Background. DKK1 antagonizes canonical Wnt signalling through high-affinity binding to LRP5/6, an essential component of the Wnt receptor complex responsible for mediating downstream canonical Wnt signalling. DKK1 overexpression is known for its pathological implications in osteoporosis, cancer, and neurodegeneration, suggesting the interaction with LRP5/6 as a potential therapeutic target. Results. We show that the small-molecule NCI8642 can efficiently displace DKK1 from LRP6 and block DKK1 inhibitory activity on canonical Wnt signalling, as shown in binding and cellular assays, respectively. We further characterize NCI8642 binding activity on LRP6 by Surface Plasmon Resonance (SPR) technology. Conclusions. This study demonstrates that the DKK1-LRP6 interaction can be the target of small molecules and unlocks the possibility of new therapeutic tools for diseases associated with DKK1 dysregulation. 1. Background DKK1 is a 29？KDa secreted protein belonging to the Dickkopf (DKK) family [1], which comprises four main glycoproteins in vertebrates (DKK1-4) [2]. DKK1 has been identified as a potent inhibitor of the canonical Wnt signaling due to its ability to bind to the Wnt coreceptor LRP5/6, thus blocking the canonical Wnt/β-catenin pathway [3]. Canonical Wnt pathway activation is initiated by the direct binding of the Wnt glycoprotein to Frizzled (Fz) membrane receptor and to the LRP5/6 coreceptor [4–6]. In the absence of Wnt, β-catenin undergoes phosphorylation-dependent ubiquitination and degradation [7, 8]. Wnt-mediated assembly of the activated Fz-LRP5/6 receptor complex is followed by the recruitment of the axin-GSK3β to the plasma membrane, resulting in the reduction of the phosphorylation and degradation of β-catenin [9, 10]. Stabilized β-catenin accumulates in the cytoplasm and translocates to the nucleus, where it interacts with DNA-bound TCF-LEF proteins and activates the transcription of target genes [11]. The Wnt pathway is involved in many stages of invertebrate and vertebrate development and in adult tissue homeostasis [8, 12]. Dysfunction within the Wnt/β-catenin signaling cascade has been associated with many human pathologies [8, 13], such as cancer [14–17] and bone disease [18, 19]. LRP5-activating mutations are mainly associated with high-bone mass, while loss-of-function mutations on LRP5 are linked to bone degeneration and osteoporosis [20, 21]. The inhibition of Wnt signaling by DKK1 has been related to bone degeneration processes and reduced bone mass [22]. In the central nervous system, DKK1 has been associated with the
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