A fragment-based approach towards understanding molecular recognition of 4’-phosphopantetheine adenylyltransferase from Mycobacterium abscessus
The work described in this thesis details the application of a fragment-based approach targeting 4’-phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). A fragment screen of the Abell laboratory 960-member library was conducted by Dr Sherine Thomas (Department of Biochemistry, University of Cambridge) and identified 15 hits validated by X-ray crystallography. The structural information presented several opportunities for rational elaboration of fragments into higher affinity probes of Mabs PPAT. A combination of synthetic chemistry and biophysical methods were employed to elaborate hits into higher quality ligands and understand their interaction with Mabs PPAT. Chapters 2 and 3 describe the development of two series of ligands. Firstly, a fragment linking approach was employed to link fragments in the ATP and phosphopantetheine substrate sites. This produced the first series of ligands to occupy both substrate sites of PPAT. Secondly, an ATP site binding ligand previously developed against Mycobacterium tuberculosis PPAT was re-purposed for Mabs PPAT and further optimised. In depth thermodynamic and structural characterisation of the interaction between ligands in this series and Mabs PPAT revealed complex binding phenomena and thermodynamic signatures of ATP site molecular recognition. Chapter 4 describes development of a ligand-based 19F NMR competition assay that enabled more complete characterisation of fragment binding affinities than previously possible for Mabs PPAT. The assay was validated by comparison with ITC and allowed ranking of affinities for several ligands for which this was previously not possible.