rsif.royalsocietypublishing.org Review Cite this article: Rahman MN, Vukomanovic D, Vlahakis JZ, Szarek WA, Nakatsu K, Jia Z. 2012 Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds. J R Soc Interface 20120697. http://dx.doi.org/10.1098/rsif.2012.0697 Received: 28 August 2012 Accepted: 3 October 2012 Subject Areas: biochemistry Keywords: heme oxygenase, inhibitor, X-ray crystallography, structure, HO-1, azole Author for correspondence: Zongchao Jia e-mail: [email protected]Structural insights into human heme oxygenase-1 inhibition by potent and selective azole-based compounds Mona N. Rahman 1 , Dragic Vukomanovic 1 , Jason Z. Vlahakis 2 , Walter A. Szarek 2 , Kanji Nakatsu 1 and Zongchao Jia 1 1 Department of Biomedical and Molecular Sciences, and 2 Department of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6 The development of heme oxygenase (HO) inhibitors, especially those that are isozyme-selective, promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties and may play a role in several disease states, making it an enticing therapeutic target. Traditionally, the metalloporphyrins have been used as competitive HO inhibitors owing to their structural similarity with the substrate, heme. However, given heme’s important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), non- selectivity is an unfortunate side-effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop novel compounds as potent, selective inhibitors of HO. This resulted in the creation of non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been eluci- dated, which provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non- competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. 1. Introduction The heme oxygenase (HO) system comprises two active isozymes, HO-1 and HO- 2, and is responsible for the regioselective, oxidative cleavage of heme at the a- meso carbon. The degradation of heme produces equimolar amounts of carbon monoxide (CO), ferrous iron (Fe 2þ ) and biliverdin, which is subsequently con- verted to bilirubin by biliverdin reductase (figure 1) [1–4]. While these products were originally viewed as ‘waste’ products, increasing evidence has shown that all three are biologically active, and have contributing as well as complementary roles to provide significant cytoprotection (reviewed in [5]). Studies in knock-out mice have shown that HO-1 deficiency is characterized by intrauterine mortality and chronic inflammation; over 95 per cent of HO-1 2 / 2 knock-out mice die in utero [6,7]. In the only two human cases of HO-1 deficiency reported to date [8,9], numerous anomalies were observed, including hemolysis, inflammation, nephritis, asplenia and early death [10]. Thus, HO-1 appears to play a critical role in normal cellular function in both laboratory animals and humans, largely due to conversion of a toxic molecule, heme, to cytoprotective molecules. The pro-oxidative, pro-inflammatory effects of excess free heme, which lead to fibrotic events, can be countered by its degradation by the HO system as well as the cyto- protective and anti-inflammatory effects of its by-products—namely CO, biliverdin (bilirubin) and Fe 2þ —making them novel targets to alleviate tissue inflammation, oxidative stress and fibrosis (reviewed in [11]). Endogenously formed CO, of which the HO system produces approxi- mately 85 per cent, has been shown to be an important gasotransmitter, with a regulatory role in a variety of cellular functions, including anti-inflammatory, & 2012 The Author(s) Published by the Royal Society. All rights reserved. on July 10, 2018 http://rsif.royalsocietypublishing.org/ Downloaded from
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Structural insights into human hemeoxygenase-1 inhibition by potent andselective azole-based compounds
Mona N. Rahman1, Dragic Vukomanovic1, Jason Z. Vlahakis2,Walter A. Szarek2, Kanji Nakatsu1 and Zongchao Jia1
1Department of Biomedical and Molecular Sciences, and 2Department of Chemistry, Queen’s University,Kingston, Ontario, Canada K7L 3N6
The development of heme oxygenase (HO) inhibitors, especially those that are
isozyme-selective, promises powerful pharmacological tools to elucidate the
regulatory characteristics of the HO system. It is already known that HO has
cytoprotective properties and may play a role in several disease states,
making it an enticing therapeutic target. Traditionally, the metalloporphyrins
have been used as competitive HO inhibitors owing to their structural
similarity with the substrate, heme. However, given heme’s important role
in several other proteins (e.g. cytochromes P450, nitric oxide synthase), non-
selectivity is an unfortunate side-effect. Reports that azalanstat and other
non-porphyrin molecules inhibited HO led to a multi-faceted effort to develop
novel compounds as potent, selective inhibitors of HO. This resulted in the
creation of non-competitive inhibitors with selectivity for HO, including a
subset with isozyme selectivity for HO-1. Using X-ray crystallography, the
structures of several complexes of HO-1 with novel inhibitors have been eluci-
dated, which provided insightful information regarding the salient features
required for inhibitor binding. This included the structural basis for non-
competitive inhibition, flexibility and adaptability of the inhibitor binding
pocket, and multiple, potential interaction subsites, all of which can be
exploited in future drug-design strategies.
