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Noncompetitive inhibitors of TNFR1 probe conformational activation states.

ABSTRACT: Tumor necrosis factor receptor 1 (TNFR1) is a central mediator of the inflammatory pathway and is associated with several autoimmune diseases such as rheumatoid arthritis. A revision to the canonical model of TNFR1 activation suggests that activation involves conformational rearrangements of preassembled receptor dimers. Here, we identified small-molecule allosteric inhibitors of TNFR1 activation and probed receptor dimerization and function. Specifically, we used a fluorescence lifetime-based high-throughput screen and biochemical, biophysical, and cellular assays to identify small molecules that noncompetitively inhibited the receptor without reducing ligand affinity or disrupting receptor dimerization. We also found that residues in the ligand-binding loop that are critical to the dynamic coupling between the extracellular and the transmembrane domains played a key gatekeeper role in the conformational dynamics associated with signal propagation. Last, using a simple structure-activity relationship analysis, we demonstrated that these newly found molecules could be further optimized for improved potency and specificity. Together, these data solidify and deepen the new model for TNFR1 activation.

SUBMITTER: Lo CH

PROVIDER: S-EPMC7008598 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Noncompetitive inhibitors of TNFR1 probe conformational activation states.

Lo Chih Hung CH Schaaf Tory M TM Grant Benjamin D BD Lim Colin Kin-Wye CK Bawaskar Prachi P Aldrich Courtney C CC Thomas David D DD Sachs Jonathan N JN

Science signaling 20190730 592

Tumor necrosis factor receptor 1 (TNFR1) is a central mediator of the inflammatory pathway and is associated with several autoimmune diseases such as rheumatoid arthritis. A revision to the canonical model of TNFR1 activation suggests that activation involves conformational rearrangements of preassembled receptor dimers. Here, we identified small-molecule allosteric inhibitors of TNFR1 activation and probed receptor dimerization and function. Specifically, we used a fluorescence lifetime-based h ...[more]

PMID: 31363069

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Project description:Human neutrophil elastase (HNE) is a serine protease that plays vital roles in inflammation, innate immune response, and tissue remodeling processes. HNE has been actively pursued as a drug target, particularly for the treatment of cardiopulmonary diseases. Although thousands of molecules have been reported to inhibit HNE, yet very few are being evaluated in early clinical trials, with sivelestat as the only approved HNE inhibitor. We report here a novel chemotype of sulfonated nonsaccharide heparin mimetics as potent and noncompetitive inhibitors of HNE. Using a chromogenic substrate hydrolysis assay, 14 sulfonated nonsaccharide heparin mimetics were tested for their inhibitory activity against HNE. Only 12 molecules inhibited HNE with IC50 values of 0.22-88.3 μM. The inhibition of HNE by these molecules was salt-dependent. Interestingly, a specific hexa-sulfonated molecule inhibited HNE with an IC50 value of 0.22 μM via noncompetitive mechanism, as demonstrated by Michaelis-Menten kinetics. The hexa-sulfonated derivative demonstrated at least 455-, 221-, 1590-, 21-, and 381-fold selectivity indices over other heparin-binding coagulation proteins including factors IIa, Xa, IXa, XIa, and FXIIIa, respectively. At the highest concentrations tested, the molecule also did not significantly inhibit other serine proteases of plasmin, trypsin, and chymotrypsin. Further supporting its selectivity, the molecule did not show heparin-like effects on clotting times of human plasma. The molecule also did not affect the proliferation of three cell lines at a concentration as high as 10 μM. Interestingly, the hexa-sulfonated molecule also inhibited cathepsin G with an IC50 value of 0.57 μM alluding to a dual anti-inflammatory action. A computational approach was exploited to identify putative binding site(s) for this novel class of HNE inhibitors. Overall, the reported hexa-sulfonated nonsaccharide heparin mimetic serves as a new platform to develop potent, selective, and noncompetitive inhibitors of HNE for therapeutic purposes.

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Noncompetitive inhibitors of TNFR1 probe conformational activation states.

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Noncompetitive inhibitors of TNFR1 probe conformational activation states.

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