| Summary: | <p>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.</p>
<p>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 PHD2<sub>181-407</sub> 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 PHD2<sub>181-407</sub>.Fe(II).2OG.HIF2α<sub>523-542</sub>-CODD complex (obtained with an acetate-based precipitant)
and the PHD2<sub>181-407</sub>.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 PHD2<sub>181-407</sub>.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 PHD2<sub>181-407</sub>.Mn(II).succinate conditions yielded the first
structure of PHD2<sub>181-407</sub> with Molidustat, a clinically applied inhibitor. The PHD2<sub>181-407</sub>.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.</p>
|
|---|