Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding

Article abstract of DOI:10.1002/chem.201804790

Human prolyl hydroxylases are involved in the modification of transcription factors, procollagen, and ribosomal proteins, and are current medicinal chemistry targets. To date, there are few reports on inhibitors selective for the different types of prolyl hydroxylases. We report a structurally informed template-based strategy for the development of inhibitors selective for the human ribosomal prolyl hydroxylase OGFOD1. These inhibitors did not target the other human oxygenases tested, including the structurally similar hypoxia-inducible transcription factor prolyl hydroxylase, PHD2.

Full text of DOI:10.1002/chem.201804790

A Journal of
Accepted Article
Title: Selective inhibitors of a human prolyl hydroxylase (OGFOD1)
involved in ribosomal decoding
Authors: Cyrille C Thinnes, Christopher T Lohans, Martine I Abboud,
Tzu-Lan Yeh, Anthony Tumber, Radosław P Nowak, Martin
Attwood, Matthew E Cockman, Udo Oppermann, Christoph
Loenarz, and Christopher Schofield
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To be cited as: Chem. Eur. J. 10.1002/chem.201804790
Link to VoR: http://dx.doi.org/10.1002/chem.201804790
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Selective inhibitors of a human prolyl hydroxylase (OGFOD1)
involved in ribosomal decoding
Cyrille C. Thinnes,[a] Christopher T. Lohans,[a] Martine I. Abboud,[a] Tzu-Lan Yeh,[a] Anthony Tumber,[a,b]
Radosław P. Nowak,[b] Martin Attwood,[c] Matthew E. Cockman,[c] Udo Oppermann,[b] Christoph
Loenarz[a] and Christopher J. Schofield*[a]
Abstract: Human prolyl hydroxylases are involved in the modification
of transcription factors, procollagen, and ribosomal proteins, and are
current medicinal chemistry targets. To date, there are few reports on
inhibitors selective for the different types of prolyl hydroxylases. Here
we report a structurally informed template-based strategy for the
development of inhibitors selective for the human ribosomal prolyl
hydroxylase OGFOD1. These inhibitors did not target the other
human oxygenases tested, including the structurally-similar hypoxia-
inducible transcription factor prolyl hydroxylase, PHD2.
Introduction
In humans and other animals, prolyl hydroxylases (PHs) play
critical roles in collagen biosynthesis and hypoxia sensing.[1–3]
The PHs are Fe(II) and 2-oxoglutarate (2OG) dependent
oxygenases, which normally produce succinate and CO2 as
coproducts.[4] The procollagen PHs (CP3H and CP4H)
hydroxylate either C3 or C4 of prolyl residues; the latter is
essential for maintenance of the collagen triple helix secondary
Figure 1. Prolyl hydroxylase reactions and structures. (A) Regio- and
stereoselectivity of hydroxylations catalyzed by different types of prolyl
structure (Figure 1A).[5,6] In humans, the prolyl hydroxylase
hydroxylases. Each hydroxylation is coupled to the conversion of 2-oxoglutarate
domain enzymes (PHD1, 2, and 3) act as oxygen sensors in the
(2OG) and O2 into succinate and CO2. OGFOD1 acts on ribosomal proteins, the
chronic response to hypoxia by catalysing oxygen-limited
CPHs act on procollagen, and the PHDs act on the hypoxia inducible factor
(HIF) transcription factors.[5] (B) Structures of 2OG and the 2OG analogue N-
oxalylglycine (NOG). (C, D) Views from the crystallographically observed active
sites of OGFOD1 (PDB 4NHX)[10] and PHD2 (PDB 5L9R)[15] showing the
interactions between active site residues, the bound metal [Mn(II) substituting
for Fe(II)], and NOG.
hydroxylation of prolyl residues in the hypoxia-inducible factor-α
(HIFα) subunits of HIF transcription factors, leading to HIFa
degradation in aerobic conditions (Figure 1A).[1]
The human 2OG oxygenase OGFOD1 has been recently
shown to hydroxylate Pro-62 of the ribosomal protein RPS23.[7–10]
Pro-62RPS23 is situated in the ribosomal decoding site, which is
responsible for ensuring the fidelity of mRNA codon recognition
by tRNA and release factor proteins during protein synthesis.[11,12]
While the role of this hydroxylation in human and animal cells is
not yet understood, the Saccharomyces cerevisiae OGFOD1
homologue Tpa1p is proposed to catalyze dihydroxylation of the
corresponding prolyl residue, and to regulate translational
accuracy in an mRNA sequence context-dependent
manner.[8,13,14]
Of the ~60-70 human 2OG oxygenases, some are current
medicinal chemistry targets, including enzymes involved in
chromatin modification and lipid metabolism.[5,16] Inhibition of the
procollagen hydroxylases is under consideration as a target to
limit the overproduction of collagen associated with certain
cancers and fibrotic diseases.[17] The PHDs are presently being
targeted for the treatment of hypoxia-related diseases, with
inhibitors in late-stage clinical trials for anaemia.[18] If OGFOD1 is
indeed involved in mRNA codon recognition, as suggested based
on studies of yeast homologues,[8,13] small molecule-mediated
inhibition of ribosomal hydroxylation could prove useful for the
treatment of diseases such as muscular dystrophy that are
caused by premature stop-codons through nonsense
suppression.[19] However, due to the uncertainty regarding the
specific roles of OGFOD1 and OGFOD1-catalysed hydroxylation
in animals, it is unclear how exactly its inhibition might manifest.
