Computational Design and Optimization of Novel Subtype Specific Frizzled Binding Proteins for Modulation of Wnt Signaling

Abstract

Wnt signaling is essential to a range of critical biologic processes including embryonic development, mature tissue maintenance, and cell proliferation. Dysregulation of the Wnt signaling pathway is conclusively implicated in a range of cancer types. Wnt interacts with its extracellular receptor Frizzed (Fz) via an essential palmitoleic acid modification, and blockade of the Fz lipid binding site results in inhibition of canonical Wnt signaling and decreased tumor growth. Therefore, molecules that can potently antagonize the Wnt-Fz interaction have significant promise as anti-cancer therapeutics. The degeneracy and functional implications of the interactions between 19 distinct Wnt ligands and 10 Fz receptors remains unresolved. Specific antagonism of individual Frizzled receptors will enable disentanglement of this complex signaling network and the roles specific Wnt ligand-receptor interactions play in different tissue and cancer types. Novel, high affinity, Fz-5/8 specific binders were therefore computationally designed and optimized for application as Wnt inhibitors. Subsequent refinement of these binders included stabilization, enhancement of functional antagonism, and interface reengineering resulting in variants with orthogonal binding specificity against alternate receptor subtypes. These proteins were also functionalized as novel, water-soluble Wnt surrogates for complementary activation of Wnt signaling. These novel, rationally designed proteins enable precise modulation of Wnt signaling as required for full elucidation of the role of Wnt signaling in clinically relevant contexts and therapeutic intervention in anti-cancer and regenerative medicine applications. In addition, the computational design of a de novo interface utilizing an idealized ankyrin scaffold provides a general method for the development of novel protein binders.