Focused Design of Novel Cyclic Peptides Endowed with GABARAP-Inhibiting Activity

Abstract

(1) Background: Disfunctions in autophagy machinery have been identified in various conditions, including neurodegenerative diseases, cancer, and inflammation. Among mammalian autophagy proteins, the Atg8 family member GABARAP has been shown to be greatly involved in the autophagy process of prostate cancer cells, supporting the idea that GABARAP inhibitors could be valuable tools to fight the progression of tumors. (2) Methods: In this paper, starting from the X-ray crystal structure of GABARAP in a complex with an AnkirinB-LIR domain, we identify two new peptides by applying in silico drug design techniques. The two ligands are synthesized, biophysically assayed, and biologically evaluated to ascertain their potential anticancer profile. (3) Results: Two cyclic peptides (WC8 and WC10) displayed promising biological activity, high conformational stability (due to the presence of disulfide bridges), and K_d values in the low micromolar range. The anticancer assays, performed on PC-3 cells, proved that both peptides exhibit antiproliferative effects comparable to those of peptide K1, a known GABARAP inhibitor. (4) Conclusions: WC8 and WC10 can be considered new GABARAP inhibitors to be employed as pharmacological tools or even templates for the rational design of new small molecules.

Keywords: Atg8; GABARAP inhibitors; LIR motif; PC-3; autophagy; cancer; peptide.

Figure 1

(A) 3D representation of the GABARAP/AnkB-LIR complex, as derived from the X-ray structure (PDB accession code 5YIR). The protein surface is colored depending on the atomic par-tial charges of the protein residues: blue for positive and red for negative charges. The AnkB-LIR peptide is represented as cyan sticks. (B) Plot of the protein and ligand (AnkB-LIR) Cα atoms RMSD over the simulation time.

Figure 2

RMSF plots of AnkB-core analogs. Backbone atoms were considered in these calculations. The residues shared by all peptides are highlighted by capital letters.

Figure 3

Depiction of the representative structure of the most populated cluster of conformations assumed by WC8 (A) and WC10 (B) in the complex with GABARAP. The GABARAP solvent-accessible surface is shown accordingly by residue charges: blue for positive and red for negative residues, respectively. The interactions between complexes are represented in colored dashes: yellow for H-bonds, purple for salt bridges, and green for cation-π.

Figure 4

Binding of K1 peptide on GABARAP. MST curve (A) and steady-state analysis obtained by fitting the proper form of the Scatchard equation for the plot of the bound RU at equilibrium vs. the ligand concentration in solution during SPR experiments (B).

Figure 5

Binding of WC8 and WC10 to GABARAP. MST and SPR curves acquired by recombinant GABARAP incubated with different concentrations of WC8 (A,B) and WC10 (C,D) peptides. In the MST plot referred to WC10 (C), the point corresponding to a concentration of 391 nM (evidenced in gray) appears to be a clear outlier, also considering the other experiments; hence, it was discarded and not included in the calculation of the K_d value.

Figure 6

Effect of K1, WC8, and WC10 on cell viability. Cell viability was determined by MTS assay on PNT2 (A) and PC-3 cell lines (B) 96 h post-treatment. The absorbance was measured with a 96-well-plate spectrophotometer (Varioskan Flash Multimode Reader) at 490 nm.