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 have 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 sonic hedgehog (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 K_D 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 = 230 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.

Figure 1

Hedgehog signaling pathway. Binding of the Hedgehog ligand(s) (HhN, corresponding to Shh, Dhh, or Ihh) to the Patched receptor relieves its inhibitory effect on Smoothened (Smo), resulting in the activation of Gli transcription factors and induction of Gli-controlled genes. Hedgehog-Interacting Protein (HHIP) inhibits the signaling pathway by competing with Patched for binding to the Hedgehog ligands. Adapted from www.phosphosite.org.

Figure 2

Macrocyclic HHIP L2 loop mimic. (a) Crystal structure of Shh (green) in complex with the extracellular domain of HHIP (pink) (pdb 3HO5). The three loop regions of HHIP involved in Shh binding are labeled and the zinc ion in the L2 binding cleft of Shh is shown as sphere model (blue). (b) Top: schematic structure of the macrocyclic peptide HL2-m1. Bottom: model of HL2-m1 (yellow, stick model) bound to Shh (green, surface model). The L2 loop of HHIP (pink, ribbon model) is superimposed to the modeled complex.

Figure 3

Shh binding affinity of linear and macrocyclic L2 mimics. (a) Dose-response curves for direct binding of the recombinantly produced FLAG tag-fused peptides to plate-immobilized GST-Shh as determined using HRP-conjugated anti-FLAG antibody. (b) Sequences and K_D values corresponding to macrocyclic L2 mimics and linear L2-based peptide. X = MDYKDDDDKGSGS-. The mutated positions in the evolved macrocyclic peptides compared to the initial cyclic peptide HL2-m1 are highlighted.

Figure 4

Overview of strategy for evolution of macrocyclic peptides. A plasmid library encoding for partially randomized peptide sequences fused to a FLAG tag and a C-terminal GyrA intein are transformed into E. coli cells and arrayed on multiwell plates. The corresponding pre-cursor polypeptides are produced via ribosomal translation and O2beY incorporation via amber stop codon (TAG) suppression. The macro-cyclic peptides are produced inside cells through ‘self-processing’ of the biosynthetic precursors via O2beY/Cys cyclization and aspartate-induced intein cleavage. After cell lysis, peptide binding to immobilized Shh is quantified colorimetrically. The variants showing improved Shh binding activity are deconvoluted via DNA sequencing. The best variant and acquired SAR data are used for the next round of affinity maturation.

Figure 5

Circular dichroism (CD) spectra corresponding to the linear HL2-pep (a) and the macrocyclic HL2-m5 peptide (b) in buffer and in the presence of trifluoroethanol (TFE) at varying concentration. The signals are reported as mean residue molar ellipticity (θ_MRE).

Figure 6

HL2-m5-induced suppression of Hh pathway signaling. (a) Dose dependent inhibition of luciferase expression (FF/Ren ratio) in Shh-stimulated NIH3T3 cells containing a dual luciferase reporter system. (b) Restoration of Hedgehog pathway signaling upon addition of purmorphamine (5 μM) to cells treated with HL2-m5 (10 μM). (c) Relative transcriptional levels of Gli1, Gli2, and Ptch1 genes in Shh-stimulated NIH3T3 cells in the presence and in the absence of HL2-m5 (10 μM) as determined by real-time PCR. mRNA levels in unstimulated cells are included for comparison.

Figure 7

Hh analog selectivity of linear and cyclic L2 mimics. Data relative to the peptides are derived from direct binding experiments to immobilized Hh proteins. Data relative to robotnikinin are derived from competition experiments (10 μM robonikinin + 400 nM FLAG-HL2-m5). For each compound, values are normalized to the highest binding response measured across the three Hedgehog analogs.

Scheme 1

Synthesis of macrocyclic peptides. (a) Synthetic route for the preparation of the diamino acid building block encompassing the Cys/O2beY thioether linkage. (b) Solid-phase syn-thesis of macrocyclic peptide HL2-m5.