Structural and mechanistic studies on PHD2 and the discovery of potential inhibitors

Human prolyl hydroxylases isoforms 1-3 (HsPHD1-3) are hypoxia sensing 2-oxoglutarate (2OG)-dependent oxygenases that catalyse post-translational modifications of hypoxia-inducible factor (HIFα). The PHDs catalyse the trans-4-prolyl hydroxylation of the oxygen degradation domains (ODDs) in HIFα isoforms, which promotes their subsequent degradation via the von-Hippel Lindau-E3-Ligase-26s proteasomal pathway, i.e., PHD catalysis suppresses expression of HIFα target genes. Molecular analyses, including a crystal structure of PHD2 in complex with a HIF1α fragment peptide, have revealed the PHDs form a discrete 2OG oxygenase subfamily with unusual kinetic properties, though the precise structural basis of the latter have been unclear. PHD inhibition is a therapeutic option for treatment of anaemia/ischaemia-related diseases. In-depth biophysical characterisation of the PHDs could provide insights into their ‘O2-sensing’ ability which may aid the development of therapeutics that differ than the current late-stage/approved 2OG-mimicking PHD inhibitors in use. The work described in this thesis aimed to reveal insights into the mechanism of the PHDs and aid in the development of improved inhibitors through the structural analyses on PHD2.
Significant efforts were made to obtain conditions suitable for producing PHD2- substrate complexes in anaerobic conditions that may enable future O2-initiated timeresolved mechanistic studies. Such studies may reflect key catalytic intermediates and active site interactions that are involved in the reaction of the PHDs and may provide useful data for novel inhibitor discovery. After initiating the reaction with O2 with timepoints exceeding 17 minutes to 5-days, the crystals were cryo-cooled in liquid-N2 to quench catalysis and allow for structural characterisation with X-ray crystallographic methods. Four previously unreported PHD2 crystal forms were obtained with a truncated form of PHD2181-407 and a total of 41 structures were refined with resolutions ranging from 1.17 Å to 3.10 Å. Of the new crystal forms, those for the anaerobic PHD2181-407.Fe(II).2OG.HIF2α523-542-CODD complex (obtained with an acetate-based precipitant) and the PHD2181-407.Mn(II).succinate complex (Mn(II) is substituted for Fe(II) and is catalytically inert) are notable as they are suitable for mechanistic and cocrystallisation/ soaking-based studies, respectively. The acetate-based crystallisation condition provides a robust-high-resolution (achieved 1.07 Å resolution) anaerobic system suitable for time-resolved work, as evidenced by the demonstration of in crystallo catalysis (without apparent crystal degradation) to give the first structure of a PHD2181-407.Fe.product complex (timepoint was 5-days O2-exposure in a 200 μm x 30 μm x 15 μm crystal). The acetate-derived crystallisation condition, from this work, has enabled further mechanistic-based crystallographic studies that are in progress, e.g., including high pressure O2 experiments. The PHD2181-407.Mn(II).succinate conditions yielded the first structure of PHD2181-407 with Molidustat, a clinically applied inhibitor. The PHD2181-407.Mn(II).succinate crystal form was optimised for high-throughput soaking of small-molecule fragments and monodentate Fe-binding compounds to explore potential allosteric and orthosteric sites within PHD2 in a structure-guided approach to drug discovery. The combined studies provide insights into PHD catalysis and a foundation for the development of novel PHD inhibitors.

Keywords: Hypoxia
Prolyl Hydroxylases
PHD2
Succinate
Mechanistic-Based Crystallography
2OG
Molidustat
HIF2-alpha
Hypoxia-Inducible Factors
Oxygenases
Takeda-17
Fumarate
XChem
IOX4
Oxidoreductase
Monodentate Binding Inhibitors
PHD Inhibitors
Peroxyproline
Fragment-Based Drug Discovery
Advisor: Schofield, C