Design, synthesis and biological evaluation of PROteolysis-TArgeting Chimeric (PROTAC) molecules as anticancer agents

Project Information

PROTACs

Grant agreement ID: 101031883

DOI

10.3030/101031883

Project terminated on 30 September 2023

EC signature date 23 April 2021

Start date 4 April 2022

End date 3 April 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 172 932,48

EU contribution

€ 172 932,48

Coordinated by

FUNDACION UNIVERSITARIA SAN PABLO-CEU
Spain

Periodic Reporting for period 1 - PROTACs (Design, synthesis and biological evaluation of PROteolysis-TArgeting Chimeric (PROTAC) molecules as anticancer agents)

Reporting period: 2022-04-04 to 2024-04-03

Summary of the context and overall objectives of the project

Cancer diseases represent critical medical challenges. According to the recent estimates, 1 958 310 new cancer cases and 609 820 cancer deaths are projected to occur in the United States in 2023 [1]. On the other hand, 2 740 000 new cancer cases and 1 290 000 cancer-related deaths were detected in European Union in 2022 [2]. These figures underline that tumours remain one of the leading causes of death worldwide. Traditional anticancer drug design based on small molecules continues to be a powerful strategy for the development of novel chemotherapeutics. However, these anticancer therapies are facing major problems such as drug resistance, especially in advanced cancers. Therefore, in this project we decided to develop new series of PROteolysis-TArgeting Chimerics (PROTACs). It is worth noting that recently PROTACs have become a promising approach to overcome resistance since they act degrading instead of inhibiting the target with the advantage of reducing the systemic drug exposure and to counteract the protein expression that often accompanies inhibition of protein function [3,4].

Main objective of the project
The main goal of the project was to develop new PROTACs targeting several enzymes, including histone deacetylases (HDACs), protein kinase 2 (CK2) and aromatase (AROM), all of which play crucial roles in cancer development and progression. Taking advantage of the host group´s experience in developing dual inhibitors for CK2/HDACs [5,6], we decided to synthesise PROTACs that simultaneously target both enzymes. To date, the reasearcher has successfully synthesized two different series of dual CK2/HDACs PROTACs based on the structure of CX-4945 (a potent CK2 inhibitor). These PROTACs were made by adding a moiety responsible for the action on HDACs to the CK2 ligand and connecting them to a Cereblon (CRBN) ligase recruiter through a linker of different lengths. Currently, we are actively evaluating the degradation capabilities of these newly synthesised CK2/HDACs PROTACs. In addition, during his research, the researcher has successfully synthesised two different series of novel PROTACs targeting AROM. The structure of these new potential AROM degraders was based on Aminoglutethimide, which is a first-generation AROM inhibitor [7]. These AROM-PROTACs were connected to two different E3 ligase recruiters, recognised by CRBN and von Hippel-Lindau (VHL) ligases, via variable-length linkers. Like the CK2/HDACs-PROTACs, the newly synthesised AROM-PROTACs are being evaluated biologically. Importantly, these types of PROTACs, belonging to both the CK2/HDACs and AROM classes, have not been previously described in the literature. For this reason, we are considering the possibility of applying for a patent after confirming their degradation capacity.

Additional objective
Since PROTACs often do not comply with the ¨Lipinski´s rule of five¨, their clinical applications may be limited due to their unfavorable pharmacokinetic properties and difficulties in crossing biological membranes. In light of these considerations, the researcher has devised synthetic methods for coupling the compounds with a folic acid (FA) molecule, which can serve as a natural carrier of biologically active substances, addressing their permeability limitations. So far, the researcher has synthesized FCs of CX-4945 and Aminoglutethimide, both of which are also used as cores of the PROTACs we have developed. Given the presence of the same functional groups, this strategy may be easily applied to obtain FCs from the synthesised PROTACs, with the aim of addressing issues related to the uptake of the compounds by cancer cells if detected. Furthermore, the synthesis of FCs from PROTACs promises to produce compounds with better activity and lower toxicity, a goal we hope to achieve in the future.

[1] Siegel, R.L.; Miller, K.D.; Wagle, N.S.; et al.; CACancer J. Clin. 2023, 73, 17.
[2] https://joint-research-centre.ec.europa.eu/jrc-news-and-updates/cancer-cases-and-deaths-rise-eu-2023-10-02_en access: 8th November, 2023.
[3] Li, M.; Zhi, Y.; Liu, B.; et al.; J. Med. Chem. 2023, 66, 2308.
[4] Han, X.; Sun, Y.; MedComm. 2023, 4, e290.
[5] Martinez, R.; Di Geronimo, B.; Pastor, M.; et al.; Molecules, 2020, 25.
[6] Rangasamy, L.; Ortin, I.; Zapico, J.M.; et al.; ACS Med. Chem. Lett., 2020, 11, 713.
[7] Sainsbury, R. Cancer Treat. Rev. 2013, 39, 507-517.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The initial step of the project involved the application of computational techniques, which played an important role in selecting the most promising molecules for the synthesis. The computational analysis aimed to identify the most suitable sites to add the PROTAC linker to the functionalized inhibitors. Based on them a series of structures for potential PROTACs, incorporating variations in linker length, type of E3 ligase recruiter, and different functional groups were designed.
The synthesis process presented certain challenges, requiring the exploration of multiple synthesis routes. After several attempts, a multistep convergent synthesis approach allowed us to generate four series of potential PROTACs, each targeting different molecular targets, with the required purity level (>95%) for subsequent biological studies. Furthermore, based on the structure of inhibitors utilized in the synthesized PROTACs, the researcher devised a synthetic method to attach inhibitors to the FA molecule, which may significantly improve their biological properties. Since the same functional groups are present in the synthesized PROTACs, this strategy can be easily applied to these PROTACs in the future.
Unfortunately, due to the early termination of the project, the final phase involving the evaluation of pharmacokinetic properties and biological activity was left incomplete. However, these studies are currently underway or planned to begin shortly. Once we have confirmation of the biological capabilities for the developed PROTACs, we will consider the possibility of patenting and publishing our findings in high-impact journals.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The statistics about cancer disease underline the pressing need to develop new anticancer therapies in our society. This PROTACs project may contribute to the creation of innovative therapeutic options to combat the global cancer challenge.
It is worth noting that, during this project, the researcher synthesised several classes of PROTACs that had not been previously reported in the literature. Some of these PROTACs have the potential to bind to two different molecular targets (CK2/HDACs), leading to their degradation and increasing their potency as anticancer agents. Additionally, the researcher synthesized a series of PROTACs targeting AROM enzyme, a previously unknown type of PROTACs. Importantly, the entire series of PROTACs obtained during the project are highly innovative and have the potential to represent a great advance in the treatment of several types of cancer. Furthermore, the researcher developed a synthetic method to link the synthesized PROTACs with the FA molecule, which may significantly improve their biological properties, making them more suitable for clinical applications. The results of this research have not yet been published and have the potential to be protected by patent.

General structure of PROTACs and their putative mechanism of action.