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

Pravin Kumar Ankush Jagtap, Tomáš Kubelka, Komal Soni, Cindy L. Will, Divita Garg, Claudia Sippel, Tobias G. Kapp, Harish Kumar Potukuchi, Kenji Schorpp, Kamyar Hadian, Horst Kessler, Reinhard Lührmann, Felix Hausch, Thorsten Bach () and Michael Sattler ()
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Pravin Kumar Ankush Jagtap: Helmholtz Zentrum München
Tomáš Kubelka: Helmholtz Zentrum München
Komal Soni: Helmholtz Zentrum München
Cindy L. Will: Max Planck Institute for Biophysical Chemistry, Cellular Biochemistry
Divita Garg: Helmholtz Zentrum München
Claudia Sippel: Max Planck Institute of Psychiatry
Tobias G. Kapp: Technische Universität München
Harish Kumar Potukuchi: Helmholtz Zentrum München
Kenji Schorpp: Helmholtz Zentrum München
Kamyar Hadian: Helmholtz Zentrum München
Horst Kessler: Technische Universität München
Reinhard Lührmann: Max Planck Institute for Biophysical Chemistry, Cellular Biochemistry
Felix Hausch: Max Planck Institute of Psychiatry
Thorsten Bach: Technische Universität München
Michael Sattler: Helmholtz Zentrum München

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-19514-1

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