The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Å. DCAF15 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and coordinates additional interactions between the two proteins. Studies with RBM39 point mutants and Indisulam analogs validated the structural model and defined the RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 and only these proteins were detectably downregulated in Indisulam-treated HCT116 cells. This work further explains how Indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade novel targets.

Lysyl oxidase has emerged as an important enzyme in cancer metastasis. Its activity has been reported to become upregulated in several types of cancer, and blocking its activity has been shown to limit the metastatic potential of various cancers. The small-molecules phenylhydrazine and β-aminopropionitrile are known to inhibit lysyl oxidase; however, issues of stability, toxicity, and poorly defined mechanisms limit their potential use in medical applications. The experiments presented herein evaluate three other families of hydrazine-derived compounds – hydrazides, alkyl hydrazines, and semicarbazides – as irreversible inhibitors of lysyl oxidase including determining the kinetic parameters and comparing the inhibition selectivities for lysyl oxidase against the topaquinone-containing diamine oxidase from lentil seedlings. The results suggest that the hydrazide group may be a useful core functionality that can be developed into potent and selective inhibitors of lysyl oxidase and eventually find application in cancer metastasis research.

The design, synthesis, and evaluation of two bifunctional molecular probes that can be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent manner are described. These probes use alkylhydrazines to irreversibly bind the target enzymes, which can then be visualized with either Western blotting or in-gel fluorescence. The results show that the Western blotting readout, which utilizes commercially available anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger readout than the fluorescein-based fluorescence readout. This visualization strategy can be used to measure the potency of enzyme inhibitors by selectively visualizing the active enzyme that remains after treatment with an inhibitor. Looking forward, this probe molecule and visualization strategy will enable activity-based protein-profiling experiments, such as determining inhibitor selectivity values within full proteome mixtures, for this family of amine oxidase enzymes.

32The anti-cancer agent Indisulam inhibits cell proliferation by causing degradation of 33 RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction 34 between RBM39 and the DCAF15 E3 ligase substrate receptor leading to RBM39 35 ubiquitination and proteasome-mediated degradation. To delineate the precise 36 mechanism by which Indisulam mediates DCAF15-RBM39 interaction, we solved the 37 DCAF15-DDB1-DDA1-Indisulam-RBM39(RRM2) complex structure to 2.3 Å. DCAF15 38 has a novel topology which embraces the RBM39(RRM2) domain largely via nonpolar 39 interactions, and Indisulam binds between DCAF15 and RBM39(RRM2) and 40 coordinates additional interactions between the two proteins. Studies with RBM39 41 point mutants and Indisulam analogs validated the structural model and defined the 42 RBM39 alpha-helical degron motif. The degron is found only in RBM23 and RBM39 43 and only these proteins were detectably downregulated in Indisulam-treated HCT116 44 cells. This work further explains how Indisulam induces RBM39 degradation and 45 defines the challenge of harnessing DCAF15 to degrade novel targets. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65E3-binding moiety, a linker, and a target-binding moiety 11 . Bifunctional degraders 92 literally tether target proteins to E3 ligases to facilitate ubiquitination and degradation. 93Auxin, a small molecule phytohormone, binds to an E3 ligase forming a new ligase 94 binding surface with increased affinity for the target protein 12 . Because auxin was 95 described as a "molecular glue" 13 , this type of TPD molecule, is known as a molecular 96 glue degrader. The IMiD drugs were recently discovered to be molecular glue 97 degraders. They bind the CRBN E3 ligase and create a new binding surface that 98 recruits beta-hairpin containing proteins 14 . Another class of TPD molecule is described by the plant hormone Gibberellin (GA). GA binds to its receptor and induces 100 a conformational change that allows receptor binding to its target protein. The 101 receptor-GA-target protein complex is recognized by the E3 ligase leading to target 102 protein degradation 12 . 103 104 Indisulam (Fig. 1a), an anti-cancer agent, was recently found to be a TPD molecule. 105Originally discovered by screening sulfonamides for cancer cell growth inhibition, 15 106Indisulam stood out by causing G1/S cell cycle arrest and demonstrating efficacy in 107 multiple tumor xenograft models 16 . Two seminal papers revealed that Indisulam 108 inhibits cell growth by degrading the essential splicing factor RBM39 17,18 . Indisulam 109 mediates an interaction between RBM39 and the E3 ligase DCAF15 leading to RBM39 110 poly-ubiquitination and proteasomal degradation. It was unclear whether Indisulam 111 acts allosterically by binding DCAF15 or RBM39 to bring about a conformational 112 change that enhances DCAF15-RBM39 interaction, whether Indisulam stabilizes a 113 weak DCAF15-RBM39 interaction, or whether Indisulam acts as a molecular glue to 114 enhance RBM39 binding to DCAF15 (Fig. 1a)...

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