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  Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly

Jagtap, P. K. A., Kubelka, T., Soni, K., Will, C. L., Garg, D., Sippel, C., et al. (2020). Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly. NATURE COMMUNICATIONS, 11(1): 5621. doi:10.1038/s41467-020-19514-1.

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 Creators:
Jagtap, Pravin Kumar Ankush, Author
Kubelka, Tomas, Author
Soni, Komal, Author
Will, Cindy L., Author
Garg, Divita, Author
Sippel, Claudia1, Author           
Kapp, Tobias G., Author
Potukuchi, Harish Kumar, Author
Schorpp, Kenji, Author
Hadian, Kamyar, Author
Kessler, Horst, Author
Luehrmann, Reinhard, Author
Hausch, Felix2, Author           
Bach, Thorsten, Author
Sattler, Michael, Author
Affiliations:
1Dept. Translational Research in Psychiatry, Max Planck Institute of Psychiatry, Max Planck Society, ou_2035295              
2Max Planck Institute of Psychiatry, Max Planck Society, Kraepelinstr. 2-10, 80804 Munich, DE, ou_1607137              

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Free keywords: U2AF HOMOLOGY MOTIF; SPLICING FACTOR; STRUCTURAL BASIS; RNA; RECOGNITION; INSIGHTS; DOMAINS; DESIGN; KINASE; TARGETScience & Technology - Other Topics;
 Abstract: Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3 splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation. So far only a few compounds have been reported as splicing modulators. Here, the authors combine high-throughput screening, chemical synthesis, NMR, X-ray crystallography with functional studies and develop phenothiazines as inhibitors for the U2AF Homology Motif (UHM) domains of proteins that regulate splicing and show that they inhibit early spliceosome assembly on pre-mRNA substrates in vitro.

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Language(s): eng - English
 Dates: 2020
 Publication Status: Published online
 Pages: 11
 Publishing info: -
 Table of Contents: -
 Rev. Type: -
 Degree: -

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Title: NATURE COMMUNICATIONS
Source Genre: Journal
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Publ. Info: HEIDELBERGER PLATZ 3, BERLIN, 14197, GERMANY : NATURE RESEARCH
Pages: - Volume / Issue: 11 (1) Sequence Number: 5621 Start / End Page: - Identifier: ISSN: 2041-1723