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

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.

Therapeutic Modulation of RNA Splicing in Malignant and Non-Malignant Disease

“…Phenothiazines (panel 3) inhibit UHM-ULM interactions between splicing factors that are important for spliceosome assembly. The consequent inhibition of this assembly is expected to result in aberrant splicing; however, the exact splicing events engendered by these compounds are yet to be described [169]. Another strategy exploits RBM39-degrading compounds (panel 4; indisulam, tasisulam, and CQS) to target the accessory RNA-binding protein RBM39 for proteasomal degradation [105].…”

Identification of a small molecule splicing inhibitor targeting UHM domains

“…We confirmed that this molecule indeed binds the UHM pocket of U2AF 65 by NMR spectroscopy experiments and we show that it can modulate splicing and cell proliferation. Interestingly, while this work was in progress, another UHM‐targeting molecule with a markedly different structure was reported [29]. Altogether, these two studies provide the proof of concept that UHM domains can be targeted with a variety of compounds to control spliceosome assembly.…”

Biology of the mRNA Splicing Machinery and Its Dysregulation in Cancer Providing Therapeutic Opportunities

“…The cyclic peptide was subsequently used to discover small molecules that target the UHM-ULM interaction. To this end, the peptide was fluorescently labeled and used with synthetic RBM17 UHM in a fluorescence polarization assay, testing competition by over 43,000 compounds [ 355 ]. A validated hit was used for SAR and structure based optimization studies.…”

“…The identified phenothiazine compounds appeared general inhibitors of UHM-ULM interactions that impair early spliceosome assembly. While most compounds inhibited complex A formation, one analogue inhibited transition to complex B, presumably by preventing docking of the U4/U6.U5-tri-snRNP [ 355 ]. Although this difference could perhaps be exploited to design more specific inhibitors, the high conservation of UHM-ULM interactions between various proteins in the spliceosome makes this a challenging task.…”