PROTAC Linker Design: towards Functionality

Project Information

PREDICT

Grant agreement ID: 101026833

DOI

10.3030/101026833

Project closed

EC signature date 23 March 2021

Start date 1 July 2021

End date 30 June 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

UNIVERSITY OF DUNDEE
United Kingdom

Periodic Reporting for period 1 - PREDICT (PROTAC Linker Design: towards Functionality)

Reporting period: 2021-07-01 to 2023-06-30

Summary of the context and overall objectives of the project

Proteolysis targeting chimeras (PROTACs) are small molecules that are designed to induce degradation of a target protein and represent a new strategy to design chemical probes and therapeutic agents of previously “undruggable” proteins. PROTACs are bifunctional molecules consisting of binding ligands to a target protein and an E3 ligase, a protein used in a cell’s tagging process of proteins with ubiquitin for recognition by the cell’s own degradation mechanism, connected by a linker. PROTACs are developed to enable the formation of a ternary complex of target protein, PROTAC and E3 ligase that results in tagging of the non-natural target protein with ubiquitin and subsequent degradation using cellular mechanisms.

Commonly, the linker portion of a PROTAC is considered to be a “spacer” of appropriate length between both ligands enabling ternary complex formation. Linkers of PROTACs are mostly optimized with regard to their length, lipophilicity and privileged conformation, e.g. being already preorganised for binding to target and E3 ligase. The goal of this project is to design, develop and assess the utility a novel linker motif that adds a new function to the linker. This function was leveraged to develop a novel hit-identification assay that is envisioned to accelerate development of degrader candidates in PROTAC drug discovery projects.

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

In the course of this project, a series of novel PROTACs targeting the von Hippel-Lindau (VHL) E3 ligase has been designed and synthesized. The performance of these novel PROTAC molecules has been studied in cellular and biophysical assays. In an initial degradation assay, four of the novel VHL-targeting PROTACs induced significant reduction of VHL protein levels. These compounds were further evaluated in dose- and time-dependent treatments in comparison to the previously reported state-of the-art VHL degrader, CM11. Two of these degraders showed improved efficacy with regard to both half-degradation times (t½) and half-maximal degradation concentration (DC50) values. Target engagement assays in live cells compared to permeabilized cells indicated improved cell permeability of these PROTACs compared to CM11, which can be attributed to the newly introduced functional motif in the PROTACs’ linkers. Furthermore, the first PROTAC degrading both isoforms of VHL has been identified within this series of novel VHL-targeting PROTACs.

Biophysical assays confirmed that degrader efficiency correlates with their ability to induce a ternary complex. To gain structural insight into the structural arrangement of the ternary complex, co-crystallization of protein with VHL-targeting PROTACs was attempted, yet unsuccessfully. As an alternative approach, cryo-EM was trialled on a VHL:PROTAC:VHL protein assembly, and a low-resolution 3D reconstruction of this protein assembly was generated, which serves as promising starting point for further sample optimization.

Finally, using this series of PROTACs featuring a novel functional motif within their linkers, a hit-identification assay has been designed and optimized, that is anticipated to identify PROTACs that efficiently form ternary complexes at an early stage in PROTAC development.

The results of this project have been presented at two international conferences and will be published in due course.

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 development of a library of novel PROTACs targeting the VHL E3 ligase led to the identification of several VHL-targeting PROTACs with increased cellular potency compared to the state-of-the-art VHL degrader, CM11. In particular, the first von-Hippel-Lindau (VHL)-targeting PROTAC degrading both isoforms of VHL was identified, which could potentially serve as valuable chemical probe to address questions around the biology of VHL and its natural substrate, the hypoxia-inducible factor 1-alpha (HIF-1α).

Furthermore, a novel early-stage in vitro hit-identification assay has been developed, that is designed to identify PROTAC scaffolds that enable efficient ternary complex formation. Implementation of this hit-identification assay into PROTAC development programs is expected to accelerate the identification of privileged degrader candidates for further optimization, thus shortening the early phase of PROTAC drug discovery programs.