1. IntroductionThe heme oxygenase (HO) system comprises two active isozymes, HO-1 and HO-
2, and is responsible for the regioselective, oxidative cleavage of heme at the a-
meso carbon. The degradation of heme produces equimolar amounts of carbon
monoxide (CO), ferrous iron (Fe2þ) and biliverdin, which is subsequently con-
verted to bilirubin by biliverdin reductase (figure 1) [1–4]. While these products
were originally viewed as ‘waste’ products, increasing evidence has shown that
all three are biologically active, and have contributing as well as complementary
roles to provide significant cytoprotection (reviewed in [5]). Studies in knock-out
mice have shown that HO-1 deficiency is characterized by intrauterine mortality
and chronic inflammation; over 95 per cent of HO-1 2/2 knock-out mice die inutero [6,7]. In the only two human cases of HO-1 deficiency reported to date
[8,9], numerous anomalies were observed, including hemolysis, inflammation,
nephritis, asplenia and early death [10]. Thus, HO-1 appears to play a critical
role in normal cellular function in both laboratory animals and humans, largely
due to conversion of a toxic molecule, heme, to cytoprotective molecules. The
pro-oxidative, pro-inflammatory effects of excess free heme, which lead to fibrotic
events, can be countered by its degradation by the HO system as well as the cyto-
protective and anti-inflammatory effects of its by-products—namely CO, biliverdin
(bilirubin) and Fe2þ—making them novel targets to alleviate tissue inflammation,
oxidative stress and fibrosis (reviewed in [11]).
Endogenously formed CO, of which the HO system produces approxi-
mately 85 per cent, has been shown to be an important gasotransmitter, with
a regulatory role in a variety of cellular functions, including anti-inflammatory,
Figure 1. The oxidative degradation of heme in the heme oxygenase/carbonmonoxide (HO/CO) pathway results in the release of equimolar amounts ofcarbon monoxide, ferrous iron and biliverdin, the latter of which is convertedto bilirubin by biliverdin reductase.
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antiapoptotic, antiproliferative, as well as vasodilatory effects
[12–15]. Many of these activities contribute to the cytoprotec-
tive characteristics of HO. In many cases, the mechanisms
underlying these effects involve an increase in the activity of a
pathway such as: synthesis of cyclic guanosine monophosphate
via activation of soluble guanylyl cyclase (sGC) [16,17], stimu-
lation of calcium-dependent potassium channels [18] and
activation of mitogen-activated protein kinase signalling path-
ways [19–22]. In other instances, CO may be inhibitory
through its interaction with a heme moiety, as has been
reported for haemoglobin, myoglobin, prostaglandin endoper-
Figure 2. (a) Sequence alignment of human HO-1 and HO-2. The coordinating His residue and the catalytically critical Asp residue are emboldened in red. Hemeregulatory motifs (HRMs) of HO-2 are indicated by maroon boxes. Ribbon diagrams illustrating the crystal structures of human (b) HO-1 and (c) HO-2. The hememoiety (orange) and critical residues mentioned in the text are depicted as stick diagrams. The heme iron is coordinated through the N-3 nitrogen of the imidazolering of the His residue (black dashes). For simplicity, only water molecules (cyan) in the distal pocket have been included with the critical distal water ligandhighlighted in blue. Structural images were created in PYMOL [57].
