Dual-action inhibitors of HIF prolyl hydroxylases that induce binding of a second iron ion

Article abstract of DOI:10.1039/c2ob26648b

Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron· inhibitor stoichiometry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2ob26648b

Organic &
Biomolecular
Chemistry
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Volume 11 | Number 5 | 7 February 2013 | Pages 689–860
ISSN 1477-0520
PAPER
Christopher J. Schofield et al.
Dual-action inhibitors of HIF prolyl hydroxylases that induce binding of
a second iron ion

Organic &
Biomolecular
Chemistry
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PAPER
Dual-action inhibitors of HIF prolyl hydroxylases that
induce binding of a second iron ion†
Cite this: Org. Biomol. Chem., 2013, 11,
732
Kar Kheng Yeoh,‡§a Mun Chiang Chan,§a Armin Thalhammer,a
Marina Demetriades,a Rasheduzzaman Chowdhury,a Ya-Min Tian,b Ineke Stolze,b
Luke A. McNeill,a Myung Kyu Lee,c Esther C. Y. Woon,¶a Mukram M. Mackeen,k
a
Akane Kawamura,a Peter J. Ratcliffe,b Jasmin Mecinović***a and
Christopher J. Schofield*a
Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest
for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the
single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active
site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identifi-
cation of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a
second iron ion at the active site. Following analysis of small-molecule iron complexes and application of
non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichiometry, selected diacyl-
hydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some com-
pounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines.
Received 21st August 2012,
Accepted 30th October 2012
DOI: 10.1039/c2ob26648b
www.rsc.org/obc
domain (CODD) in human HIF-α) signals for the degradation
of HIF-α via the ubiquitin-proteasome pathway. When oxygen
Introduction
In humans and other animals, the effects of limiting oxygen availability becomes limiting, HIF prolyl hydroxylase activity is
are counteracted by the hypoxic response, which is regulated reduced, resulting in accumulation of HIF-α, which dimerizes
by the oxygen-dependent post-translational hydroxylation with constitutively expressed HIF-β to stimulate expression of
(Fig. 1A) of the α-subunit of hypoxia inducible factor (HIF). genes with hypoxia-responsive element (HRE)-containing pro-
Hydroxylation of either of two prolyl residues (Pro402 at the moters. In humans, there are three isoforms of the HIF prolyl
N-terminal oxygen-dependent degradation domain (NODD) hydroxylases (PHD1-3 or EGLN1-3 enzymes). Additionally,
and Pro564 at the C-terminal oxygen-dependent degradation factor inhibiting HIF (FIH) catalyzes asparaginyl hydroxylation
of HIF-α (Asn803 in HIF-1α) in the C-terminal transactivation
domain (CAD) to reduce its interaction with the transcriptional
coactivator p300 in an oxygen-dependent manner. PHD2
(EGLN1) is probably the most important of the HIF hydroxy-
lases for oxygen sensing in healthy human tissues (for reviews
see ref. 1–5). Both the PHD and FIH enzymes are proposed to
contribute to the oxygen sensing capacity of the HIF system
and belong to the large family of Fe(II) and 2-oxoglutarate
(2OG) dependent oxygenases (2OG oxygenases6–8), which share
common structural and mechanistic features (Fig. 1A and 1B).
Inhibition of the PHDs has potential for the treatment of
anemia, ischemia-related diseases, and other diseases9,10 by
enabling a range of cellular and systemic responses that
enhance oxygen delivery or reduce oxygen demand. Early
studies demonstrated that iron chelators, such as deferox-
amine (DFO, Fig. 1C), and transition metals ions, such as
Co(II), also induce the hypoxic response,11,12 with the latter
likely to act (at least in part) via competition with Fe(II) for
binding to the active sites of the HIF hydroxylases. Several
aChemistry Research Laboratory, Department of Chemistry, University of Oxford,
12 Mansfield Road, Oxford OX1 3TA, UK. Fax: +44 (0) 1865 275625;
Tel: +44 (0) 1865 275674; E-mail: christopher.schofield@chem.ox.ac.uk
bHenry Wellcome Building for Molecular Physiology, University of Oxford, Oxford
OX3 7BN, UK
cBioNanotechnology Research Centre, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon
305–806, Republic of Korea
†Electronic supplementary information (ESI) available: Additional experimental
information, including synthetic procedures and full characterization of all new
compounds. See DOI: 10.1039/c2ob26648b
‡Current address: School of Chemical Sciences, Universiti Sains Malaysia, 11800
USM, Pulau Pinang, Malaysia.
§These authors contributed equally to this study.
¶Current address: Department of Pharmacy, National University of Singapore,
18 Science Drive 4, Singapore 117543, Singapore.
kCurrent address: School of Chemical Sciences, Faculty of Science & Technology,
Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
**Current address: Institute for Molecules and Materials, Radboud University
Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
E-mail: j.mecinovic@science.ru.nl
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Fig. 1 Structural considerations for the design of diacylhydrazines as ‘dual-function’ inhibitors of the HIF prolyl hydroxylases. (A) The reaction catalyzed by the HIF
prolyl hydroxylases (PHD enzymes). (B) View from a crystal structure of PHD2 in complex with a bidentate iron chelating inhibitor 5.26 (C) Structures of inhibitors of
2OG dependent oxygenases that act via generic iron chelation, such as deferoxamine 2, or by competition with the 2OG co-substrate, such as N-oxalyl amino acids
including N-oxalyl glycine 3,37 and isoquinoline compound 5.26 Dimethyloxalyl glycine (DMOG) 4 is a cell-penetrating form of 3. Ligand atoms involved in Fe(II) che-
lation are shown in bold. (D–G) Views derived from representative small-molecule crystal structures of diacylhydrazine-iron complexes.27,55 (D, E) Views derived from
a crystal structure of a diacylhydrazine–iron complex with tridentate iron chelation (CSD ID OKUGIK27). (F, G) View derived from a crystal structure of a diacylhydra-
zine–iron complex with concomitant bidentate and tridentate iron chelation (CSD ID QEVBEY55).