Thus, such OGFOD1 inhibitors are also of interest as chemical
probes to decipher the biological role of RPS23 hydroxylation, as
[a]
Dr. C. C. Thinnes, Dr. C. T. Lohans, Dr. M. I. Abboud, Dr. T.-L. Yeh,
Dr. A. Tumber, Prof. Dr. C. Loenarz, Prof. Dr. C. J. Schofield
Department of Chemistry, University of Oxford
Oxford, OX1 3TA (UK)
E-mail: christopher.schofield@chem.ox.ac.uk
Dr. A. Tumber, Dr. R. P. Nowak, Prof. Dr. U. Oppermann
Structural Genomics Consortium, University of Oxford
Headington, OX3 7DQ (UK)
[b]
[c]
Dr. M. Attwood, Dr. M. E. Cockman
Centre for Cellular and Molecular Physiology, University of Oxford
Oxford, OX3 7BN (UK)
Supporting information for this article is given via a link at the end of
the document.
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well as those of other recently reported ribosome-associated
hydroxylations.[20–23]
As the 2OG oxygenases are involved in diverse biological
processes, developing inhibitors selective for
a particular
oxygenase is an important therapeutic consideration. The
available biophysical evidence, principally from crystallography,
implies that key features in the active sites of the different types
of human PHs are substantially, but not completely, conserved.[4]
Therefore, there is the potential that inhibitors targeting OGFOD1
could also interfere with hypoxia sensing and collagen
biosynthesis through inhibition of other PHs (or vice versa). To
date, no detailed evidence for inhibitors selective for the different
types of human PHs have been reported. Here, we establish the
viability of a structure-guided template-based approach for the
development of selective OGFOD1 inhibitors which do not target
the other human oxygenases tested, including the human
hypoxia-sensing enzyme PHD2.
Results
In order to assess the viability of developing inhibitors
selective for particular PHs, with a focus on OGFOD1, we first
compared crystal structures of OGFOD1 and PHD2.[10,24,25]
Although there are differences in the OGFOD1 and PHD2 active
sites, the binding modes of 2OG [and 2OG analogue N-
oxalylglycine (NOG)] are conserved (Figure 1B-1D). The 2-
oxoacid group of 2OG (or NOG) binds to the metal in a bidentate
manner, while the 2OG C-5 carboxylate is positioned to interact
with conserved tyrosine and arginine residues (Tyr169 and
Arg230 in OGFOD1, Tyr329 and Arg383 in PHD2; Figure 1C, 1D).
These comparisons, along with those shown for other human
oxygenases,[4] suggest that inadvertent inhibition of the PHDs
may represent a challenge in developing selective OGFOD1
inhibitors, and vice versa.
Figure 2. Oxygenase inhibitors and their binding modes. (A) Structures of
inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) and PHD2. Like
OGFOD1 and PHD2, HPPD is an Fe(II)-dependent 2-oxoacid oxygenase, but
from a different structural family. (B) Comparison of the common chelation
motifs of sulcotrione 2, GSK1278863 6, and FG2216 7. The structures of
sulcotrione 2 and GSK1278863 6 were modelled onto the crystallographically
observed structure of PHD2 with bound FG2216 7 (PDB 3HQU).[25] A larger
version is shown in Figure S5. (C) In silico binding model of GSK1278863 6 with
OGFOD1 (PDB 4NHX)[10] and PHD2 (PDB 5L9R).[15] The tricarbonyl is expected
to interact with the bound metal, while the N-cyclohexyl groups likely occupy the
substrate binding position.
Given the conserved elements of the OGFOD1 and PHD2
active sites,[10] and evidence for induced fit and conformational
movements in prolyl hydroxylase catalysis,[15] we contemplated a
structurally informed template-derivatization approach for
developing OGFOD1 inhibitors.[26] We first considered the 4-
hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor nitisinone
1 (which is clinically used for the treatment of tyrosinaemia)[27] and
related plant growth inhibitors sulcotrione 2, mesotrione 3,
prohexadione-calcium 4, and trinexapac-ethyl 5 (Figure 2A).[28]
These compounds are related to the ‘tricarbonyl’ chemotype
found in the PHD inhibitor GSK1278863 6, which is currently in
clinical trials for the treatment of anaemia.[18] In addition to
GSK1278863 6, we tested PHD inhibitors FG2216 7, FG4592 8,
and IOX2 9.[29,30]
These inhibitors were screened for binding to OGFOD1 and
PHD2 by differential scanning fluorimetry (DSF) and for inhibition
using matrix-assisted laser desorption ionisation time-of-flight
mass spectrometry (MALDI-TOF MS) (Figure S1-S4).[31,32] As
expected based on previous studies,[33] PHD inhibitors FG2216 7,
FG4592 8, and IOX2 9 inhibited OGFOD1 (Figure S1). Notably,
triketone-based HPPD and plant growth inhibitors 1-5 (Figure 2A),
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related to GSK1278863 6, displayed moderate inhibition of
OGFOD1, while not inhibiting the activity of PHD2 within our limits
of detection (Figure S2). Additionally, these inhibitors increased
the apparent thermal stability of OGFOD1, as observed by DSF
(Figure S3, S4). The inhibition of OGFOD1 by prohexadione-
calcium 4 and trinexapac-ethyl 5 (Figure S2) suggests that further
investigation of these agrochemicals is warranted, given their
potential interactions with other human 2OG oxygenases.
