biostudies-literatureAnzai MMEXT | Japan Society for the Promotion of ScienceMEXT | Japan Science and Technology Agency (JST)MEXT | Japan Society for the Promotion of Science (JSPS)University of TokyoMEXT | Japan Science and Technology Agency643https://www.ebi.ac.uk/biostudies/studies/S-EPMC12015293biostudies-literatureUnknown8(1)Bone-destructive diseases are caused by dysregulated osteoclastogenesis. Osteoclast differentiation is positively regulated by the ligand for receptor activator of nuclear factor kappa B (RANKL) binding to the RANK on progenitor cells. RANK then forms a multivalent interaction with an adapter molecule, tumor necrosis factor receptor-associated factor 6 (TRAF6), to transduce various downstream signals. We used affinity-based screening of a multivalent random-peptide library to identify a tetravalent peptide, WHD-tet, that binds to the RANK-binding region of TRAF6 through a multivalent interaction. CR4-WHD-tet, a cell-permeable form of WHD-tet, efficiently inhibited the RANKL-induced differentiation of bone-marrow cells to osteoclasts and osteoclastogenesis in a mouse model. CR4-WHD-tet specifically inhibited the recruitment of MAPK kinase 3 to TRAF6 without affecting other signal transducers in a late stage of differentiation, inhibiting the activation of p38-MAPK, which promotes the final stage. Thus, the interaction modulator CR4-WHD-tet fine-tunes the formation of a critical signaling complex to inhibit osteoclastogenesis.Communications biologyClustered peptide regulating the multivalent interaction between RANK and TRAF6 inhibits osteoclastogenesis by fine-tuning signals.PMC12015293JPMJSP2129JP22J11001Kawabata HWaku TIkegami AInoue JIAnzai MWatanabe-Takahashi MOkuda YSakurai HNishikawa KMasuda YfalseClustered peptide regulating the multivalent interaction between RANK and TRAF6 inhibits osteoclastogenesis by fine-tuning signals.Bone-destructive diseases are caused by dysregulated osteoclastogenesis. Osteoclast differentiation is positively regulated by the ligand for receptor activator of nuclear factor kappa B (RANKL) binding to the RANK on progenitor cells. RANK then forms a multivalent interaction with an adapter molecule, tumor necrosis factor receptor-associated factor 6 (TRAF6), to transduce various downstream signals. We used affinity-based screening of a multivalent random-peptide library to identify a tetravalent peptide, WHD-tet, that binds to the RANK-binding region of TRAF6 through a multivalent interaction. CR4-WHD-tet, a cell-permeable form of WHD-tet, efficiently inhibited the RANKL-induced differentiation of bone-marrow cells to osteoclasts and osteoclastogenesis in a mouse model. CR4-WHD-tet specifically inhibited the recruitment of MAPK kinase 3 to TRAF6 without affecting other signal transducers in a late stage of differentiation, inhibiting the activation of p38-MAPK, which promotes the final stage. Thus, the interaction modulator CR4-WHD-tet fine-tunes the formation of a critical signaling complex to inhibit osteoclastogenesis.2025-01-01T00:00:00Z2025 Apr2025-07-01T03:05:14.16Z2025-07-01T03:05:14.16ZS-EPMC120152934026355610.1038/s42003-025-08047-2