Pin1-PROTACs: Degradation versus Inhibition, GEneration of tools for anticancer therapy – PRODIGE

Designing and predicting the structure of a surely effective PROTAC can be challenging. Indeed, PROTAC’s activity and selectivity depend not only on the affinity of the ligand for the POI but also on: 1) the choice of the POI’s binding site; 2) the type of ligase; 3) the nature of the combination POI-E3 ligands; 4) the linker length/composition; 5) the geometry of the entire construct. The formation of the ternary complex is essential to have an efficient degrader and it depends on all of these factors. For this a combinatorial approach to screen as much as possible all these variables and to maximise the chances to generate active Pin1-PROTACs was adopted.

To create the 1st library of Pin1-PROTACs, different Pin1 ligands incorporating a terminal alkyne were envisaged. On the other hand, we planned to synthesise diverse E3 ligands accessorised with a terminal carboxylic acid. In parallel, an assortment of azide-amine terminal linkers was designed. After that, we assembled the 3 building blocks by means of a CuAAC (Copper-catalysed Alkyne-AzideCycloaddition) and an amide bond formation reaction, to obtain all the different combinations. Since the synthesised Pin1 ligands were new molecules, their affinity towards Pin1 was tested using the Chemical Shift Perturbation NMR method (CSP). It should be noted, however, that the binding affinity between the PROTACs and the target often does not correlate to how effectively the PROTAC will degrade that POI. Subsequently, the synthesised Pin1-PROTACs were subjected to a preliminarybiological evaluation: cytotoxicity tests, cell proliferation and viability assessment and degradation assay were performed.

In 2020, we also envisaged to create a 2nd library of Pin1-PROTACs with two different goals: 1) perform the in-cell self-synthesis of PROTACs; 2) enhance the probabilities to have active PROTACs changing the “click” reaction and, consequently, the degrader’s geometry. The in-cell self-assembly of PROTACs could permit to overcome some well-known problems that restrict the enormous therapeutic potential of this strategy. PROTACs possess high molecular weight and polar surface area which can limit cellular permeation, solubility and compromise bioavailability and pharmacokinetics. On the other side, the two smaller PROTACs precursors are expected to be more cell-permeable. To reach both goals, we envisaged to take advantage of the highly biorthogonal IEDDA (Inverse Electron Demand Diels-Alder) cycloaddition between Pin1 ligands, bearing a trans-cyclo-octene (TCO) and E3 ligands, tagged with a tetrazine.

Currently, we are still investigating the degradation activity of the 1st library of Pin1-PROTACs; therefore, the in-cell approach has not yet been conducted and will be developed in the near future.

1) Synthesis and evaluation of Pin1 Ligands

To synthesise the 1st library of Pin1-PROTACs, we developed a library of clickable, cell-permeable pseudopeptide Pin1 ligands. This part of PRODIGE led to a scientific publication in a peer-reviewed journal(DOI:10.1039/d4cc05968a). The KD of the ligands were determined using NMR (Chemical Shift Perturbation method). Compound 4a demonstrated the highest affinity (KDWW: 44 ± 6 μM), identifying its SATE-protected version 4b as the lead compound for biological studies and PROTACs synthesis. To assess the cell permeability of 4b, a fluorescent analogue (4b-Rhod) was synthesised. Its permeability was confirmed in 2 Pin1-expressing ovarian cancer cell lines, SKOV3 and IGROV1. The effect of 4b on cancer cells (MM1.R) was evaluated using an MTT viability assay. 4b caused a concentration-dependent reduction in cell proliferation with an IC50 of 79 ± 13 μM. To confirm cell permeability, MM1.R cells were incubated with unprotected analogues 4a and 4c. Neither compound affected cell viability, suggesting that 4b’s SATE group is needed to have permeability. A SensoLytes Green Pin1 Assay was conducted to evaluate the inhibitory activity of 4a and 4b. 4a exhibited inhibition while 4b showed no inhibition due to the SATE group blocking Pin1 binding.