GSK1278863 6 and sulcotrione 2 were modelled into the
crystallographically-observed OGFOD1 and PHD2 active sites,
with consideration for potential metal-chelating properties, salt
bridge interactions, and crystallographic studies of PHD2 with
inhibitors (such as FG2216 7; PDB 3HQU) (Figure 2B, S5).[25]
These analyses imply that both 6 and 2 will engage in Fe(II)
chelation via an enolate form of their 1,3-diketone motif (Figure
S5). Similarly to the C-5 carboxylate of 2OG/NOG,[10] and the
carboxylate of FG2216 7,[25] the GSK1278863 6 carboxylate is
predicted to interact with Tyr169 and Arg230 of OGFOD1. The
OGFOD1:6 model suggests that the inhibitor ring systems bind in
two approximately perpendicular planes, one comprising the
diketone ring and glycinamide side chain, and the other formed
from the cyclohexyl rings (Figure 2C). The model also implies that
the cyclohexyl rings engage differently with the OGFOD1 and
PHD2 active sites (Figure 2C). By contrast, compounds based on
an isoquinoline chemotype (e.g., FG2216 7) are predicted to bind
in a more co-planar manner[25] and were considered less likely to
readily lead to OGFOD1-selective inhibitors.
Both the barbiturate-based PHD inhibitors (e.g., 6) and the
HPPD/plant growth inhibitors (e.g., 1-5) have a tri-carbonyl motif.
However, triketones 1-5 are likely more conformationally flexible
than the barbiturates. Notably, the DSF results suggest that the
triketones stabilize OGFOD1 more than PHD2 (Figure S3),
whereas the glycinamide-containing PHD2 inhibitors (e.g., 6-9)
stabilize PHD2 more than OGFOD1 (Figure S4). We thus
explored whether modification of the barbiturate/cyclohexane-1,3-
dione-based ring scaffolds could be exploited in the development
of selective OGFOD1 inhibitors.
While many reported PHD2 inhibitors possess a glycinamide
side chain, the triketone plant growth/HPPD inhibitors do not
(Figure 2A). Modelling results suggest that the sulcotrione 2
methyl sulfonyl group and the nitro group of mesotrione 3 and
nitisinone 1 may mimic the 2OG/glycinamide side chain binding
at their active sites (Figure 2B). It is possible that the enzyme
active site may accommodate the side chains of nitisinone 1,
sulcotrione 2, and mesotrione 3, which are bulkier than the
glycinamide side chain of FG2216 7; however, in the absence of
structural information, we cannot preclude the possibility that
these inhibitors with bulky side chains interact with the enzyme in
an alternate orientation.
Figure 3. Fragment-based screening approach for OGFOD1 inhibition. (A)
Structures of diketones, triketones, and structurally related compounds used for
fragment-based screening. (B) Inhibitory effect of the fragments on the
hydroxylation activity of OGFOD1 (1 µM). The plotted data represent the mean
percentage inhibition for the experiment performed in triplicate, while the error
bars indicate the standard deviation.
Diketones
(e.g.,
diethyl
malonate
10
and
2-
acetylcyclohexanone 11), triketones (e.g., triacetylmethane 12
and substituted 1,3-cyclohexanediones 13-15), and structurally-
related compounds (e.g., 2¢,6¢-dihydroxyacetophenone 16 and
barbituric acid 17) were screened for OGFOD1 inhibition (Figure
3). The preliminary results suggested that the degree of OGFOD1
inhibition may in part relate to the propensity for inhibitor
enolization. Thus, while the diketones tested (i.e., 10, 11) were
poor inhibitors, the triketones (e.g., 12-15) were more potent
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(Figure 3B). However, the phenolic triketone ‘mimic’ 2¢,6¢-
dihydroxyacetophenone 16 was a relatively poor OGFOD1
inhibitor. The most potent template identified for OGFOD1
inhibition was manifested in the C-5 substituted barbiturate
derivatives (18-20); such compounds are readily enolized, which
is likely beneficial for metal chelation. N-Methylation of the
barbiturate ring (e.g., 20) improved potency, as did the
introduction of an acyl substituent on C-5 of the barbiturate core
(e.g., 18 compared to 17). Replacement of the C-2 oxygen with
sulfur (thiobarbituric acid 19) did not substantially increase the
potency relative to the oxygen analogue (i.e., 18), while
replacement of the oxygen at C-4 with a nitrogen (e.g., 21)
abolished any inhibitory activity (Figure 3B). We therefore focused
on obtaining selective OGFOD1 inhibitors by modifying the C-5
position of a 1,3-dimethyl barbiturate core. Notably, compounds
22 and 23, which combine a barbiturate core with a glycinamide
ethyl ester side chain, did not manifest selectivity for OGFOD1
over PHD2 (Figure 4; Figure S6).