transition metal ions have also been shown to act as 2OG oxy-
genase inhibitors.11,13
We therefore aimed to develop inhibitors that bind to the
PHD active sites and simultaneously deplete iron levels in
Following earlier studies on procollagen prolyl hydroxy- cells, i.e. have a ‘specific’ and a ‘non-specific’ component to
lases, small-molecule inhibitors of the PHDs were their mode of action. Here, as a proof of principle, we report
identified,10,14–21 most of which compete with the 2OG co-sub- the successful design of such dual-action inhibitors based on
strate for binding to the enzyme active site (for review, see ref. the analysis of small molecule iron complexes along with their
22). Most, if not all, of the reported compounds that inhibit synthesis, structure–activity analyses and cell-based evaluation.
2OG oxygenases bind to the single active site metal in a biden- ‘Non-denaturing’ electrospray ionization mass spectrometry
tate manner as observed by crystallographic analyses.6,7,15,23–25 (ESI-MS) was used as a principal analytical tool in identifying
Representative examples of 2OG-competitive inhibitors dis- inhibitors leading to the desired metal binding stoichiometry.
playing bidentate iron chelation include the generic 2OG oxy-
genase inhibitor N-oxalyl glycine (NOG, its cell-penetrating
ester form is dimethyl oxalyl glycine, DMOG), as well as bicyc-
Results and discussion
lic inhibitors such as 2-(1-chloro-4-hydroxyisoquinoline-3-car-
boxamido)acetic acid, BIQ26 (Fig. 1C).
The objective of our study was to design PHD2 inhibitors that
Overall, previous studies have demonstrated that PHD inhi-
bition can be achieved by limiting iron availability (e.g. as with
the iron chelator DFO) or by specific binding of inhibitors to
the active sites of HIF hydroxylases. It is unclear whether a
selective and complete inhibition of all HIF hydroxylases is
desirable to better mimic the ‘natural’ hypoxic response, due
to the potential involvement of oxygenases other than the HIF
hydroxylases and the lack of understanding of the precise roles
of individual PHD isoforms and FIH. Indeed, relatively non-
specific compounds may better mimic the ‘natural’ hypoxic
response.
bind ferrous iron at its active site and deplete cellular iron
levels by inducing the binding of a second iron ion. We began
by the analysis of reported crystal structures of small molecule-
iron complexes27 and more complex supramolecular struc-
tures.28 These structures revealed that diacylhydrazines can
form octahedral complexes with iron characterized by a triden-
tate chelation mode27 (Fig. 1D and E). Notably, these struc-
tures demonstrate that a single diacylhydrazine moiety has the
potential to simultaneously chelate a second transition metal
ion28 when appropriately functionalized with additional metal-
chelating groups (e.g. phenolic hydroxyl groups or
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heteroaromatic sp2 nitrogens) as shown in Fig. 1F and G. We
thus envisaged that, in contrast to the bidentate chelation
ESI-MS studies
mode observed with all previously reported PHD2 inhibitors, ESI-MS studies on PHD2 in the presence of two equivalents
diacylhydrazines may also enable the binding of a second tran- of ferrous sulfate implied that hydrazide forms
1
a
sition metal ion at the active site in an analogous manner to PHD2·Fe2·1 complex as the major observed new species
the iron chelation observed with functionalized diacylhydra- under standard assay conditions (Fig. 3A). The PHD2·Fe2·1
zines in the small-molecule crystal structures (Fig. 1F and G). complex peak (27 990 Da, peak D, Fig. 3A) has a molecular
We conceived that the chelation of a ‘first’ transition metal by mass of 290 Da larger than that of the no inhibitor control
the possibly deprotonated diacylhydrazine motif might struc- (PHD2.Fe, 27 700 Da, peak B, Fig. 3A), corresponding to the
turally pre-organize the inhibitor to support chelation of a mass of diacylhydrazide 1 (237 Da) and a second iron
second metal ion.
(56 Da). Note that there is a small peak (27 755 Da, peak C,
Initial docking studies using the GOLD software29,30 Fig. 3A) corresponding to a PHD2.Fe2 complex in the
suggested that the diacylhydrazine 1 (Fig. 2) may bind at the no inhibitor control which may result from non-specific
PHD2.Fe active site. Therefore, compound 1 was prepared and binding of Fe(II) ions to the PHD2 protein under the stan-
tested for binding to the catalytic domain of PHD2 (residues dard assay conditions. In contrast, the 2OG co-substrate
181–426, hereafter PHD2) using the ESI-MS (which has pre- or 2OG-competitive inhibitors such as NOG or BIQ do
viously been found to be useful for assaying ligand binding to the not induce binding of
catalytic domain of PHD231–33 and other 2OG oxygenases).24,34–36 Fig. S1†).37
a second iron to PHD2 (ESI
Fig. 2 Scaffolds used for the development of diacylhydrazine derivatives as PHD2 inhibitors. Cell-based activity assays were carried out for selected diacylhydrazines
(in their methyl or ethyl ester forms, compounds highlighted in blue). In vitro potency and cell-based activity data are summarized in Table 1. Some compounds
were not tested, including those with limited solubility or due to interference with the biochemical assays.
