Psoromic Acid Derivatives: A New Family of Small‐Molecule Pre‐mRNA Splicing Inhibitors Discovered by a Stage‐Specific High‐Throughput in Vitro Splicing Assay

Abstract

Splicing of pre-mRNA is catalyzed by the spliceosome, which is composed of the U1, U2, U4/U6, and U5 small nuclear ribonucleoproteins (snRNPs) and numerous non-snRNP proteins. The spliceosome assembles in a stepwise fashion, with U1 and U2 interacting initially with the pre-mRNA (to form the A complex), followed by the U4/U6.U5 tri-snRNP (to generate the pre-catalytic B complex). The activated spliceosome (B) is subsequently formed by major rearrangements leading to the displacement of U1 and U4. After additional restructuring, step 1 of splicing occurs, during which the cleaved 5’-exon of the pre-mRNA and intron-3’-exon lariat intermediates are generated. The spliceosomal C complex is formed at this time; it catalyzes step 2 of splicing, during which the 5’and 3’-exons are ligated to form mature mRNA. Many pre-mRNAs (>88% of human genes) are alternatively spliced, in a process in which different sets of exons from the same pre-mRNA are spliced together, often in a regulated and/or tissue-/cell-specific manner. Alternative splicing is therefore a major means of generating protein diversity in an organism. Furthermore, disruption/misregulation of alternative and constitutive splicing are causes or severity modulators of many human diseases, including, among others, cancer and neurodegenerative and autoimmune diseases. The spliceosome is thus a highly attractive drug target. Small-molecule inhibitors capable of blocking discrete steps of the extremely dynamic functional cycle of the spliceosome would also be highly useful for the detailed investigation of the structure and function of the spliceosome. However, only a limited number of small-molecule inhibitors that specifically target the pre-mRNA splicing machinery have been identified. Although several cell-based high-throughput splicing assays have been developed, there is a great need for an in vitro high-throughput splicing assay that would be free of all the constraints imposed by the use of living cells. As the basis for a robust, rapid, and sensitive high-throughput in vitro splicing assay, we took advantage of the fact that some spliceosomal proteins are first recruited to the spliceosome and required at a late stage—that is, during/after step 1 of splicing. By monitoring the presence of such a protein in the spliceosome, we could search for inhibitors affecting spliceosome assembly, activation, and/or step 1 catalysis. We thus prepared nuclear extract from a HeLa cell line stably expressing a FLAG-tagged version of the DEAD box ATPase Abstrakt, which is a C-complex-specific protein. For the ultimate detection of the presence of FLAG-tagged protein in spliceosomes, tagged protein was bound with anti-FLAG antibody conjugated to horse radish peroxidase (Figure 1A). We then immobilized a preformed complex of the PM5 pre-mRNA substrate (containing three MS2 protein aptamers at its 5’-end) and the phage MS2-coat protein fused to maltose binding protein (MS2-MBP) in the wells of a microplate coated with anti-MBP antibodies. Splicing active nuclear extract was added and the microplate was incubated at 30 8C (typically 90 min) to allow spliceosome assembly and step 1 of splicing (PM5 lacks a 3’exon, preventing step 2 of splicing). After washing and addition of luminescent substrate, luminescence produced by the peroxidase was measured, reflecting the amount of protein bound and thus the extent of spliceosomal complexes formed. A splicing time course demonstrates that the signal obtained with extract containing FLAG-tagged Abstrakt, consistently with its association with the C complex, appeared much later than extract containing FLAG-tagged hSmu-1 (used as a control), which binds at an earlier stage (i.e. , in the B complex; Figure 1B). In addition, the initial appearance of the tagged hSmu-1 and Abstrakt luminescence signals corresponded to the known times of appearance of the B and C complexes, respectively, during in vitro splicing. No signal was observed with a control extract lacking FLAG-tagged protein. These results clearly indicate that the luminescence signal is proteinspecific and thus that this assay can be used to monitor the splicing reaction. [a] Dr. T. R. Samatov, Dr. P. Odenw lder, Dr. S. Bessonov, Prof. Dr. R. L hrmann Max-Planck-Institut f r biophysikalische Chemie Abt. Zellul re Biochemie Am Fasberg 11, 37077 Gcttingen (Germany) E-mail : Reinhard.Luehrmann@mpi-bpc.mpg.de [b] Dr. A. Wolf Lead Discovery Center GmbH Emil-Figge-Strasse 76a, 44227 Dortmund (Germany) [c] Dr. C. Deraeve, Dr. R. S. Bon, Prof. Dr. H. Waldmann Max-Planck-Institut f r molekulare Physiologie, Abt. Chemische Biologie Otto-Hahn-Strasse 11, 44227 Dortmund (Germany) [d] Prof. Dr. H. Waldmann TU Dortmund, Fakult t Chemie 44227 Dortmund (Germany) [e] Dr. P. Odenw lder Current address: McKinsey and Company Am Sandtorkai 77, 20457 Hamburg (Germany) [f] Dr. C. Deraeve Current address: Laboratoire de Chimie de Coordination du CNRS 205 Route de Narbonne, 31077 Toulouse, Cedex 4 (France) [g] Dr. R. S. Bon Current address: School of Chemistry and Biomedical and Health Research Centre University of Leeds Leeds, LS2 9JT (UK) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201100790.