Design and Evolution of a Macrocyclic Peptide Inhibitor of the Sonic Hedgehog/Patched Interaction

Abstract

The Hedgehog (Hh) signaling pathway plays a central role during embryonic development and its aberrant activation has been implicated in the development and progression of several human cancers. Major efforts toward the identification of chemical modulators of the Hedgehog pathway has yielded several antagonists of the GPCR-like Smoothened receptor. In contrast, potent inhibitors of the Sonic Hedgehog/Patched interaction, the most upstream event in ligand-induced activation of this signaling pathway, have been elusive. To address this gap, a genetically encoded cyclic peptide was designed based on the Shh-binding loop of Hedgehog-Interacting Protein (HHIP) and subjected to multiple rounds of affinity maturation through the screening of macrocyclic peptide libraries produced in E. coli cells. Using this approach, an optimized macrocyclic peptide inhibitor (HL2-m5) was obtained that binds Shh with a KD of 170 nM, which corresponds to a 120-fold affinity improvement compared to the parent molecule. Importantly, HL2-m5 is able to effectively suppress Shh-mediated Hedgehog signaling and Gli-controlled gene transcription in living cells (IC50 = 250 nM), providing the most potent inhibitor of the Sonic Hedgehog/Patched interaction reported to date. This first-in-class macrocyclic peptide modulator of the Hedgehog pathway is expected to provide a valuable probe for investigating and targeting ligand-dependent Hedgehog pathway activation in cancer and other pathologies. This work also introduces a general strategy for the development of cyclopeptide inhibitors of protein-protein interactions.

Selection, Addiction and Catalysis: Emerging Trends for the Incorporation of Noncanonical Amino Acids into Peptides and Proteins in Vivo

“…O2beY features a bromine as leaving group, which is readily displaced by a nearby cysteine residue to yield small to medium‐sized MPs ( n =1 to 9 in Figure B). Again, the authors were not only able to demonstrate efficient macrocyclization in vivo, but also showcased the potential of this approach by the design and affinity maturation of a MP inhibitor for the sonic hedgehog/patched interaction . The matured peptide HL2‐m5 (Figure D) underwent quantitative macrocyclization in vivo and showed a 120‐fold increased affinity, when compared to the parent, linear peptide.…”

Peptide Nanomaterials Designed from Natural Supramolecular Systems

“…The peptides inhibited the infection of various cell lines by HIV‐1. Moreover, recently, a cyclic peptide was developed to inhibit sonic hedgehog (Shh) signaling, which plays an important role in embryonic development and cancer progression . The cyclic peptide was designed based on the Shh‐binding loop of hedgehog‐interacting protein (HHIP), which is a membrane protein that acts as a negative regulator of the hedgehog pathway (Figure b).…”

Structure of Sonic Hedgehog protein in complex with zinc(II) and magnesium(II) reveals ion-coordination plasticity relevant to peptide drug design

“…1d; Richards & Degnan, 2009). The latter molecular-recognition event led to the development of macrocyclic peptides that inhibit the Hh pathway (Owens et al, 2017).…”

“…The importance of Hedgehog signaling pathways in human health and disease has stimulated efforts to manipulate Shh-N ligand-receptor interactions (Stanton et al, 2009;Owens et al, 2017). Many cancers exhibit abnormal activation by Hh ligands (Heretsch et al, 2010).…”