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Fig. 3 Mass-spectrometric analysis of the monocyclic diacylhydrazines that apparently induce binding of a second iron ion to PHD2. (A, B) Deconvoluted ESI-MS
spectra under non-denaturing conditions for in the presence of 2 equiv. of Fe(II) ions and 1 equiv. of monocyclic diacylhydrazine derivatives 1–10. There is a small
peak (peak C) in the control (without inhibitors) corresponding to a PHD2.Fe2 complex which may result from non-specific binding of Fe(II) ions to the PHD2 under
the standard assay conditions. (C) Deconvoluted ESI-MS spectra for five active site variants of PHD2 in the presence of 2 equiv. of ferrous ions and 1 equiv. of com-
pound 1; (a) Y303A, (b) Y310F, (c) D254A, (d) M299V and (e) Y329F PHD2. PHD2 variants were purified as N-terminally His6-tagged proteins; removal of the His6-
tag with thrombin leaves six additional residues (GSHMAS) at the N-terminus meaning the variants have higher molecular masses than the untagged proteins used
in (A, B).
To investigate the role of iron in the binding of 1 to PHD2, that under standard conditions, ferrous ions, but not any
we conducted binding experiments in the presence of apo- other tested transition metal ions (except possibly Zn(II)), are
PHD2 (PHD2 without the ferrous iron) and two equivalents of required for the formation of di-metal complexes between di-
Zn(II), Cd(II), Ni(II) or Mn(II), respectively. Notably, only Zn(II) acylhydrazine 1 and PHD2.
gave rise to small peaks that may correspond to PHD2·Zn·1
To explore the molecular features that are required for the
and PHD2·Zn2·1 complexes (ESI Fig. S2†). These results imply binding of the second iron ion, analogs of diacylhydrazine 1
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Scheme 1 Synthetic procedures for preparation of diacylhydrazine derivatives as potential PHD2 inhibitors. Method A: carboxylic acid anhydride, EtOAc. Method
B: monomethyl adipate, 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI), Et3N, THF, then NaOH, THF. Method C: (i) ethyl
pentafluorophenylfumarate, THF : EtOAc 1 : 1; (ii) LiOH, THF : H2O 1 : 2. See Fig. 2 for full structures of individual compounds.
(Fig. 2) were then synthesized. Variants of the sidechain (2–5) of 1 (compound 7) might increase the binding affinity for a
and the pyridine ring (6–10) of hydrazine 1 were prepared. second iron. However, this substitution led to a decreased for-
Compounds 1–10 were obtained from monoacylhydrazines mation of the two iron binding complex (peak K, Fig. 3B) and
(Scheme 1) either via ring opening of anhydrides (Method A), an increased formation of a single iron complex (peak J,
1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylami- Fig. 3B). We hypothesized that this may be due to the stronger
nopropyl) carbodiimide (EDCI) coupling with the monomethyl iron chelating ability of 7 which may result in the binding of 7
adipate followed by basic hydrolysis (Method B), or by nucleo- to free Fe(II) in the solution, therefore limiting the amount of
philic substitution with ethyl pentafluorophenyl fumarate fol- Fe(II) available for two iron binding complex formation. This is
lowed by basic hydrolysis (Method C).
supported by subsequent ESI-MS experiments (Fig. 4) with
ESI-MS studies imply that the binding affinities of diacylhy- varying concentrations of Fe(II) and 7. The results imply that
drazines decrease with increasing size of the side chains the single iron binding complex (PHD2.Fe.7, peak C, Fig. 4) is
(Fig. 3). Glutarate derivative 2 formed a two-iron complex with predominantly formed when Fe(II) is limiting, whereas two
PHD2 (peak E, Fig. 3A); however, no detectable complex was iron binding complex (PHD2.Fe2.7, peak D, Fig. 4) is predomi-
observed with adipate derivative 3, possibly due to the nantly observed when Fe(II) is present in excess.
extended aliphatic side chain of 3 which might hinder efficient
In general, appropriately functionalized bicyclic inhibitors
binding of the compound into the PHD2 2OG binding pocket. have been reported to be more potent against PHD2 than their
Interestingly, the maleate derivative 4 was able to induce monocyclic analogs (provided they fit into the enzyme active
binding of a second iron to PHD2 (peak G, Fig. 3A), whereas site), in part likely because the second ring of the inhibitors
the isomeric fumarate derivative 5 only bound to one iron can form hydrophobic interactions with the amino acid resi-
(peak H, Fig. 3B), revealing that the stereochemistry of the ole- dues located at the entrance of the PHD2 active site (Fig. 1B).38
finic side chain affects the binding of the second iron ion. We therefore prepared bicyclic (11–24, Fig. 2) and tricyclic
When the 2-pyridyl group of the parent compound 1 was (25–28, Fig. 2) analogs of 1 using similar methods to those
replaced by a phenyl (6), 3-pyridyl (8), or 4-pyridyl (10) group, described above. Consistent with the ESI-MS results of the
the analogues lost their ability to chelate a second iron monocyclic derivatives, most of the bicyclic and tricyclic com-
(Fig. 3B), suggesting a specific regiochemical requirement of pounds (11–28) are able to induce the binding of a second
the pyridyl-N to bind a second iron.
iron to PHD2 (Table 1). Substantial single iron complex peaks
We considered that the introduction of an electron-donat- were also detected in some of the two iron binding compounds
ing dimethylamino group to the para-position of aromatic ring under standard conditions (i.e. succinates 15, 25 and maleate
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Fig. 4 Non-denaturing ESI-MS studies demonstrate differential binding modes of 7 under different Fe(II) concentrations. (A) When 7 is in excess (5 equiv.) and Fe(II)
is limiting (0.5 equiv.), an increase of apo-PHD2 (peak A) and PHD2.Fe.7 (peak C) is observed, demonstrating the ability of the 7 to chelate free Fe(II) in the solution.
