A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein–Protein Interaction Inhibitors

Weber, Elisabeth; Rothenaigner, Ina; Brandner, S; Hadian, Kamyar; Schorpp, Kenji

doi:10.1177/1087057116681408

Cited by 8 publications

(4 citation statements)

References 20 publications

(24 reference statements)

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“…These proteins were used for 2D NMR and in vitro ubiquitination experiments. FLAG-His-tagged Ubc13 protein (Ubc13-FH) and GST proteins (including GST-OTUB1 and GST-Ubc13) were produced as described previously (51).…”

Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Brenke

Popowicz

Schorpp

et al. 2018

Journal of Biological Chemistry

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Constitutive NF-κB signaling represents a hallmark of chronic inflammation and autoimmune diseases. The E3 ligase TNF receptor-associated factor 6 (TRAF6) acts as a key regulator bridging innate immunity, pro-inflammatory cytokines, and antigen receptors to the canonical NF-κB pathway. Structural analysis and point mutations have unraveled the essential role of TRAF6 binding to the E2-conjugating enzyme ubiquitin-conjugating enzyme E2 N (Ubc13 or UBE2N) to generate Lys-linked ubiquitin chains for inflammatory and immune signal propagation. Genetic mutations disrupting TRAF6-Ubc13 binding have been shown to reduce TRAF6 activity and, consequently, NF-κB activation. However, to date, no small-molecule modulator is available to inhibit the TRAF6-Ubc13 interaction and thereby counteract NF-κB signaling and associated diseases. Here, using a high-throughput small-molecule screening approach, we discovered an inhibitor of the TRAF6-Ubc13 interaction that reduces TRAF6-Ubc13 activity both and in cells. We found that this compound, C25-140, impedes NF-κB activation in various immune and inflammatory signaling pathways also in primary human and murine cells. Importantly, C25-140 ameliorated inflammation and improved disease outcomes of autoimmune psoriasis and rheumatoid arthritis in preclinical mouse models. Hence, the first-in-class TRAF6-Ubc13 inhibitor C25-140 expands the toolbox for studying the impact of the ubiquitin system on immune signaling and underscores the importance of TRAF6 E3 ligase activity in psoriasis and rheumatoid arthritis. We propose that inhibition of TRAF6 activity by small molecules represents a promising novel strategy for targeting autoimmune and chronic inflammatory diseases.

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Section: Recombinant Protein Expression and Purificationmentioning

confidence: 99%

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Brenke

Popowicz

Schorpp

et al. 2018

Journal of Biological Chemistry

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“…These compound interference can be produced solely by compounds themselves, such as fluorescent compounds, or by their interaction with biological components in assay system (14). One of the powerful method to solve this problem is to use orthogonal assay systems (15)(16)(17). Here, we setup 2 orthogonal assays for bromodomain inhibitor screening, alpha-screen and homogeneous time resolved fluorescence assay.…”

Section: Discussionmentioning

confidence: 99%

Novel brd4 inhibitors with a unique scaffold exhibit antitumor effects

Kim

et al. 2021

Oncol Lett

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Since bromodomain containing 4 (brd4) has been considered as a prominent cancer target, numerous attempts have been made to develop potent brd4 bromodomain inhibitors. The present study provided a novel chemical scaffold which inhibited brd4 activity. Mid-throughput screening against brd4 bromodomain was performed using alpha-screen and homogeneous time-resolved fluorescence assays. Furthermore, cell cytotoxicity and xenograft assays were performed to examine if the compound was effective both in vitro and in vivo . As a result, it was revealed that compounds having naphthalene-1,4-dione scaffold inhibited the binding of bromodomain to acetylated histone. The compounds with naphthalene-1,4-dione had cytotoxic effects against the Ty82 cell line, a NUT midline carcinoma cell line, whose proliferation is dependent on brd4 activity. A10, one of the compounds with naphthalene-1,4-dione scaffold, also exhibited tumor growth inhibition effects in the xenograft assay. In addition, the compounds exhibited cytotoxic effects against gastric cancer cell lines which were resistant to I-BET-762, a BET bromodomain inhibitor. In conclusion, the novel scaffold to suppress brd4 activity was effective against cancer cells both in vitro and in vivo .

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“…Chemoinformatics filters can help identify these frequent hitters (Baell & Holloway, 2010; Brenke et al., 2016; Schorpp et al., 2014). Hit evaluation in orthogonal and counter screens is mandatory to confirm activity and specificity (Rothenaigner & Hadian, 2021; Weber et al., 2017).…”

Section: Commentarymentioning

confidence: 99%

“…Reaction conditions for other E3 RING ligases can be altered accordingly to meet specific requirements. Besides TRAF6-Ubc13, AlphaScreen has also been shown to be suitable to detect, e.g., RNF8-Ubc13 interaction (Weber, Rothenaigner, Brandner, Hadian, & Schorpp, 2017).…”

Section: Introductionmentioning

confidence: 99%

Methods to Detect Small Molecule Inhibition of RING E3 Ligase Activity

et al. 2022

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Protein ubiquitination is an essential post‐translational modification that regulates a large number of cellular processes. This reaction is facilitated by the consecutive action of three central enzymes, i.e., E1 activating enzyme, E2 conjugating enzyme, and the E3 ligase. More than 600 E3 enzymes guarantee the specificity and selectivity of these reactions and thus represent an exciting, while highly underrepresented, class of drug targets. Specific methods can be employed to monitor their activity and thus query compound libraries for inhibitory small molecules. Here, we describe two protocols—one high‐throughput and one low‐throughput method—to detect E3 ligase activity and test small molecule modulation. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AlphaScreen assay to measure TRAF6‐Ubc13 interaction Basic Protocol 2: Gel‐based in vitro ubiquitination assay (K63‐linked chains)

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A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein–Protein Interaction Inhibitors

Cited by 8 publications

References 20 publications

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

Novel brd4 inhibitors with a unique scaffold exhibit antitumor effects

Methods to Detect Small Molecule Inhibition of RING E3 Ligase Activity

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