Investigating the ligandability of plant homeodomains

Thesis

Plant homeodomains (PHDs) are protein domains which bind to the N-terminal region of histone 3 (H3) in a manner dependent on the post-transitional modification state of residues near the N-terminus of H3. For example some PHDs will only bind H3 if lysine 4 (K4) is trimethylated, whereas other PHDs will only bind H3 if K4 is unmodified. PHDs play a crucial role in gene regulation in cells and mutations and translocations of PHDs have been linked to disease states.

A search of the human proteome revealed the presence of 173 PHDs. These were aligned using structural information and the alignment used to create a PHD phylogenetic tree, the first time such a tree has been reported for this domain family.

All PHD structures in the Protein Data Bank were computationally analysed using SiteMap in order to assess ligandability. Single PHDs were generally found to be poorly ligandable, but some were predicted to be at the lower threshold of ligandability. The study was expanded and found that PHDs found in tandem with another PHD or other epigenetic reader domain were generally more ligandable due to the formation of ligandable pockets at domain-domain interfaces.

Based on the results from the SiteMap analysis, experimental work was carried out to discover ligands of the tandem PHD-PHD of double PHD finger protein 2 (DPF2) and the PHD-JmjC of PHD finger protein 8 (PHF8). Assays were designed and both virtual and experimental screens conducted. No hits were identified for DPF2, but a single fragment hit for PHF8 was found with an IC₅₀ of 260 μM. Analogues of this fragment were synthesised and tested, but none were more potent than the initial hit.

Authors: Bowkett, D

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