(B) When Fe(II) is equimolar to compound 7 (1 equiv.), 7 binds to PHD2 with either single Fe (peak C) and dual Fe (peak D) (top spectra). (C) When Fe(II) is in excess
(5 equiv.), 7 binds to PHD2 with dual Fe (peak D, top).
27), however, when Fe(II) is present in excess, only two iron Compounds that bind PHD2 (based on ESI-MS), such as the
binding complex peaks were observed (data not shown). As succinate (1), glutarate (2, not shown), maleate (4) and fuma-
anticipated, 3-quinolinyl derivatives (21–23) (scaffold G, Fig. 2) rate (5) derivatives, gave rise to at least one predicted docking
and fumarate derivatives (24, 28) only bind to PHD2 with a mode displaying bidentate coordination of the catalytic Fe(II)
single iron (Table 1). These results show that the ability of a atom with concomitant salt bridge formation between Arg383
diacylhydrazine derivative to induce a second iron binding to of PHD2 and the carboxylate group of a ligand (Fig. 5). In con-
PHD2 is strongly affected by its side chain and the orientation trast, adipate 3, which did not bind to PHD2, failed to dock
of the aromatic ring.
into the binding site.
Further studies were carried out to compare the PHD2
For compounds capable of a second iron binding, at least
binding affinities of selected diacylhydrazide derivatives with one binding mode with three hydrogen bond acceptor atoms
those of the unreactive 2OG analogue NOG and BIQ (Fig. 1). (typically an sp2-hybridized nitrogen atom, a diacylhydrazine
The ESI-MS competition experiments using equimolar of NOG oxygen atom and diacylhydrazine nitrogen atom) located on
or BIQ revealed that they could substantially compete with the one side of the ligand is apparently required, as illustrated for
diacylhydrazides for binding to the PHD2 active site (ESI the maleate derivative 4 (Fig. 5C). Inhibitor binding to the
Fig. S1†). These results therefore indicate that the diacylhydra- active site iron could cause structural pre-organization of these
zides bind specifically to the 2OG binding site in PHD2 as acceptor atoms to support binding of the second iron. Interest-
reported for NOG,37 BIQ37 and related compounds.38 The ingly, the trans-geometry of the side chain in the fumarate
single iron binding 2-pyridine derivatives (8, 9) and 3-quino- derivative 5 resulted in docking poses with the amide carbonyl
line derivatives (21, 24), as well as the two iron binding 3-iso- of the fumarate side chain pointing towards the entrance of
quinoline 11 are among the few diacylhydrazines that were the active site (Fig. 5D), thus ablating the alignment of three
shown to compete effectively with NOG for binding to PHD2 acceptor atoms which we propose to be required for second
under standard conditions (ESI Fig. S1†).
iron binding. This is consistent with the ESI-MS data demon-
strating that none of the fumarate derivatives 5, 9, 14 and 28
were able to induce the binding of a second iron binding
(Table 1).
Docking studies
To validate the docking data and to investigate residues
We have been unable to obtain crystal structure of PHD2 that may contribute to binding of the second iron ion, we per-
formed site-directed mutagenesis on PHD2. Residues that
complex with a diacylhydrazine, so to investigate the potential
binding modes of the diacylhydrazines, we performed docking could potentially be involved in the induction of the second
studies (ESI†) with selected compounds using the GOLD soft- iron based on crystal structures of PHD2-inhibitor complexes
(PDB IDs: 2HBT, 2G1M,39 3HQR37 were identified and replaced
ware.29,30 A proposed binding mode for the diacylhydrazines
that induce binding of a second iron ion is shown in Fig. 5A. with non-chelating analogs. In particular, the active site
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Table 1 Summary of the in vitro and cell-based potencies of the diacylhydrazines as PHD inhibitors
PHD2·Fen·Cmpd.
Complexes in ESI-MSd
Cell-based activityc
Scaffolda
Core
Sidechain
PHD2 IC50
HIF-1α induction
n = 1
n = 2
b
Cmpd.
