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An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

ABSTRACT: Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced I?B? degradation and NF-?B activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

SUBMITTER: Lo CH

PROVIDER: S-EPMC5899890 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Lo Chih Hung CH Vunnam Nagamani N Lewis Andrew K AK Chiu Ting-Lan TL Brummel Benjamin E BE Schaaf Tory M TM Grant Benjamin D BD Bawaskar Prachi P Thomas David D DD Sachs Jonathan N JN

SLAS discovery : advancing life sciences R & D 20170522 8

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules t ...[more]

PMID: 28530838

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Project description:The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.

Dataset Information

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

Publications

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors.

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