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Unfortunately, the strong inhibitory efficacy of the met-
alloporphyrins, due to their structural similarity to heme, is
marred by undesired side-effects, notably non-selectivity,
given the significant role heme has in a number of physiologi-
cally relevant and important enzymes such as cytochrome
P450, NOS and sGC. As such, there have been concerns
raised regarding the validity of interpretations and con-
clusions derived from studies using these compounds
[52–54]. However, it has also been demonstrated that some
of these metalloporphyrins, when used over a specific
concentration range, do maintain selectivity against HO
in vitro; notably, chromium mesoporphyrin IX is the most
useful of the metalloporphyrins, selectively inhibiting HO
but not NOS or sGC at concentrations restricted to 5 mM or
less [55]. Thus, there is a recognizable and urgent need to cul-
tivate isozyme-selective HO inhibitors in order to develop
pharmacological tools and explore novel therapeutics in the
HO field. One of the means to explore this potential is to
look at the differences between the three-dimensional struc-
tures of the isozymes, which can be exploited in this regard.
1.3. Structural conservation of hemeoxygenase isozymes
The HO system comprises two active isozymes [4]. HO-1 is
an approximately 32 kDa protein that is predominantly
expressed in the spleen and can be induced by a variety of
stimuli, including heat-shock, heavy metals, heme and ROS.
Thus, it is known as the inducible HO. By contrast, HO-2,
the constitutive enzyme, is an approximately 36.5 kDa
protein that has a wider distribution in the body, but with
its highest concentration being in the brain and testes. A
third isozyme, HO-3, has been demarcated as a pseudogene
and is inactive, despite sharing approximately 90 per cent
sequence identity with HO-2 [56]. Sequence alignment
between HO-1 and HO-2 shows 45 per cent identity between
the full-length sequences, with 55 per cent identity in the con-
served core region, especially that of the conserved heme
pocket regions (figure 2a) [58]. Structural alignment of the
crystal structures of the truncated, heme-conjugated human
HO-1 and HO-2 shows remarkable structural conservation
Figure 3. Structures of representative QC-inhibitors used for X-ray crystallographic studies, including azalanstat (QC-1) depicting regions of interest for SAR studies.
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atom. The most potent and selective compound towards HO-1
inhibition was (2R,4R)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imi-
Figure 4. Ribbon diagrams depicting the structures of HO-1 in complex with the respective QC-inhibitors. Heme (orange) and inhibitors ( purple) are depicted asstick diagrams. Black dashes illustrate metal coordination via the nitrogen atom of the azole group of either His25 or the QC compound. Hydrogen bonds aredepicted as yellow dashed lines. Selected residues mentioned in the text are depicted as stick diagrams and labelled for clarity. For simplicity, only water moleculesin the distal pocket are shown. Electrostatic surface potentials, as calculated using PYMOL [57], depict positively (blue) and negatively (red) charged areas, and revealhydrophobic pockets (white) into which the western and northeastern regions of the QC-inhibitors fit. All images were prepared using PYMOL [57]. (a) rHO-1 incomplex with QC-15 (PDB #2DY5), (b) hHO-1 in complex with QC-82 (PDB #3CZY—Chain A), (c) hHO-1 in complex with QC-86 (PDB #3K4F), (d ) hHO-1 in complexwith QC-80 (PDB #3HOK—Chain B), (e) hHO-1 in complex with QC-308 (PDB #3TGM—Chain B). The two distal hydrophobic pockets (18 HP and 28 HP) thatstabilize the two phenyl moieties of QC-308 allow a ‘double-clamp’ mode of binding.
