Structural Basis of Digoxin That Antagonizes RORγt Receptor Activity and Suppresses Th17 Cell Differentiation and Interleukin (IL)-17 Production

Abstract

The retinoic acid-related orphan nuclear receptor ␥t (ROR␥t)/ROR␥2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of ROR␥t and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the ROR␥t ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited ROR␥t activity and decreased IL-17 production but not ROR␣ activity. Digoxin inhibited IL-17 production in CD4 ؉ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that ROR␥t is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel ROR␥t antagonists.

Isoflavones enhance interleukin-17 gene expression via retinoic acid receptor-related orphan receptors α and γ

“…This implies that RORs contain an activation function (AF-2) located at the C terminus of its LBD, and agonist binding induces the AF-2 helix to adopt a conformation that encourages interactions with the LXXLL motifs of co-activator proteins such as SRCs (Fujita-Sato et al, 2011; Jin et al, 2010; Kurebayashi et al, 2004). On the basis of the results from two-hybrid assay, we showed that the LBDs of RORα and RORγ can interact with the LXXLL motifs of EBIP96 even in the absence of any exogenous ligands (Fig.…”

Deficiency of β-arrestin1 ameliorates collagen-induced arthritis with impaired TH17 cell differentiation

“…In addition, the up-regulation of Arrb1 expression in T H 17 cell differentiation was inhibited by Digoxin (Fig. 5B), a RORγt-specific inhibitor in T H 17 cells (26,27). Further investigation is needed for clarification of β-arrestin1 expression regulated by RORγt.…”

Inhibiting the Th17/IL-17A–Related Inflammatory Responses With Digoxin Confers Protection Against Experimental Abdominal Aortic Aneurysm

“…Surprisingly, recent studies ascertained that digoxin and its derivatives can inhibit Th17 cell differentiation and IL-17A production by selectively antagonizing the activity of RORγt. 24,25 In an experimental autoimmune encephalomyelitis model, which is a canonical IL-17A-mediated autoimmune disease model, digoxin conspicuously inhibits IL-17A production, attenuating the severity of experimental autoimmune encephalomyelitis. …”

“…24,25 In an experimental autoimmune encephalomyelitis model, which is a canonical IL-17A-mediated autoimmune disease model, digoxin conspicuously inhibits IL-17A production, attenuating the severity of experimental autoimmune encephalomyelitis. 24 In our previous study, we found that digoxin significantly prolonged allograft survival time in the BALB/cto-B6 mouse cardiac transplantation model by suppressing Th17 cell differentiation. 26 However, it is unknown whether the specific RORγt antagonist could attenuate experimental AAA (EAAA).…”