Peptide-based allosteric inhibitor targets TNFR1 conformationally active region and disables receptor-ligand signaling complex

Tumor necrosis factor (TNF) receptor 1 (TNFR1) plays a pivotal role in mediating TNF induced downstream signaling and regulating inflammatory response. Recent studies have suggested that TNFR1 activation involves conformational rearrangements of preligand assembled receptor dimers and targeting receptor conformational dynamics is a viable strategy to modulate TNFR1 signaling. Here, we used a combination of biophysical, biochemical, and cellular assays, as well as molecular dynamics simulation to show that an anti-inflammatory peptide (FKCRRWQWRMKK), which we termed FKC, inhibits TNFR1 activation allosterically by altering the conformational states of the receptor dimer without blocking receptor-ligand interaction or disrupting receptor dimerization. We also demonstrated the efficacy of FKC by showing that the peptide inhibits TNFR1 signaling in HEK293 cells and attenuates inflammation in mice with intraperitoneal TNF injection. Mechanistically, we found that FKC binds to TNFR1 cysteine-rich domains (CRD2/3) and perturbs the conformational dynamics required for receptor activation. Importantly, FKC increases the frequency in the opening of both CRD2/3 and CRD4 in the receptor dimer, as well as induces a conformational opening in the cytosolic regions of the receptor. This results in an inhibitory conformational state that impedes the recruitment of downstream signaling molecules. Together, these data provide evidence on the feasibility of targeting TNFR1 conformationally active region and open new avenues for receptor-specific inhibition of TNFR1 signaling.

TNFR1 signaling; anti-inflammatory; conformational dynamics; noncompetitive inhibition; receptor-specific inhibition.