Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and Crohn's disease. The biological effects of TNF are mediated by binding to TNF receptors, TNF receptor 1 (TNFR1), or TNF receptor 2 (TNFR2), and this coupling makes TNFR1-specific inhibition by small-molecule therapies essential to avoid deleterious side effects. Recently, we engineered a time-resolved fluorescence resonance energy transfer biosensor for high-throughput screening of small molecules that modulate TNFR1 conformational states and identified zafirlukast as a compound that inhibits receptor activation, albeit at low potency. Here, we synthesized 16 analogues of zafirlukast and tested their potency and specificity for TNFR1 signaling. Using cell-based functional assays, we identified three analogues with significantly improved efficacy and potency, each of which induces a conformational change in the receptor (as measured by fluorescence resonance energy transfer (FRET) in cells). The best analogue decreased NF-κB activation by 2.2-fold, IκBα efficiency by 3.3-fold, and relative potency by two orders of magnitude. Importantly, we showed that the analogues do not block TNF binding to TNFR1 and that binding to the receptor's extracellular domain is strongly cooperative. Despite these improvements, the best candidate's maximum inhibition of NF-κB is only 63%, leaving room for further improvements to the zafirlukast scaffold to achieve full inhibition and prove its potential as a therapeutic lead. Interestingly, while we find that the analogues also bind to TNFR2 in vitro, they do not inhibit TNFR2 function in cells or cause any conformational changes upon binding. Thus, these lead compounds should also be used as reagents to study conformational-dependent activation of TNF receptors.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †.

Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.

Conformational states of TNFR1 as a molecular switch for receptor function.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that plays a key role in the regulation of the inflammatory pathway. While inhibition of TNFR1 has been the focus of many studies for the treatment of autoimmune diseases such as rheumatoid arthritis, activation of the receptor is important for the treatment of immunodeficiency diseases such as HIV and neurodegenerative diseases such as Alzheimer's disease where a boost in immune signaling is required. In addition, activation of other TNF receptors such as death receptor 5 or FAS receptor is important for cancer therapy. Here, we used a previously established TNFR1 fluorescence resonance energy transfer (FRET) biosensor together with a fluorescence lifetime technology as a high-throughput screening platform to identify a novel small molecule that activates TNFR1 by increasing inter-monomeric spacing in a ligand-independent manner. This shows that the conformational rearrangement of pre-ligand assembled receptor dimers can determine the activity of the receptor. By probing the interaction between the receptor and its downstream signaling molecule (TRADD) our findings support a new model of TNFR1 activation in which varying conformational states of the receptor act as a molecular switch in determining receptor function.