Therefore, 24 was prepared, in which structural features of 22
are ‘merged’ with HPPD/plant growth inhibitors 1-3, by introducing
a C-5 aromatic acyl substituent in place of a glycinamide. To
investigate the optimal spacing between the barbiturate and the
aromatic side chain, the initial screen encompassed a 1,3-
dimethyl barbiturate core bearing acetyl 20, benzoyl 24,
phenylacetyl 25, and hydrocinnamoyl 26 (or CCT3) substituents.
The resulting aromatic compounds potently inhibited OGFOD1,
with an increase in potency being observed upon extending the
carbon chain length from 24 (IC50 = 30 μM) to 25 (IC50 = 2 μM),
and from 25 to 26 (CCT3) (IC50 = 0.7 μM; assays performed using
1 μM OGFOD1; Figure S7). Extending the side chain further (e.g.,
27, 28) did not provide a substantial increase in potency.
Importantly, this series displayed no observable PHD2 inhibition
at concentrations up to 100 μM (Figure S8). These results suggest
that the C-5 substituent is important for obtaining selectivity
between OGFOD1 and PHD2.
The importance of the N-alkyl substituents on inhibition was
examined with barbiturates bearing C-5 cyclopentyl substituents
(Figure 4C). Analogues bearing N,N¢-dimethyl (29), N,N¢-diethyl
(30), and N,N¢-dicyclohexyl (31) groups were prepared (Figure
4C); these analogues were less potent, suggesting that
substituents larger than methyl groups may not be favourable for
OGFOD1 inhibition (Figure S9). Note, however, that
GSK1278863 6 potently inhibits OGFOD1 despite the presence
of N,N¢-dicyclohexyl groups (Figure 2A).[33]
Figure 5. Selectivity of lead compounds against 2OG oxygenases. (A)
Comparison of inhibition data for compounds CCT3 and CCT4 against
OGFOD1 and other human 2OG oxygenases. NI: no inhibition observed. (B)
Western blot (antibody specific for HIFα) showing the lack of impact of CCT3
and CCT4 on the activity of PHD enzymes in HeLa cells.[33] By comparison, the
known PHD inhibitor IOX4 inhibits the hydroxylation of HIFα, preventing
proteasomal degradation.[34] (C) Extent of displacement of 2OG from the active
Figure 4. Optimization studies of barbiturate inhibitors. (A) Structures of the
barbiturate glycinamide ethyl esters tested against OGFOD1 and PHD2. (B)
Panel of phenyl-substituted N,N¢-dimethylbarbiturates, demonstrating the
impact of increasing acyl chain length on OGFOD1 inhibition. (C) Panel of
barbiturates synthesized with different N-alkyl substituents. (D) Panel of C-5
substituted N,N¢-dimethylbarbiturates based on lead compound CCT3 (26).
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site of OGFOD1 (blue) and PHD2 (black) by CCT3 and CCT4 as observed by
compounds do not inhibit the PHDs in cells. Based on a MDR1-
MDCK assay (performed by Cyprotex, UK; see the Supporting
Information), CCT3 and CCT4 demonstrate good cell permeability
properties, and are predicted to be permeable to the blood brain
barrier. Liver microsome stability studies indicate only low levels
of clearance of CCT3 and CCT4 (Cyprotex, UK).
CPMG-edited 1H-NMR. See Figure S15 for additional details.
Modelling suggests that the aryl side chains of 25 and CCT3
likely do not fit in the OGFOD1 2OG binding site due to steric
constraints; instead, the aryl ring may bind in the substrate binding
site, potentially contributing to the selectivity of these inhibitors.
Varying the C-5 side chain with different mono- and bicyclic
aromatic and saturated substituents (32-45) did not have a
substantial impact on potency (Figure 4D, Figure S10). Similarly,
changing the nature of the C-5 link from a ketone to an amide,
and extending the carbon chain length beyond two carbons
between the carbonyl and the substituent (i.e., 46) had little effect.
