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

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 Lys63-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 in vitro 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 in vivo 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.

Heterogenous nuclear ribonucleoprotein Q increases protein expression from HIV-1 Rev-dependent transcripts.

BACKGROUND: Heterogenous nuclear ribonucleoproteins (hnRNPs) control many processes of the gene expression machinery including mRNA transcription, splicing, export, stability and translation. Recent data show interaction of the HIV-1 Rev regulatory protein with a subset of hnRNP proteins, that includes hnRNP Q, suggesting that hnRNPs can contribute to regulation of HIV-1 gene expression by Rev. FINDINGS: In this work we address the effect of hnRNP Q on Rev-dependent gene expression. We show that hnRNP Q overexpression increased levels of proteins produced from a Rev-dependent reporter gene in the presence of Rev. Increased protein levels did not correlate with changes in either the levels or the nucleocytoplasmic distribution of Rev-dependent reporter mRNAs. Similar observations were made in persistently HIV-1 infected HeLa cells. In these cells, hnRNP Q overexpression increased levels of the HIV-1 Gag-p24 protein, while levels of viral Rev-dependent mRNAs were not affected. CONCLUSION: Our data indicate that hnRNP Q can stimulate the protein production of Rev-dependent mRNAs without changing mRNA levels and mRNA export, respectively. This suggests that hnRNP Q can boost HIV gene expression at the level of protein production.

Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase-Glutathione Interaction.

In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni(2+)-NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results.

Inhibition of Canonical NF-κB Signaling by a Small Molecule Targeting NEMO-Ubiquitin Interaction.

The IκB kinase (IKK) complex acts as the gatekeeper of canonical NF-κB signaling, thereby regulating immunity, inflammation and cancer. It consists of the catalytic subunits IKKα and IKKß and the regulatory subunit NEMO/IKKγ. Here, we show that the ubiquitin binding domain (UBAN) in NEMO is essential for IKK/NF-κB activation in response to TNFα, but not IL-1ß stimulation. By screening a natural compound library we identified an anthraquinone derivative that acts as an inhibitor of NEMO-ubiquitin binding (iNUB). Using biochemical and NMR experiments we demonstrate that iNUB binds to NEMOUBAN and competes for interaction with methionine-1-linked linear ubiquitin chains. iNUB inhibited NF-κB activation upon UBAN-dependent TNFα and TCR/CD28, but not UBAN-independent IL-1ß stimulation. Moreover, iNUB was selectively killing lymphoma cells that are addicted to chronic B-cell receptor triggered IKK/NF-κB activation. Thus, iNUB disrupts the NEMO-ubiquitin protein-protein interaction interface and thereby inhibits physiological and pathological NF-κB signaling.

Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens.

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)-compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni(2+)-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.