Synthesis and development of molecular probes for proximity induction of proteins in cancer

Thesis

Induced proximity is a therapeutic strategy that utilises small molecules to bring two proteins close together. The proteins and the small molecule form a ternary complex, resulting in modulation of the target protein function and activation of cellular processes such as degradation and post-translational modifications. Molecular glues and heterobifunctional molecules have emerged as novel strategies to induce proximity. Molecular glues are small molecules that bind to a neomorphic surface between two proteins by stabilising or facilitating new protein-protein interactions. Heterobifunctional molecules consist of two ligands that bind two different proteins, connected by a linker. In this thesis, both strategies were used to target two different oncogenic proteins.

In part I, a structure-activity-relationship (SAR) campaign was conducted to optimise a potential molecular glue degrader of PD-L1. Compound AK-087 was discovered in a screen for activators of the E3 ligase, ITCH. AK-087 was hypothesised to stabilise the interactions between ITCH, and its E2 ligase, UBCH7, resulting in enhanced degradation of PD-L1 in melanoma cells. PD-L1 is overexpressed in melanoma cells as a strategy for immune evasion. PD-L1 binds to PD-1 on T-cells, leading to T-cell inactivation and apoptosis, preventing T-cell mediated killing of the cancer cell. Decreasing PD-L1 on the cancer cell surface has been shown to lead to enhanced response to mitogen activated protein kinase (MAPK) inhibitor treatments and immunotherapy. The SAR campaign led to the development of compound 36, which was approximately 40 times more potent in degrading PD-L1, compared to AK-087. Current work is ongoing to test the compounds in vivo, improve the metabolic stability of the compounds and further validate the target(s) of compound 36.

In part II, heterobifunctional molecules were synthesised to induce proximity between TRIM24 and BRD4. TRIM24 is an epigenetic protein that is highly expressed in liver and breast cancer, and is associated with poor patient survival rates, making it an attractive drug target for cancer treatment. High affinity ligands for the bromodomain of TRIM24 have been previously designed by other groups but bromodomain inhibition did not result in decreased cancer cell proliferation. TRIM24 has known E3 ligase activity, and it was hypothesised that recruiting TRIM24 to BRD4 using PROTACs would cause ubiquitination and degradation of BRD4. ITC and AlphaScreen® provided evidence for binding and ternary complex formation induced by the bifunctional molecules. The molecules were then tested for PROTAC and RIPTAC activity in cells but were shown to be inactive despite displaying cooperative binding and the ability to induce a ternary complex. For future work, co-crystallisation of compounds with the two proteins could allow us to understand why the compounds are not inducing productive ternary complexes.

Authors: Oei, KZ

• DOI: 10.5287/ora-j20vg99oq
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English