Targeted degradation of Pin1 by protein-destabilizing compounds

Abstract

The concept of targeted protein degradation is at the forefront of modern drug discovery, which aims to eliminate disease-causing proteins using specific molecules. In this paper, we explored the idea to design protein degraders based on the section of ligands that cause protein destabilization, hence that facilitate the cellular breakdown of the target. Our studies present covalent agents targeting Pin1, a cis-trans prolyl isomerase that plays a crucial role in tumorigenesis. Our design strategy entailed iterative optimizations of agents for potency and Pin1 destabilization in vitro. Biophysical and cellular studies suggest that the agents may act like molecular crowbars, displacing protein-stabilizing interactions that open the structure for recognition by the proteasome degradation machinery. This approach resulted in a series of potent and effective Pin1 degraders with potential applications in target validation and in therapeutic development. We propose that our design strategy can identify molecular degraders without engineering bifunctional agents that artificially create interactions between a disease-causing protein and a ubiquitin ligase.

Keywords: PIN1; drug discovery; molecular crowbars; protein degradation.

Fig. 1.

Expression of Pin1 in cells and comparison of Pin1 degradation in response to Pin1 inhibitors in BxPC3 cells. (A) Different cell lines were probed for endogenous expression of Pin1. (B) BxPC3 cells were treated with 0.1% DMSO (dimethyl sulfoxide) or 25 µM of known Pin1 inhibitors for 24 h and Pin1 levels were quantified.

Fig. 2.

Comparative properties of selected Pin1 binding agents. (A) Chemical structure of D-peptide, BJP-07-017-3, and 158D11. (B) Associated DELFIA displacement dose–response curves (SD values are obtained from duplicate measurements) and (C) Denaturation thermal shift curves relative to the indicated agents. Curves are normalized in the Y axis for better illustration; original data are reported as SI Appendix, Fig. S2. (D) Surface representation of Pin1 in complex with BJP-07-017-3 (PDB ID 6O34). Residue Cys 113 is highlighted.

Fig. 3.

Effect of selected agents on Pin1 degradation in cell. Agents selected based on DELFIA and ΔTm values were tested for Pin1 degradation in the pancreatic cancer cell line BxPC3. BxPC3 treated with 5 μM or 15 μM of different compounds for 24 h.

Fig. 4.

Denaturation thermal shift data and western blot data of selected agents. (A) Name, structure, IC₅₀ values, and ΔTm values for agents 158G6, 158G12, 164A1, 164A2, 164A3, and 164A5. For these agents the major denaturation shift is reported, and a minor denaturation thermal shift transition was also observed (not reported). Degrading agents 158G12 and 164A4 ΔTm values are highlighted in bold. (B) BxPC3 cells were treated with 0.1% DMSO or 15 µM of the reported agents for 24 h and Pin1 levels were quantified.

Fig. 5.

Effect of 158H9 and 164A10 on Pin1 degradation in a panel of human cancer cell lines. Agents selected based on DELFIA and ΔTm values were tested for Pin1 degradation in (A) BxPC3, (B) MIA PaCa-2, (C) PANC-1, (D) MDA-MB-231, (E) PC3 and (F) A549 NucLight Red. Cells were treated with increasing concentrations of each compound for 24 h.

Fig. 6.

Pin1 targeting agents cause Pin1 degradation via the proteasome. BxPC3 cells were treated with (A) 158H9 or (B) 164A10 in the presence or absence of autophagy (Baf A1 and HCQ) or proteasome (BTZ or CFZ) inhibitors for 24 h.

Fig. 7.

Time-dependent Pin1 degradation and rescue by a proteasome inhibitor. BxPC3 cells were treated with (A) 158H9 or (B) 158H9 and BTZ and Pin1 levels were assessed at the indicated time points.

Fig. 8.

Characterization of ligand binding to Pin1 by solution NMR spectroscopy. (A) Overlay of 2D [¹⁵N, ¹H] correlation spectra for Pin1 (50 μM in buffer 50 mM phosphate pH = 7.5, 150 mM NaCl, and 1 mM DTT (Dithiothreitol)) collected in absence (blue) and presence (red) of compound 164A9 (250 μM). Resonance assignments are reported as SI Appendix, Fig. S3. (B) Ribbon representation of Pin1 in complex with BJP-07-017-3 (stick model) PDB ID 6O34. The secondary structure elements are labeled, and the blue areas of the protein represent backbone residues that experienced significant chemical shift perturbations upon complex formation with compound 164A9. (C) Overlay of 2D [¹⁵N, ¹H] correlation spectra for Pin1 (50 μM in buffer 50 mM phosphate pH = 7.5, 150 mM NaCl, and 1 mM DTT) collected in absence (blue) and presence of various compounds, each at 250 μM: red, sulfopin; yellow, 164A9; green, 158F10 (SI Appendix, Fig. S4). The Inset highlights the chemical shift perturbations induced by the ligands on the side chain ¹⁵N^ε, ¹H^ε indole resonances of residue Trp 73.

Fig. 9.

Overall structure of hPin1 R14A and the feature-enhanced 2mF_o-DF_c electron density of Cys113 and the covalently bound 158A10, 158D9, and 158F10 compounds. (A) The overall structure of hPin1 R14A with bound PEG (polyethylene glycol)400 within the asymmetric unit of the P3₁21 crystal form. The PPIase and WW domains of hPin1 are shown in blue and pink, respectively. The dashed line outlines the regions featured in the remaining panels. (B–D) Feature-enhanced 2mF_o-DF_c electron density of Cys113 and the covalently bound 164A10 (PDB ID 8VJG), 158D9 (PDB ID 8VJF), and 158F10 (PDB ID 8VJE) compounds contoured at 1σ, represent as blue/green mesh. The inhibitors are shown in ball-and-stick representation, with the carbon, nitrogen, and oxygen atoms displayed in orange, blue, and red, respectively.

Fig. 10.

The 158F10-induced structural changes of Pin1. Structure of Pin1 with bound 158F10 as seen in the single copy (A) and the two copies (B and C) of the inhibitor-bound protein of the P3121 crystal and the P212121 crystal, respectively. Note the conformational flexibility of 158F10 and of helix D and loop FG. See also SI Appendix, Fig. S5 (PDB ID 8VJE and PDB ID 8VJD for the 158F10 protomer).

Fig. 11.

Schematic illustration of the proposed mechanism of how a molecular crowbar induces Pin1 degradation. Binding of our agents to Pin1 brings the reactive acetyl group of the inhibitor close to Cys113. Upon formation of a covalent bond between the inhibitor to Cys113, the inhibitor-bound Cys113 acts like a molecular crowbar. It destabilizes the short helix D causing the helix to unfold and loop FG to move its position exposing hydrophobic residues such as Trp 73. Hence, our inhibitor acts as a molecular crowbar that opens up and destabilizes Pin and subsequent cellular degradation.