inactive compounds. Time course experiments clearly imply that SF3B inhibitors exchange more slowly than inactive analogs. Lastly, we show positions C1 and C6 of herboxidiene are important for binding and inhibiting SF3B1 in the binding pocket. We also model drug-protein interaction. Our work will help elucidate how SF3B1 selects the correct branch point sequence and give medicinal chemists new insights to make small molecule drugs more potent.