N-oxalyl glycine, NOG (DMOG)
2-(1-Chloro-4-hydroxyisoquinoline-3-
carboxamido)acetic acid, BIQ
10 μM
0.3 μM
Active
Active
+
+
1
2
3
4
A
A
2-Pyridyl
2-Pyridyl
Succinate
Glutarate
Adipate
>300 μM
>300 μM
>300 μM
>300 μM
47 μM
>300 μM
0.3 μMe
40 μM
0.082 μM
>300 μM
18.4 μMe
24.6 μMe
>300 μM
9.7 μM
>300 μM
>300 μM
>300 μM
>300 μM
>300 μM
>300 μM
21 μM
—
—
—
—
Active
—
Active
—
Active
—
Active
Active
—
Active
Active
Active
—
Active
Active
—
Inactive
Inactive
—
Inactive
ND
+
+
A
A
2-Pyridyl
2-Pyridyl
No binding
Maleate
+
5 (29)
6
7 (30)
8
9 (31)
10
11 (32)
12 (33)
13
14 (34)
15 (35)
16 (36)
17
18 (37)
19 (38)
20
21 (39)
22 (40)
23
24 (41)
25 (42)
26 (43)
27
A
2-Pyridyl
Phenyl
4-Dimethyl-amino-pyridin-2-yl
3-Pyridyl
3-Pyridyl
Fumarate
Succinate
Succinate
Succinate
Fumarate
Succinate
Succinate
Glutarate
Maleate
Fumarate
Succinate
Glutarate
Maleate
Succinate
Glutarate
Maleate
Succinate
Glutarate
Maleate
+
+
B
B
C
C
C
D
D
D
D
E
+
+
+
+
4-Pyridyl
3-Isoquinolinyl
3-Isoquinolinyl
3-Isoquinolinyl
3-Isoquinolinyl
2-Quinolinyl
+
+
+
+
+
+
+
+
+
+
E
E
2-Quinolinyl
2-Quinolinyl
F
F
F
1-Isoquinolinyl
1-Isoquinolinyl
1-Isoquinolinyl
3-Quinolinyl
3-Quinolinyl
3-Quinolinyl
G
G
G
G
H
H
H
H
+
+
+
+
>300 μM
>300 μM
>300 μM
0.4 μMe
0.13 μMe
ND
3-Quinolinyl
Fumarate
Succinate
Glutarate
Maleate
3-9H-Pyrido [3,4-b]indolyl
3-9H-Pyrido [3,4-b]indolyl
3-9H-Pyrido [3,4-b]indolyl
3-9H-Pyrido [3,4-b]indolyl
+
+
+
ND
—
ND
28 (44)
Fumarate
54 μM
+
a See Fig. 2 for structures of inhibitor scaffolds. b Determined by AlphaScreen assay in the presence of 1 nM recombinant PHD2 enzyme and 2 μM
of 2OG co-substrate. IC50 values were determined by fitting the dose–response curve as described in the methods section. c The corresponding
methyl or ethyl esters (compound numbers in parentheses) of the diacylhydrazines were tested in the cell-based assays. ND, not determined
(including compounds that were not well soluble, or interfered with the assay). d Number of iron ions bound in the most abundant
PHD2·Fen·compound complex in non-denaturing protein ESI-MS experiments under standard conditions (n = 1, 2). e Compounds that display
bimodal inhibition curve. IC50 values were determined as described in the main text.
variants Y303A, Y310F, D254A, M299V and Y329F were pre- (Table 1). Initial two-concentration screening at 10 μM and
pared and analyzed for binding with 1 by ESI-MS (Fig. 3C). All 300 μM of compounds revealed that the two iron binding
tested variants had some affinity for the binding of 1 with two monocyclic diacylhydrazines 1, 2 and 4 and bicyclic diacylhy-
iron ions, supporting the proposal that chelation of the second drazines 15–20 are weak PHD2 inhibitors. These results are in
iron results from the diacylhydrazines. However, the Y310F, broad concordance with the ESI-MS competition studies,
and in particular Y303A variant resulted in a substantial which indicate their weak binding to PHD2 in the presence of
reduction in the binding affinity of 1. This observation sup- equimolar of the unreactive 2OG analog NOG. Subsequent
ports a potential role for Y303 in binding of the second iron investigation to determine the half maximal inhibitory concen-
ion, in accordance with the binding mode proposed in Fig. 5A, tration (IC50) of the active compounds revealed that while all
perhaps via coordination of its phenolic oxygen. In the case of the single iron binding diacylhydrazides including the fuma-
Y310, it is unlikely that the phenolic oxygen directly coordi- rate derivatives [5 (IC50 47 μM), 9 (IC50 0.082 μM), 14 (IC50
nates to the ‘second’ iron due to its position in the PHD2 9.7 μM), and 28 (IC50 54 μM)], the 3-pyridine 8 (IC50 40 μM),
active site.
and the 3-quinoline 21 (IC50 21 μM) display the typical sigmoi-
dal inhibition curves, the active two-iron binding compounds
exhibit an atypical bimodal type of inhibition curves (Fig. 6).
Some compounds interfere with the AlphaScreen assay, par-
ticularly when used at high concentrations; however, controls
revealed that this does not appear to be the case for these two-
iron binding compounds (data not shown). The fact that the
bimodal curve was only observed with the active two-iron
Inhibitory potencies of the diacylhydrazines
In vitro PHD2 inhibition studies were then carried out using
an amplified luminescent proximity homogeneous assay
(ALPHA), hereafter referred to as the AlphaScreen assay40
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Fig. 5 Possible structural explanation for diacylhydrazine-induced binding of a second iron at the PHD2 active site. (A) Proposed ligand interaction diagram for dia-
cylhydrazines that induce binding of a second iron in the PHD2 active site. The model for this potential binding mode was derived from structure–activity data
(Table 1) followed by docking analyses. (B, C and D) Representative binding modes for inhibitors 1, 4 and 5 obtained from docking analyses using the GOLD soft-
ware. Selected residues of the PHD2 active site and the active site catalytic iron atom (red) are highlighted. For docking, protein coordinates were derived from the
co-crystal structure of PHD2 with the bicyclic isoquinoline inhibitor BIQ (PDB ID 2HBT). Proposed locations for binding of the second iron (green) are indicated. Note
that the experimental results reveal that the succinate-(1) and maleate-(4) derivatives bind with two irons, but the fumarate-(5) derivative with only one iron.