On the basis of these SAR studies, in particular those
examining the impact of the C-5 and barbiturate N-alkyl
substituents, the sub-micromolar potency inhibitors CCT3 (26)
and CCT4 (42) were selected for further characterization (Figure
4, S11). These two inhibitors were screened for inhibitory activity
against a panel of human 2OG oxygenases (Figure 5A). Of the
human enzymes screened, including the HIF prolyl hydroxylase
PHD2, the ribosomal oxygenases MINA53 and NO66 (which
hydroxylate histidinyl residues in ribosomal proteins)[20] and the
asparaginyl hydroxylase factor-inhibiting HIF (FIH), CCT3 and
CCT4 only potently inhibited OGFOD1 (with IC50 values of 0.73
µM and 0.69 µM, respectively). Additionally, these compounds
showed poor, or no, inhibition of more distantly related 2OG
oxygenases, such as the histone demethylases KDM4A,
JARID1B, and JARID1C (Figure 5A). CCT3 and CCT4 did
potently inhibit the structurally close yeast OGFOD1 homologues
Tpa1p and Ofd1 (which hydroxylate RPS23 prolyl residues;
Figure S12), consistent with the high levels of similarity between
the homologous active sites.[10] It is also notable that the
sensitivity of the current hydroxylation assay is a limitation for
ranking the activities of the potent inhibitors, as the IC50 values
obtained are close to the OGFOD1 concentration used (i.e., the
minimum IC50 value that can be measured is 0.5 µM); thus, these
inhibitors may be more potent than represented by the currently
reported IC50 values.
Discussion
Our results demonstrate the viability of a template-based
approach for the development of selective 2OG oxygenase/prolyl
hydroxylase inhibitors capable of differentiating between closely
related active sites, such as those of the human prolyl
hydroxylases OGFOD1 and PHD2. Furthermore, the optimized
inhibitors also did not inhibit the other 2OG oxygenases tested,
including other ribosomal oxygenases, as well as histone
demethylases. The results suggest that specific inhibitor
‘templates’ may be preferred for certain oxygenases or
oxygenase subfamilies, as supported by work implying differential
selectivity between PH and JmjC histone demethylase
inhibitors.[37] This preference for particular templates may even
extend to enzymes with closely related active sites. Appropriately
modified barbiturate-based inhibitors may selectively inhibit
OGFOD1 because of their ability to support substituents which
extend towards active site residues present in OGFOD1 but not
in PHD2. By contrast, the glycinamide side chain present in many
PHD2 inhibitors (including several compounds in clinical trials,
e.g., 6 and 7) is clearly not required for potent OGFOD1 inhibition.
It should also be noted that potent PHD2 inhibitors without a
glycinamide side chain are known.[33,34] In future work, it will be of
interest to further explore the selectivity of the compounds
reported here. In this regard, studies with the procollagen C-4
(and C-3) prolyl hydroxylases are of particular interest, especially
as the procollagen C-4 PHs are potential therapeutic targets.[38]
There is considerable academic and pharmaceutical interest
in developing chemical probe compounds to investigate the
biological functions of 2OG oxygenases.[39] Our results suggest
that the development of leads based on known pharmaceutical
and agrochemical ‘templates’ (some of which can penetrate the
blood-brain barrier) with well-studied physicochemical properties,
To validate the in vitro inhibition results, direct binding
interactions between inhibitors CCT3 and CCT4 and OGFOD1
and PHD2 were assessed by NMR. While both inhibitors were
observed to strongly bind to OGFOD1, as observed using 1H
Carr-Purcell-Meiboom-Gill (CPMG) analyses (Figure S13), only
weak binding to PHD2 was observed by water-Ligand Observe
Gradient Spectroscopy (wLOGSY) experiments (Figure S14).
Competition experiments between the inhibitors and enzyme-
bound 2OG were conducted by monitoring the recovery of the
enzyme-free 2OG methylene peak at 2.35 ppm using CPMG-
edited 1H-NMR upon addition of the inhibitor.[35] The results
indicate that CCT3 and CCT4 are capable of displacing bound
2OG from the active site of OGFOD1, but not from that of PHD2
(Figure 5C, S15).
such as barbiturates, will be
a productive strategy. The
combination of the tricarbonyl barbiturate template of the PHD2
inhibitor GSK1278863 6 with the side chains of agrochemical
oxygenase inhibitors (e.g., prohexadione-calcium), followed by
subsequent optimization, yielded potent and selective OGFOD1
inhibitors. Future work can now be focused on applying these
OGFOD1 inhibitors to investigate the biological roles of OGFOD1,
and applying a similar inhibitor development strategy to other
ribosomal oxygenases. Based on what is observed in these
functional studies, further optimization of these inhibitors may be
warranted (e.g., if penetration of the blood-brain barrier is
desirable).