2) Synthesis and evaluation of Pin1-PROTACs

Based on the 4b scaffold, 12 potential Pin1-PROTACs were synthesised varying linker length and composition. Additional Pin1 ligands were alsoused to generate PROTACs (degradation efficiency depends on more than just KD). Pin1-PROTAC stability was assessed in six different cell culture media at 37°C over 72 hours using HPLC. Among the tested media, RPMI showed the best results in terms of stability, making it the preferred mediumfor cell-based assays. Pin1-PROTACs were tested on ovarian cancer (IGROV1,SKOV3), lung cancer (A549), breast cancer (MCF7) and a multiple myeloma (MM1.R) cell lines. The presence of Pin1 and CRBN in these cell lines was confirmed via immunofluorescence staining. Cytotoxicity (LDH assay) and viability (trypan blue assay) tests revealed that Pin1-PROTACs are, in general, not cytotoxic but exhibit antiproliferative activity, similar to 4b. This effect could result from Pin1 degradation or inhibition. To determine whether Pin1-PROTACs induce Pin1 degradation, Western Blot assays were performed. No degradation was observed following incubation of P1 with SKOV3, MCF7, and MM1.R for 6, 24, or 72 h at concentrations up to 10 μM, and higher. Similar results were obtained for P5 and P10. Conversely, P8 demonstrated a 35% reduction in Pin1 levels in MM1.R and MCF7 cells at 40 μM before showing a Hook effect. We are currently testing the remaining 8 Pin1-PROTACs to evaluate their degradation potential.

This project has laid the foundation for a novel approach to target Pin1, a key oncogenic protein considered undruggable. By leveraging the PROTAC strategy, we have initiated the development of the first generation of Pin1-directed degraders, along with a library of cell-permeable clickable pseudopeptide inhibitors with imaging and therapeutic potential. The results obtained so far highlight multiple promising directions for future exploration.

1. Optimisation of Degradation Efficiency

The preliminary identification of a partially active compound opens the way for scaffold optimisation and alternative E3 ligase recruitment strategies to enhance potency and selectivity.

2. Subcellular Targeting Studies

Ongoing compartment-specific degradation assays will provide crucial insights into how Pin1’s localisation affects its degradation, enabling more rational PROTAC design in the future.

3. In-Cell Self-Assembly

The planned development of in-cell PROTAC self-assembly could significantly improve cellular uptake and therapeutic efficacy. This innovative direction may help overcome limitations related to size, polarity, and pharmacokinetics inherent to conventional PROTACs.

4. Tool for Functional Biology.

Beyond therapy, Pin1-PROTACs represent a powerful research tool for acute, reversible knockdown in complex biological models, includinglarge animals, where genetic methods face limitations.

5. Therapeutic Expansion

While cancer remains the primary focus, the involvement of Pin1 in neurodegenerative and viral diseases creates opportunities for broader therapeutic applications in the future.

Overall, this project positions Pin1-PROTACs at the intersection of chemical biology, drug discovery, and translational medicine. It opens new perspectives not only for the development of anti-cancer therapies but also for understanding fundamentalbiological mechanisms and enhancing the druggability of challenging targets.

Targeted protein degradation is a powerful new modality in chemical biology and drug discovery, emerging in the last decade, as a promising therapeutic strategy. PROteolysis TArgeting Chimeras (PROTACs) are bifunctional molecules made up of a ligand for the target Protein of Interest (POI) and a ligand for an E3 ubiquitin ligase, which are covalently joined by a flexible linker. Mechanistically, PROTAC promotes the recruitment of the E3 ligase in close proximity to the POI forming a ternary complex. This proximity enables E3 ligase-mediated ubiquitination of the POI, followed by its consecutive recognition and degradation by the Ubiquitin Proteasome System (UPS). Thanks to their unique catalytic mode of action, PROTACs present different advantages over the competitive-and-occupancy driven process of traditional small molecule-based inhibitors. In particular, PROTACs can target “undruggable” proteins.
Pin1 is a multifunctional Peptidyl-Prolyl Isomerase (PPiase), which catalyses the cis-trans isomerisation of Xaa-Pro amide ?-bonds in proteins. In cells, Pin1 serves as a “molecular timer”, switching “on” or “off” the functions of its multiple signalling phosphoprotein substrates, in order to control the amplitude and the duration of many cellular responses or processes. Pin1 is overexpressed in most cancers and it is a potential target for cancer therapy. However, although many Pin1’s inhibitors have been identified, the majority of them lack the required potency, specificity, cell permeability and efficacy. Pin1, like the others PPIases, is indeed considered “undruggable”.
To overcome the problems related to the development of Pin1 inhibitors, while taking advantage of all the unique strengths of PROTACs strategy over existing targeting modalities (monoclonal antibodies and small molecule inhibitors above all), this project envisages the development of a library of Pin1-PROTACs. Pin1-PROTACs will be built with the pioneering and innovative goal to intentionally degrade Pin1 in a controlled manner in order to treat cancer. Beyond their use as potential drugs, other remarkable and versatile applications will be explored for Pin1-PROTACs. To this end, they may be used as a new research tool to respond the unanswered questions regarding the numerous different roles of Pin1 in physiological processes and pathological conditions. In addition, Pin1-PROTACs will bring a novel, convenient, fast and reversible method to degrade Pin1 globally and quickly in living animals, for which gene-targeted knockout strategies may remain unfeasible.