binding compounds led to the hypothesis that this could induce the binding of a second iron to the enzyme active sites
result from two (or more) general mechanisms of PHD2 inhi- may, at least to an extent, reflect their inhibitory potencies (i.e.
bition by these compounds: one via binding to the enzyme a stronger ligand is generally expected to be a more potent
active site and another via chelation of free Fe(II). We propose inhibitor as demonstrated by compounds 8, 9, 11, and 21);
that the bimodal inhibition curve is a result of the changing although it is not always the case. For instance, compound 24
relative concentrations of equilibrating apo-PHD2, PHD2.Fe, (a single-iron binding compound) which substantially com-
PHD2.Fe.inhibitor, PHD2.Fe2.inhibitor and inhibitorn.Fe com- petes with NOG in the ESI-MS studies only showed weak
plexes. The ESI-MS data for 7 provide some support for this inhibitory potency (IC50 > 300 μM). The differences observed
proposal (Fig. 4). When Fe(II) is present in excess of the com- in the binding affinities and inhibitory potencies of the inhibi-
pound 7 (Fig. 4C), the inactive PHD2.Fe2.7 complex is observed tors may reflect the different assay conditions used in the
but as the Fe(II) concentration is lowered in comparison to the ESI-MS (i.e. gas phase) and AlphaScreen (i.e. aqueous phase)
compound concentration (Fig. 4B), the active PHD2.Fe experiments.
complex is observed, in concordance with the apparently
reduced inhibition observed in the AlphaScreen assay at
higher compound concentrations (phase 2 of the bimodal
curve, Fig. 6). However, when the compound is present in
Cell-based studies
excess over Fe(II) (Fig. 4A), the amount of inactive apo-PHD2 We proceeded to investigate the cell-based activity of selected
increases with increasing 7 (possibly consistent with phase 3 diacylhydrazide compounds, using HIF-1α stabilization as an
of inhibition curve observed in the AlphaScreen assay, Fig. 6), indicator for cellular PHD2 inhibition (Fig. 7). Initially, we con-
most likely due to the chelation of free Fe(II) in solution by 7. sidered the possibility that at least some of the compounds
The operation of the two inhibition mechanisms results in the might be cell-permeable in their free acid form due to their
difficulty in determining the IC50 values for the active two-iron structural similarity to the reported cell-based HIF prolyl
binding compounds. To allow a rough estimation of inhibitory hydroxylase inhibitor BIQ (Fig. 1C).41 Diacylhydrazine deriva-
potencies, we calculated the IC50s by analyzing the first phase tives bearing a free carboxylate group with in vitro PHD2
(sigmoidal) of the inhibition curve [for compounds 7 (IC50 inhibitory activity (compounds 7, 11 and 12, Table 1) did not
0.3 μM) and 25 (IC50 0.4 μM)], or by removing the two outlying up-regulate HIF-1α levels in human hepatoma Hep3B cells
points [for compounds 11 (IC50 18.4 μM) and 12 (IC50 (data not shown), suggesting that the diacylhydrazine or car-
24.6 μM)], as indicated in Fig. 6.
boxylic acid moieties negatively affect cell-penetrating ability.
Overall, our ESI-MS binding studies indicate that the PHD2 Therefore, for the subsequent cell-based testings, we used
binding affinities of the diacylhydrazines and their abilities to ester derivatives, which in general are more cell-penetrating
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Fig. 6 Compounds that bind to single Fe display typical inhibition curve, whereas dual Fe binding compounds display atypical bimodal type of inhibition curve.
Results are from AlphaScreen, n ≥ 3. Points removed for rough estimation of IC50 values are highlighted in dashed boxes. The three phases of the bimodal inhibition
curve are indicated by black bars (phase 1, 2 and 3).
than their respective free acids.42 The requisite methyl or ethyl
esters (29–44) were obtained from the corresponding acylhy- bution of iron chelating ability to their cellular 2OG oxygenase
drazines by acylation (Scheme 1). inhibitory potency in Hep3B cells. For this purpose, cells were
For selected compounds, we then investigated the contri-
Hep3B cells were exposed to the esters at two concen- incubated with the iron chelator DFO or DMOG (as positive
trations (0.1 mM and 1 mM) under normoxic conditions for controls) as well as compound 30, in the absence or presence
6 h. The cell-based HIF hydroxylase inhibitor dimethyl oxalyl of equimolar Fe(II), which were added either at the beginning
glycine16,43 (DMOG, Fig. 1C) was used as a positive control or during the final 2 h of a 6 h incubation period. As expected,
and DMSO as a negative control. Consistent with in vitro DFO, DMOG and 30 induced HIF-1α stabilization (Fig. 7B);
inhibition data of their corresponding acids (i.e. 7, 11, and however, in the presence of an equimolar amount of Fe(II),
12) we observed a clear induction of HIF-1α levels by com- HIF-1α stabilization was not observed with DFO or 30 (Fig. 7B).