We examined the potential inhibition of PHD enzymes by
CCT3 and CCT4 in the HeLa human cell line.[36] As compared to
the known PHD inhibitor IOX4,[34] CCT3 and CCT4 did not
stabilize HIFα (Figure 5B). PHD-catalyzed hydroxylation targets
HIFα for proteasomal degradation, indicating that these
It is important to note that many PHD2 inhibitors reported in
the literature, including those screened in this work, and those
currently in clinical trials, also inhibit OGFOD1.[33] Indeed, they
may also inhibit other human prolyl hydroxylases and 2OG
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[12] W. V. Gilbert, Trends Biochem. Sci. 2011, 36, 127-132.
oxygenases, including those for which assays are currently not
available.[10,40] From a clinical perspective, it is also important to
note that the barbiturate-related ‘triketone’ HPPD inhibitor
nitisinone, which is used in the treatment of type I tyrosinaemia,[27]
is an OGFOD1 inhibitor, something that might be taken into
consideration if nitisinone successors with improved properties
are pursued. In the present work, we have demonstrated that it is
possible to attain selectivity between different 2OG oxygenases,
with lead compounds that inhibit OGFOD1, but not PHD2. Such
‘biochemical selectivity’ is not necessarily an issue with clinical
applications as the desired pharmacological effect/safety profile
may be achieved by controlling metabolism and tissue distribution.
However, we propose that, at least for chronic applications,
biochemical selectivity could and should be optimized during the
development of 2OG oxygenase inhibitors. We also hope that
inhibitors selective for particular 2OG oxygenases may enable
their biological roles to be deciphered.
[13] M. V. Nesterchuk, P. V. Sergiev, O. A. Dontsova, Acta Naturae 2011, 3,
22-33.
[14] L. E. Alksne, R. A. Anthony, S. W. Liebman, J. R. Warner, Proc. Natl.
Acad. Sci. U. S. A. 1993, 90, 9538-9541.
[15] R. Chowdhury, I. K. Leung, Y. M. Tian, M. I. Abboud, W. Ge, C. Domene,
F. X. Cantrelle, I. Landrieu, A. P. Hardy, C. W. Pugh, P. J. Ratcliffe, T. D.
Claridge, C. J. Schofield, Nat. Commun. 2016, 7, 12673.
[16] N. R. Rose, M. A. McDonough, O. N. King, A. Kawamura, C. J. Schofield,
Chem. Soc. Rev. 2011, 40, 4364-4397.
[17] J. D. Vasta, K. A. Andersen, K. M. Deck, C. P. Nizzi, R. S. Eisenstein, R.
T. Raines, ACS Chem. Biol. 2016, 11, 193-199.
[18] M. C. Chan, J. P. Holt-Martyn, C. J. Schofield, P. J. Ratcliffe, Mol.
Aspects Med. 2016, 47-48, 54-75.
[19] K. M. Keeling, X. Xue, G. Gunn, D. M. Bedwell, Annu. Rev. Genomics
Hum. Genet. 2014, 15, 371-394.
[20] W. Ge, A. Wolf, T. Feng, C. H. Ho, R. Sekirnik, A. Zayer, N. Granatino,
M. E. Cockman, C. Loenarz, N. D. Loik, A. P. Hardy, T. D. Claridge, R.
B. Hamed, R. Chowdhury, L. Gong, C. V. Robinson, D. C. Trudgian, M.
Jiang, M. M. Mackeen, J. S. McCullagh, Y. Gordiyenko, A. Thalhammer,
A. Yamamoto, M. Yang, P. Liu-Yi, Z. Zhang, M. Schmidt-Zachmann, B.
M. Kessler, P. J. Ratcliffe, G. M. Preston, M. L. Coleman, C. J. Schofield,
Nat. Chem. Biol. 2012, 8, 960-962.
Acknowledgements
[21] R. Chowdhury, R. Sekirnik, N. C. Brissett, T. Krojer, C. H. Ho, S. S. Ng,
I. J. Clifton, W. Ge, N. J. Kershaw, G. C. Fox, J. R. C. Muniz, M. Vollmar,
C. Phillips, E. S. Pilka, K. L. Kavanagh, F. von Delft, U. Oppermann, M.
A. McDonough, A. J. Doherty, C. J. Schofield, Nature 2014, 510, 422-
426.
We thank the Wellcome Trust, Cancer Research UK, and the
Biotechnology and Biological Sciences Research Council
(BBSRC) for supporting our research on 2OG-dependent
oxygenases. C.L. thanks the Leverhulme Trust for an Early
Career Fellowship.
[22] S. Markolovic, Q. Zhuang, S. E. Wilkins, C. D. Eaton, M. I. Abboud, M. J.
Katz, H. E. McNeil, R. K. Leśniak, C. Hall, W. B. Struwe, R. Konietzny,
S. Davis, M. Yang, W. Ge, J. L. P. Benesch, B. M. Kessler, P. J. Ratcliffe,
M. E. Cockman, R. Fischer, P. Wappner, R. Chowdhury, M. L. Coleman,
C. J. Schofield, Nat Chem Biol 2018, 14, 688-695.
Keywords: barbiturate • inhibitors • medicinal chemistry •
OGFOD1 • oxygenase
[23] T. Feng, A. Yamamoto, S. E. Wilkins, E. Sokolova, L. A. Yates, M.
Münzel, P. Singh, R. J. Hopkinson, R. Fischer, M. E. Cockman, J.
Shelley, D. C. Trudgian, J. Schödel, J. S. McCullagh, W. Ge, B. M.
