Chemical probe development for the KLHL12 E3 ligase

Thesis

E3 ligases in the ubiquitin-proteasome system regulate protein homeostasis through the selective degradation of substrate proteins. However, their diverse functions and assembly mechanisms remain incompletely understood. I aimed to develop structures, assays, and chemical probes to better understand and modulate two atypical E3 assemblies, CUL3^KLHL12 and CUL2/CUL4^NRBP1, implicated in developmental disorders and cancer. A crystal structure of the KLHL12 Kelch domain bound to antagonist PLEKHA4 revealed a conserved binding mode for substrates, co-factors, and inhibitors supporting spatial sequestration as a control mechanism. Substrate displacement assays enabled identification of small molecule inhibitors, including fragment EVD-1 that selectively inhibited KLHL12 at sub-micromolar concentrations over other family members. Covalent binding to Cys511 was observed by crystallography and key determinants of compound reactivity established by structure-activity relationships. A tractable synthetic route for future analogues was identified as well as exit vectors for elaboration and potential PROTAC development. Proteins, binding assays and preliminary cryo-EM studies were also advanced for characterisation of putative CUL2/CUL4^NRBP1 complexes and a molecular glue-induced KLHL12-USP21 complex. Together, these studies expand our understanding of E3 ligase assemblies. EVD-1 further establishes a powerful tool to investigate KLHL12 biology, or for functionalisation into KLHL12-based PROTACs. In summary, this work expands the repertoire of chemically tractable E3 ligases, positioning KLHL12 as a promising target for small-molecule inhibitors or in the context of targeted protein degradation strategies.

Authors: Dalietou, EV

• DOI: 10.5287/ora-7eae0rjmb
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: PROTAC; E3 ligase; ubiquitin-proteasome system; structural biology; small molecule-inhibitor; KLHL12; chemical biology; NRBP1; ubiquitin
• Subjects: Pharmaceutical chemistry; Ubiquitin; Chemical biology; Biophysical screening; Structural biology; Chemical probe