Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling

Nagamani Vunnam; Mu Yang; Chih Hung Lo; Carolyn Paulson; William D Fiers; Evan Huber; MaryJane Been; David M Ferguson; Jonathan N Sachs

doi:10.1021/acsbiomedchemau.2c00048

Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling

ACS Bio Med Chem Au. 2023 Apr 7;3(3):270-282. doi: 10.1021/acsbiomedchemau.2c00048. eCollection 2023 Jun 21.

Authors

Nagamani Vunnam¹, Mu Yang², Chih Hung Lo¹, Carolyn Paulson¹, William D Fiers², Evan Huber¹, MaryJane Been¹, David M Ferguson³, Jonathan N Sachs¹

Affiliations

¹ Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States.
² Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
³ Department of Medicinal Chemistry and Center for Drug Design, University of Minnesota, Minneapolis, Minnesota 55455, United States.

Abstract

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.