Kessler, R. J. Gilbert, L. Y. Frolova, E. Alkalaeva, P. J. Ratcliffe, C. J.
Schofield, M. L. Coleman, Mol Cell 2014, 53, 645-654.
[1]
[2]
C. J. Schofield, P. J. Ratcliffe, Nat. Rev. Mol. Cell Biol. 2004, 5, 343-354.
S. E. Wilkins, M. I. Abboud, R. L. Hancock, C. J. Schofield,
ChemMedChem 2016, 11, 773-786.
[3]
[4]
J. D. Webb, M. L. Coleman, C. W. Pugh, Cell. Mol. Life Sci. 2009, 66,
3539-3554.
[24] M. A. McDonough, C. Loenarz, R. Chowdhury, I. J. Clifton, C. J.
Schofield, Curr. Opin. Struct. Biol. 2010, 20, 659-672.
W. S. Aik, R. Chowdhury, I. J. Clifton, R. J. Hopkinson, T. Leissing, M. A.
McDonough, R. Nowak, C. J. Schofield, L. J. Walport, Introduction to
structural studies on 2-oxoglutarate-dependent oxygenases and related
enzymes, Royal Society of Chemistry, 2015, p. 59.
[25] R. Chowdhury, M. A. McDonough, J. Mecinović, C. Loenarz, E.
Flashman, K. S. Hewitson, C. Domene, C. J. Schofield, Structure 2009,
17, 981-989.
[26] C. C. Thinnes, A. Tumber, C. Yapp, G. Scozzafava, T. Yeh, M. C. Chan,
T. A. Tran, K. Hsu, H. Tarhonskaya, L. J. Walport, S. E. Wilkins, E. D.
Martinez, S. Müller, C. W. Pugh, P. J. Ratcliffe, P. E. Brennan, A.
Kawamura, C. J. Schofield, Chem. Commun. 2015, 51, 15458-15461.
[27] P. J. McKiernan, Drugs 2006, 66, 743-750.
[5]
[6]
C. Loenarz, C. J. Schofield, Nat. Chem. Biol. 2008, 4, 152-156.
K. L. Gorres, R. T. Raines, Crit. Rev. Biochem. Mol. Biol. 2010, 45, 106-
124.
[7]
R. S. Singleton, P. Liu-Yi, F. Formenti, W. Ge, R. Sekirnik, R. Fischer, J.
Adam, P. J. Pollard, A. Wolf, A. Thalhammer, C. Loenarz, E. Flashman,
A. Yamamoto, M. L. Coleman, B. M. Kessler, P. Wappner, C. J.
Schofield, P. J. Ratcliffe, M. E. Cockman, Proc. Natl. Acad. Sci. U. S. A.
2014, 111, 4031-4036.
[28] G. R. Moran, Arch Biochem Biophys 2005, 433, 117-128.
[29] M. J. Koury, V. H. Haase, Nat. Rev. Nephrol. 2015, 11, 394.
[30] R. Chowdhury, J. I. Candela-Lena, M. C. Chan, D. J. Greenald, K. K.
Yeoh, Y. M. Tian, M. A. McDonough, A. Tumber, N. R. Rose, A. Conejo-
Garcia, M. Demetriades, S. Mathavan, A. Kawamura, M. K. Lee, F. van
Eeden, C. W. Pugh, P. J. Ratcliffe, C. J. Schofield, ACS Chem Biol 2013,
8, 1488-1496.
[8]
[9]
C. Loenarz, R. Sekirnik, A. Thalhammer, W. Ge, E. Spivakovsky, M. M.
Mackeen, M. A. McDonough, M. E. Cockman, B. M. Kessler, P. J.
Ratcliffe, A. Wolf, C. J. Schofield, Proc. Natl. Acad. Sci. U. S. A. 2014,
111, 4019-4024.
[31] R. Chowdhury, K. K. Yeoh, Y. M. Tian, L. Hillringhaus, E. A. Bagg, N. R.
Rose, I. K. Leung, X. S. Li, E. C. Woon, M. Yang, M. A. McDonough, O.
N. King, I. J. Clifton, R. J. Klose, T. D. Claridge, P. J. Ratcliffe, C. J.
Schofield, A. Kawamura, EMBO Rep. 2011, 12, 463-469.
M. J. Katz, J. M. Acevedo, C. Loenarz, D. Galagovsky, P. Liu-Yi, M.
Pérez-Pepe, A. Thalhammer, R. Sekirnik, W. Ge, M. Melani, M. G.
Thomas, S. Simonetta, G. L. Boccaccio, C. J. Schofield, M. E. Cockman,
P. J. Ratcliffe, P. Wappner, Proc. Natl. Acad. Sci. U. S. A. 2014, 111,
4025-4030.
[32] F. H. Niesen, H. Berglund, M. Vedadi, Nat. Protoc. 2007, 2, 2212-2221.
[33] T.-L. Yeh, T. M. Leissing, M. I. Abboud, C. C. Thinnes, O. Atasoylu, J. P.