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greater shift to accommodate the adamantanyl group of
QC-82 in the hHO-1 complex resulted in an apparent helical
break from Ser142 to Gly144. Thus, the adamantanyl group
may represent the upper limit of what can be accommodated
by shifts in the distal helix.
While the imidazolyl group serves as an anchor by coor-
dinating with the heme iron moiety, the binding of the
western region is stabilized through hydrophobic interactions
involving residues lining the distal hydrophobic pocket, i.e.
Phe33, Met34, Phe37, Val50, Leu54, Leu147, Phe167 and
Phe214. The extent of stabilization of the western region by
the hydrophobic pocket depends on the nature of the western
region as a whole. For example, QC-15 was found to be a
more potent inhibitor than QC-82 when spectral analyses
were used to assay inhibitor binding to the purified, recombi-
nant, truncated hHO-1 used for crystallization, despite the
greater hydrophobicity of the latter’s adamantanyl group
relative to the former’s chlorophenyl moiety in the western
Figure 5. Structural alignments of hHO-1 complexes with QC-inhibitor (green) with the native holoenzyme (PDB #1N45—Chain A) (cyan), both depicted as ribbondiagrams. QC-inhibitors are coloured purple and shown in stick form. (a) QC-82 complex (PDB #3CZY—Chain A). The distal helix is shifted with a maximaldisplacement of Gly144 (3.92 A) as highlighted by comparison of the complex (black label) and native protein (italicized blue label). The resultant helical break isindicated (black arrow). (b) QC-80 complex (PDB #3HOK—Chain B). The proximal helix is shifted with a rearrangement of residues, highlighted as wheat-colouredstick diagrams (black labels) in the complex in comparison with their relative positions in the native protein, highlighted as teal stick diagrams (italicized bluelabels). Structural alignments were performed using SUPERPOSE in CCP4 [79] and the images prepared using PYMOL [57].
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region. Further observation revealed that the shorter, central
linker of QC-82 resulted in this group not extending as far
into the hydrophobic pocket, hence making fewer contacts.
Moreover, in addition to hydrophobic contacts, the chloro-
phenyl group of QC-15 may also be stabilized by p–p
stacking interactions attributable to the prevalence of aro-
matic residues in this region. More information regarding
the structural basis of inhibitor binding was gleaned by
using the known structure data to interpret previous func-
tional data. Previously, a negative correlation was found
between potency and electronegativity of the halogen substi-
tuent in the western region within a series of alcohol
derivatives of 2-oxy-substituted 1-(1H-imidazol-1-yl-4-phe-
nylbutanes) [75,77]. For example, the potency of the alcohol
derivative of QC-15 (IC50 ¼ 0.5 + 0.1 mM) increased upon
substitution of the chlorophenyl group by bromophenyl
(IC50 ¼ 0.14 + 0.06 mM) or the more electronegative iodo-
phenyl group (IC50 ¼ 0.06 + 0.03 mM), and decreased
when replaced by the less electronegative fluorophenyl
(IC50 ¼ 1.4 + 1.1 mM) or a non-substituted phenyl group
(IC50 ¼ 6.2 + 0.8 mM). A close inspection of the hydrophobic
pocket revealed the presence of a polar thiol group (Met34),
which may explain why electronegative moieties in the wes-
tern region still give rise to potent inhibitors. Alternatively,
the larger halogens may provide more points and better
contact with the distal hydrophobic pocket.