pounds 30 (data not shown), 32, and 33 (Fig. 7A and B) in a Titrations using different Fe(II) concentrations demonstrated
concentration-dependent manner. Compounds 35–38 were that induction of HIF-1α by 30 was reduced as the iron concen-
also shown to effectively upregulate HIF-1α at higher concen- tration was increased (Fig. 7C). These observations suggest
tration (1 mM), despite the high IC50 values for their cor- that, at least in part, the cellular activity of 30 may be due to
responding acids (i.e. 15, 16, 18, 19)
>
300 μM. iron chelation. Interestingly, however, we observed that
Notwithstanding with its relatively strong binding affinity addition of an equimolar amount of Fe(II) with DMOG also
and inhibition potency of its corresponding acid 21 (IC50 resulted in a reduction in HIF-1α stabilization, compared to
21μM), the ester 39 failed to induce HIF-1α accumulation DMOG alone. NOG and DMOG are relatively poor iron chela-
(data not shown). Compounds 42 and 43 were not tested in tors, hence these results suggest a more complex relationship
cell-based assays due to their low solubility in DMSO between iron availability and the HIF system than currently
(Table 1).
understood. We also cannot rule out the possibility that the
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Fig. 7 Effects of diacylhydrazine-based compounds on cellular HIF-1α levels. (A, D) Immunoblots demonstrating HIF-1α stabilisation in the presence of diacylhydra-
zine derivatives (1 mM and 0.1 mM) in Hep3B cells after 6 h incubation. (B, C) Effects of the addition of Fe(II) ions on the HIF-1α induction caused by 30 after 6 h
incubation. No Fe(II) indicates no addition of extra Fe(II). (E) Dose-dependent HIF-1α induction caused by 29 and 34 after 6 h incubation. (F) Concentration-depen-
dent stabilisation of HIF-1α in RCC4/VHL cells by diacylhydrazine derivatives 5, 14, 29, 30 and 34. (G) Effects of 29 and 30 on HIF prolyl and asparaginyl hydroxy-
lation in VHL-defective RCC4 cells after 5 h incubation (HIF-1α band intensities were used to normalize hydroxylation signals, tubulin/β-actin protein levels were used
as loading controls). DMSO (1%) was used as a negative control; dimethyloxalylglycine (DMOG) and bicyclic isoquinoline inhibitor (BIQ) were used as positive
controls.
addition of iron may shift the equilibrium between uncom- these compounds with factors working to destabilize HIF-1α
plexed compound and (charged) compound-Fe(II) complexes, at higher concentration. Notably, 34 gave rise to detectable
thus altering the amount of compound that is available HIF-1α induction at concentrations as low as 25 μM,
for cellular uptake.27,44
and HIF-1α levels obtained at 50 μM of 34 were in fact
We then tested diacylhydrazine derivatives containing the greater than those obtained with the established cell-active
fumarate side chains (29, 31, 34, 41 and 44) in Hep3B cells. In HIF hydroxylase inhibitor DMOG at 100 μM. On the other
contrast to non-olefinic derivatives which stabilized HIF-1α in hand, 29 showed similar level of HIF-1α stabilization with
a concentration-dependent manner (Fig. 7A), the fumarate DMOG at 100 μM. The ester form of 9 (IC50 0.082 μM) (31) was
derivatives, 29 and 34 displayed better HIF-1α stabilization only slightly active in the tested cells, possibly due to the low
at lower concentration (i.e. HIF-1α stabilization at 0.1 mM com- cell-permeability of the monocyclic compound. Compound 44
pound ≫ HIF-1α stabilization at 1 mM compound) in Hep3B has low solubility in DMSO and was therefore not tested in
cells (Fig. 7D and E), possibly due to the interaction of cells.
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Notably, in RCC4/VHL cells (RCC4 cells are renal carcinoma at the active site.45 However, in the case of the diacylhydrazine-
cells lacking VHL; RCC4/VHL cells are RCC4 cells that have based HIF hydroxylase inhibitors, depletion of cellular ion has
been engineered to produce VHL), some compounds including functional consequences because non-specific iron chelators
30 displayed better HIF-1α upregulation at higher (1 mM) con- can induce HIF-1α and thereby other HIF target genes, includ-
centration, while others including 29 and 34 gave rise to ing two of the three PHD isoforms (PHD2 and PHD3).1,3–5
higher HIF-1α levels when used at the lower concentration Thus, the diacylhydrazine compounds have the potential to act
(200 μM) (consistent with the observation in Hep3B cells) as dual-action inhibitors in cells, by directly inhibiting the HIF
(Fig. 7F). In the RCC4/VHL cell line, 29 displayed better HIF-1α hydroxylases (and at least with the current compounds, likely
upregulation than 34; in contrast in Hep3B cells where these inhibiting other 2OG oxygenases as well) and generally deplet-
compounds displayed similar potency, suggesting the cell-type ing iron by enabling its sequestration at the active sites of the
dependent effect of these compounds. The free acid of 29 (5) PHDs and other 2OG oxygenases. Further structure–activity
at 1 mM, also upregulated HIF-1α, but to a lesser extent than studies should enable the balance between these inhibitor
its ester, likely due to reduced cell permeability. Similarly, the modes to be altered, e.g. by increasing binding to specific 2OG
free acid of 34 (14) showed almost no HIF-1α induction oxygenases. Although there may be concerns as to the stability
(Fig. 7F). To test whether the observed HIF-1α upregulation in of acylhydrazines in vivo with associated safety issues, we note
cells is due to the inhibition of the HIF prolyl and asparaginyl that monoacylhydrazines derivatives have found applications
hydroxylases, we utilized hydroxyl-residue-specific HIF-1α anti- as peptide deformylase inhibitors,46,47 and diacylhydrazines
bodies.41 Experiments were performed in VHL-defective renal have been extensively used as insecticides because of their
carcinoma cells carrying an empty vector (RCC4) using com- structural similarities to the insect molting hormone 20-
pounds 29 and 30, along with DMOG as positive control hydroxyecdysone.48–50 Although we are aware that some diacyl-
(Fig. 7G). In RCC4 cells, the VHL-mediated HIF-1α degradation hydrazines may have toxicity issues in mammalian cells, the
pathway is blocked, allowing simultaneous measurement of work on insecticides48 suggests that modification of the substi-
inhibition of HIF prolyl- and asparaginyl-hydroxylase activity tution patterns may give rise to compounds with acceptable
in cells. In contrast to DMOG which clearly shows a preference safety profiles and without significant toxicity to mammals.
