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Title: Inhibition of KDM4D and stabilisation of the PHF8 plant homeodomain's transient structural states using antibodies
Author: Wolfreys, Finn
ISNI:       0000 0004 7231 7082
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Though antibodies as therapeutics are limited to extracellular targets, their repertoire of molecular interactions has particular relevance to the many intracellular cellular proteins for which small molecule screening has reached impasse. For such proteins there is little recourse to theory, since molecular recognition is, in practical terms, still not well understood. Here I apply antibody discovery to the lysine demthylases KDM4D and PHF8, two proteins difficult to inhibit selectively due to the similarity of their binding pockets to those of the larger family. With a selective, picomolar affinity antibody, dependent on residues distal to the KDM4D active site, I present what is likely the first example of allosteric inhibition of a KDM4 lysine demethylase, demonstrating that there is opportunity outside active sites oversubscribed with pan inhibitors. Antibody discovery for PHF8, however, was plagued by a familiar problem: antibodies that bound when their antigen was immobilised directly to a surface, but barely bound at all when it was free in solution. The common explanation is that the partial denaturation that accompanies immobilisation reveals epitopes unavailable in solution, but examining the problem in detail for the Plant Homeodomain of PHF8 revealed a connection to its rarely sampled conformations. The prominence these antibodies in the immune responses to PHF8, and to some extent KDM4D, motivates two hypotheses on their origin: either the states are very immunogenic or there is a connection between states of irreversible damage and those sampled reversibly, but rarely, by a protein in solution.
Supervisor: Schofield, Christopher ; Brennan, Paul ; Lawson, Alastair Sponsor: Clarendon Fund
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Monoclonal antibodies ; Drug Discovery ; Chemical Biology ; Structural Biology ; Epigenetics ; Antibody-Inspired drug discovery ; Plant homeodomain ; PHF8 ; Protein dynamics ; KDM4D