Efficient development of stable and highly functionalised peptides targeting the CK2α/CK2β protein-protein interaction

Abstract

The discovery of new Protein-Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability in vivo and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. The lead peptide, CAM7117, prevents the formation of the holoenzyme assembly in vitro, slows down proliferation, induces apoptosis in cancer cells and is stable in human serum. CAM7117 could aid the development of novel CK2 inhibitors acting at the interface and help to fully understand the intracellular pathways involving CK2. Importantly, the approach adopted herein could be applied to many PPI targets and has the potential to ease the study of PPIs by efficiently providing access to functionalised peptides.

Fig. 1. (a) Importance of the holoenzyme to the functions of CK2 (PDB: 1JWH). The catalytic α subunits are shown in grey and green, the regulatory β subunits in yellow and pink. The binding site on CK2α for inhibitors of the PPI is shown on the right. (b) A comparison of the peptides developed prior to this work (Pc and TAT-Pc),, and the lead peptide CAM7117 developed in this work.

Fig. 2. Sequence of the portion of CK2β binding to CK2α, Pc peptide and structure of the constrained peptides reported in this work with calculated enthalpic values of binding and percentage of CK2β-like probe displacement. NA = not measured. A detailed table with the structures of all the peptides presented in this work is provided in the ESI (Table S2†). Pc, P1, and P1-Cn (where n = 1–5) peptides feature an amide at the C-terminus and an acetyl cap at the N-terminus. All the amino acids are the l isomers.

Fig. 3. Crystallographic structures of conformationally constrained peptides. (a) P1-C4 (green, PDB: ; 6Q38) and residues 186-193 of CK2β (yellow, PDB: ; 4NH1 (ref. 44)) in complex with CK2α. The image is shown as a cross-section of the interface site. (b) A comparison of the binding mode of the two different linkers in Pc (cyan, PDB: ; 4IB5 (ref. 34)) and P1-C4 (green). (c) Difference in the distance between the α-carbons of the amino acids involved in the constraint in Pc (cyan) and P1-C4 (green). (d) Crystallographic structure of P1-C4 (green) binding at the interface site. The image is shown as a cross-section of the interface site. (e) Crystallographic structure of P2-C4 (cyan, PDB: ; 6Q4Q) binding at the interface site. Stacking of the Trp residue with the constraint is highlighted in red. The image is shown as a cross-section of the interface site. (f) Comparison of the binding mode of P1-C4 (green) and P2-C4 (cyan) bound to the interface site.

Fig. 4. In vitro assessment of P1-C4 and P2-C4. (a) ITC binding curves of P1-C4 and P2-C4. A schematic representation of the peptide association with CK2α (green) is shown on the top of the ITC curves. (b) Ability of P2-C4 to cause inhibition of the association of the CK2α/CK2β-like holoenzyme using CK2α (green) and CK2β-RAD (pink and blue). (c) Ability of P2-C4 to cause inhibition of the catalytic activity of CK2α (green) towards a CK2β-dependent substrate eIF2β by P2-C4. Starting CK2 holoenzyme is shown in green and pink; peptide is shown as spheres.

Fig. 5. Structures of the multi-functionalised peptides. The peptides are constrained with a multifunctional linker containing a linkage core that locks the peptide in its binding conformation and enhances stability to proteases (pink), a spacer to avoid steric clashes (blue), a protease-resistant poly(d)arginine tag to gain cellular permeability (green) and a fluorescent tag to monitor intracellular localisation (yellow).

Fig. 6. Biological evaluation of CAM7117 and its analogues in cancer cells. (a) Cellular uptake in U2OS cell line for P2-F2C4 and P2-F3C4. Cell nuclei are stained with Hoechst 33342 stain (blue). (b) GI₅₀ curves of the antiproliferative activity of CAM7117 (solid and dashed blue lines) and CX4945 (solid and dashed black line) in U2OS and HCT116 cells. (c) Immunofluorescence experiments in the presence of FITC-labelled peptide P2-F2C4 (green), stained with antibodies against markers of different organelles (all in red): EEA1 (early endosome), Rab7 (late endosome), LAMP1 (lysosomes), ZFLP1 (cis Golgi), TGN46 (trans Golgi) and calnexin (endoplasmic reticulum). Details of antibodies are found in Table S11. Nuclei are stained with Hoechst 33342 (blue). (d) Change in intracellular localisation of CK2β (red) following treatment with CAM7117 (30 μM) for 15 and 120 minutes. Nuclei are stained with Hoechst 33342 (blue).