for inhibition of FIH over the PHDs, 29 inhibits HIF prolyl-
Finally, non-heme iron-dependent oxygenases can be cate-
hydroxylation (Hyp564) more than asparaginyl-hydroxylation gorized into those that employ one or two active site ions.
(HyAsn803). Similarly, 30 showed partial selectivity for HIF The identification of compounds that induce binding of a
prolyl-hydroxylation over asparaginyl-hydroxylation.
second ion to a mono-iron oxygenase provides a link between
Interestingly, despite the similar level of HIF-1α induction the two subfamilies, and may help to enable protein-engineer-
by DMOG (1 mM), 29 (0.2 mM) and 30 (1 mM) (Fig. 7F) in ing studies aimed at the development of new catalytic
RCC4/VHL cells, these compounds showed different levels of activities.
inhibition on HIF prolyl-hydroxylation in RCC4 cells (Fig. 7G).
HIF prolyl-hydroxylation was only partially inhibited by DMOG
and 30 at high concentrations (1 mM and 2 mM), compared to
29, which almost completely inhibit HIF prolyl-hydroxylation
at concentration as low as 0.2 mM. We therefore speculate that
Experimental
Non-denaturing ESI-MS experiments
the upregulation of HIF-1α by DMOG and 30 may not be solely The catalytic domain of PHD2 (residues 181–426) was desalted
due to the inhibition of HIF prolyl-hydroxylation.
using a Bio-Spin 6 Column (Bio-Rad, Hemel Hempstead, UK)
into 15 mM ammonium acetate (pH 7.5).31,32 This stock solu-
tion was diluted with the same buffer to a final concentration
of 100 μM. FeSO4·7H2O was dissolved in 20 mM HCl to a con-
centration of 100 mM. This was then diluted with deionized
Conclusions
The results demonstrate that appropriately substituted aryl- water to give a final working concentration of 100 μM. The
diacylhydrazines can act as HIF hydroxylase inhibitors, as indi- protein was mixed with Fe(II) and a potential inhibitor to give
cated by results with isolated PHD2 and by the application of final concentrations of 15 μM PHD2, 30 μM Fe(II), and 15 μM
modification specific antibodies in cells. The ESI-MS exper- inhibitor. In the ESI-MS studies with varying Fe(II) and inhibi-
iments with recombinant PHD2 imply that some, but not all, tor concentrations, concentrations of these reagents were
of the diacylhydrazine-based inhibitors can induce binding of adjusted according to n equivalent of PHD2 concentration.
a second iron ion at the active site of PHD2. Whether or not The solution was then incubated for 10 minutes at room temp-
binding of a second ion occurs is dependent both on the sub- erature prior to ESI-MS analysis. Data were acquired on a
stitution pattern on the aromatic ring and the side chain Q-TOF mass spectrometer (Q-TOF micro, Micromass, Altrinc-
structure.
ham, UK) interfaced with a NanoMate device (Advion Bio-
In terms of their mode of action the diacylhydrazine PHD- sciences, Ithaca, NY, USA) with a chip voltage of 1.70 kV and a
inhibitors are related to a family of serine protease inhibitors delivery pressure 0.5 psi (1 psi = 6.81 kPa). The sample cone
that bind to their target enzymes and achieve selectivity by voltage was typically 80 V with a source temperature of 40 °C
inducing the binding of a zinc ion that is not normally present and with an acquisition/scan time of 10 s/1 s. Calibration and
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sample acquisition were performed in the positive ion mode
in the range of 500–5000 m/z. The pressure at the interface
Acknowledgements
between the atmospheric source and the high vacuum region Our work is supported by the Newton-Abraham Fund (J.M.),
was fixed at 6.60 mbar. External instrument calibration was Malaysian government and Universiti Sains Malaysia (K.K.Y.),
achieved using sodium iodide. Data were processed Khazanah Foundation Merdeka Scholarship (M.C.C.), Cancer
with MASSLYNX 4.0 (Waters).
Research UK (A.T.), the British Heart Foundation, the Com-
monwealth Scholarship Commission (R.C.), the European
Union, the Biotechnology and Biological Sciences Research
Council, and the Wellcome Trust. We thank Kai-Hsuan Chang
for synthetic efforts.
AlphaScreen PHD2 inhibition assay
Full method for PHD2 inhibition assay will be reported else-
where. Standard reaction consisted of PHD2 (1 nM), Fe(II)
(20 μM), ascorbate (200 μM), 2OG (2 μM), biotinylated 19-mer
CODD peptide (DLDLE MLAPYIPMDDDFQL) (60 nM) and
inhibitor (varies) in 2% DMSO, 50 mM HEPES, 0.01% Tween-
20, 0.1% BSA. Details of the N-terminally truncated PHD2 was
prepared as reported.39,51 The IC50 values were calculated
using nonlinear regression with normalized dose-response fit
on Prism GraphPad.
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