Holt-Martyn, D. Zhang, A. Tumber, K. Lippl, C. T. Lohans, I. K. H. Leung,
H. Morcrette, I. J. Clifton, T. D. W. Claridge, A. Kawamura, E. Flashman,
X. Lu, P. J. Ratcliffe, R. Chowdhury, C. W. Pugh, C. J. Schofield, Chem.
Sci. 2017, 8, 7651-7668.
[10] S. Horita, J. S. Scotti, C. Thinnes, Y. S. Mottaghi-Taromsari, A.
Thalhammer, W. Ge, W. Aik, C. Loenarz, C. J. Schofield, M. A.
McDonough, Structure 2015, 23, 639-652.
[11] T. M. Schmeing, V. Ramakrishnan, Nature 2009, 461, 1234-1242.
This article is protected by copyright. All rights reserved.

10.1002/chem.201804790
Chemistry - A European Journal
FULL PAPER
[34] M. C. Chan, O. Atasoylu, E. Hodson, A. Tumber, I. K. Leung, R.
Chowdhury, V. Gómez-Pérez, M. Demetriades, A. M. Rydzik, J. Holt-
Martyn, Y. M. Tian, T. Bishop, T. D. Claridge, A. Kawamura, C. W. Pugh,
P. J. Ratcliffe, C. J. Schofield, PLoS One 2015, 10, e0132004.
[35] M. I. Abboud, T. E. McAllister, I. K. H. Leung, R. Chowdhury, C.
Jorgensen, C. Domene, J. Mecinović, K. Lippl, R. L. Hancock, R. J.
Hopkinson, A. Kawamura, T. D. W. Claridge, C. J. Schofield, Chem
Commun (Camb) 2018, 54, 3130-3133.
[39] C. H. Arrowsmith, J. E. Audia, C. Austin, J. Baell, J. Bennett, J. Blagg, C.
Bountra, P. E. Brennan, P. J. Brown, M. E. Bunnage, C. Buser-Doepner,
R. M. Campbell, A. J. Carter, P. Cohen, R. A. Copeland, B. Cravatt, J. L.
Dahlin, D. Dhanak, A. M. Edwards, M. Frederiksen, S. V. Frye, N. Gray,
C. E. Grimshaw, D. Hepworth, T. Howe, K. V. Huber, J. Jin, S. Knapp, J.
D. Kotz, R. G. Kruger, D. Lowe, M. M. Mader, B. Marsden, A. Mueller-
Fahrnow, S. Müller, R. C. O’Hagan, J. P. Overington, D. R. Owen, S. H.
Rosenberg, B. Roth, B. Roth, R. Ross, M. Schapira, S. L. Schreiber, B.
Shoichet, M. Sundström, G. Superti-Furga, J. Taunton, L. Toledo-
Sherman, C. Walpole, M. A. Walters, T. M. Willson, P. Workman, R. N.
Young, W. J. Zuercher, Nat Chem Biol 2015, 11, 536-541.
[36] Y. M. Tian, K. K. Yeoh, M. K. Lee, T. Eriksson, B. M. Kessler, H. B.
Kramer, M. J. Edelmann, C. Willam, C. W. Pugh, C. J. Schofield, P. J.
Ratcliffe, J. Biol. Chem. 2011, 286, 13041-13051.
[37] C. C. Thinnes, K. S. England, A. Kawamura, R. Chowdhury, C. J.
Schofield, R. J. Hopkinson, Biochim. Biophys. Acta 2014, 1839, 1416-
1432.
[40] F. McMurray, M. Demetriades, W. Aik, M. Merkestein, H. Kramer, D. S.
Andrew, C. L. Scudamore, T. A. Hough, S. Wells, F. M. Ashcroft, M. A.
McDonough, C. J. Schofield, R. D. Cox, PLoS One 2015, 10, e0121829.
[38] J. D. Vasta, R. T. Raines, J Med Chem 2018,
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Entry for the Table of Contents
FULL PAPER
Dr. Cyrille C. Thinnes, Dr. Christopher T.
Lohans, Dr. Martine I. Abboud, Dr. Tzu-
Lan Yeh, Dr. Anthony Tumber, Dr.
Radosław P. Nowak, Dr. Martin Attwood,
Dr. Matthew E. Cockman, Prof. Dr. Udo
Oppermann, Prof. Dr. Christoph Loenarz,
Prof. Dr. Christopher J. Schofield*
Humans have 60-70 2OG-dependent oxygenases, of which some are important
therapeutic targets. A priority in the development of oxygenase inhibitors is to
identify scaffolds selective for a particular enzyme, made challenging by the highly-
conserved oxygenase active sites. Here, we describe the development of potent
inhibitors for the oxygenase OGFOD1 which do not inhibit related enzymes, such as
the physiologically important oxygenase PHD2.
Page No. – Page No.
Selective inhibitors of a human prolyl
hydroxylase (OGFOD1) involved in
ribosomal decoding
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