One of the key differences between QC-15 and QC-82 is
the central linker region, which contains a dioxolane and
ketone group, respectively. Analysis of the differences in
interaction with HO-1 provided insights into potential fea-
tures, which could be exploited in future drug design. The
dioxolane group of QC-15 is involved in a hydrogen bond
network involving a water molecule as well as the carbonyl
group of Thr135 [76]. A subsequent structure of hHO-1 in
complex with QC-80 (IC50 ¼ 2.1 + 0.6 mM), which also con-
tains a central dioxolane component, revealed a similar
stabilizing interaction [80]. However, this network appears
not to be essential for binding the central region of these
imidazole-based compounds, as there is no analogous
water molecule available for a similar hydrogen-bond inter-
action with the ketone group of QC-82 in one of the
molecules in the asymmetric unit. By contrast, in the crystal
structure of hHO-1 in complex with QC-86 (IC50 ¼ 2.5 +0.4 mM), another azole-based compound with a central
ketone moiety, it appears that the carbonyl group is stabilized
by a hydrogen bond involving an active site water molecule
that is a part of a potential hydrogen-bond network involving
another water molecule and the Thr135 carbonyl group [81];
the water molecule may also be involved in the Asp140
hydrogen-bond network. Given the more open conformation
of the distal pocket to accommodate the bulky adamantanyl
group of QC-82 relative to the other compounds for which
structures have been determined, it is likely that this struc-
tural expansion acts to impede its ability to trap water
molecules, resulting in greater fluidity of the solvent struc-
ture. Further insight into this central region was gained by
looking at previous functional data involving the series
of 2-oxy-substituted 1-(1H-imidazol-1-yl-4-phenylbutanes),
which showed an interesting trend. Comparison of IC50
values showed that, generally, compounds with a central
hydroxyl group tended to be more potent than their dioxo-
lane or ketone counterparts. Substitution of the central
dioxolane group of QC-15 with a ketone group did not
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should not be ignored in future designs, especially in influen-
cing isozyme selectivity. Indeed, the determination of the
crystal structure of both full-length hHO-1 and hHO-2 would
provide valuable information in this regard.
The quest to build a better inhibitor involves much
imagination and constant exploring. While we have gained
experience and knowledge regarding this particular class of
azole-based non-competitive inhibitors for HO-1, we are
always searching for new lead compounds to develop new
classes of inhibitors for various applications. The crystal
structure of the truncated hHO-2 has already provided us
with valuable insights with regard to our complementary
goal of developing selective HO-2 inhibitors. Ideally, it
would also be useful to develop a series of competitive inhibi-
tors that prevent binding of heme altogether. Theoretically,
one would presume that a selective, competitive inhibitor
would have powerful therapeutic applications to greatly
minimize any HO activity if the substrate is not available
for degradation. Recently, caveolin-1 was discovered to com-
petitively inhibit rat HO-1 via the caveolin scaffolding
domain (residues 82–101), with a five amino acid sequence
(residues 97–101: YWFYR) identified as a minimum
sequence for binding. Two aromatic residues, Phe207 and
Phe214, in HO-1 are thought to be essential in the association
with caveolin-1 through p–p stacking and hydrophobic
interactions [96]. Further analysis of this peptide structure
may provide insights into new lead compounds to develop
a series of potential competitive inhibitors.
We hope that through this exciting process, using both
medicinal chemistry SAR and X-ray crystallography, we
will be able to develop powerful pharmacological tools for
delving deeper into the understanding of the mechanisms
by which the HO/CO system exerts its many effects and, in
turn, providing a foundation for the development of novel
therapeutic approaches for the myriad pathologies with
which it is involved.
This research was supported by CIHR. Dr Zongchao Jia holds aCanada Research Chair in Structural Biology and a Killam ResearchFellowship. We thank Dr Paul Ortiz de Montellano and Dr JohnEvans (both from the University of San Francisco) for the generousgift of the hHO1-t233 plasmid and advice regarding its expression.We are grateful to Dr Qilu Ye, Dr Jimin Zheng and Dr Frederick Fau-cher for technical expertise in structural determination, as well as MrBrian MacLaughlin, Ms Tracy Gifford and Mr Wallace Lee for assist-ance in the biological evaluations. Finally, we would like to expressour appreciation to the staff at the Advanced Photon Source (APS)at Argonne National Laboratory (Chicago, IL, USA) as well as atthe Cornell High Energy Synchrotron Source (Ithaca, NY, USA) fortheir support in X-ray data collection.
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