Theme 2
The initial phase of this project has focused on synthesising a first generation of RNA-binding protein degraders to reproduce and validate results previously reported in the literature (Figure 1A).1,2 These degraders consist of three components: a von Hippel-Lindau (VHL) E3 ligase recruiter, a linker, and an oligonucleotide (ON) designed to bind the target RNA-binding protein, Lin28A.
Two VHL-recruiting strategies were employed based on the published design: a peptide derived from hypoxia-inducible factor-1α (HIF-1α) and the small-molecule ligand VH032. Both recruit the VHL E3 ligase, promoting ubiquitination and subsequent degradation of the target protein. To target Lin28A, two previously reported oligonucleotide sequences of different lengths were used. To improve nuclease resistance and stability, the ONs were modified with phosphorothioate backbones and 2′-O-methoxyethyl (MOE) substitutions (Figure 1B).
Negative control degraders were also incorporated, containing non-binding variants of either the VHL recruiter or the oligonucleotide component. Most VHL recruiters were commercially available, except for one peptide-based negative control, which was synthesised via amide coupling. The oligonucleotides were prepared by solid-phase synthesis and functionalised at the 5′ terminus to enable conjugation. Peptide conjugates were assembled using an N-terminal cysteine-maleimide strategy, while VH032 based conjugates were synthesised through amide coupling using amino-modified oligonucleotides.
To date, eight degrader constructs have been synthesised, including four active degraders targeting Lin28A and four negative controls. These constructs combine different VHL recruiters, oligonucleotide lengths, and conjugation chemistries to assess the contribution of each component to degrader activity. Purification of the synthesised conjugates have been proved to be challenging, and method development is currently underway to streamline the process for the next set of degraders.
Figure 1. A) RNA Binding Protein degrader mechanism. B) RNA Binding Protein degraders synthesised comprising a E3 ligase recruiter and an oligonucleotide sequence targeting the RNA binding domain of Lin28A.
1 A. Ghidini, A. Cléry, F. Halloy, F. H. T. Allain, J. Hall Angew. Chem. Int. Ed. 2021, 60, 3163 – 3169. 2 Jianfei Xu, Xingxing Liang, Zhaopeng Yan, Yuejie Zhu, Jing Wang, Qian Wang, Zhenjun Yang, and Xinjing Tang J. Med. Chem. 2026 69, 4567-4578.