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ReviewCite 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: jia@queensu.ca

& 2012 The Author(s) Published by the Royal Society. All rights reserved.

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,

oxygen

NADP+ + H2ONADPH

hemeoxygenase

heme

NADPHreductase

biliverdinCO + Fe2+

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-

oxide synthase, nitric oxide synthase (NOS), catalase,

peroxidases, respiratory burst oxidase, pyrrolases, cytochrome

c oxidase, cytochrome P450 and tryptophan dioxygenase. This

is further complicated by cross-talk between the NOS and HO

systems via a common interaction of nitric oxide (NO) and CO

with sGC [22].

In keeping with the cytoprotective role of HO, both bili-

verdin and its proximal product, bilirubin, have antioxidant

properties, and are important scavengers for free radicals,

such as superoxide, peroxides, hydroxides, hypochlorous

acid, singlet oxygen, nitroxides and peroxynitrite [23–27].

Although seemingly counterintuitive, free iron, which pro-

motes production of intracellular reactive oxygen species

(ROS) [28], ultimately triggers the activation of redox-sensi-

tive signalling pathways to result in cytoprotective benefits

with respect to inflammation, mitochondrial biogenesis,

apoptosis and cell survival [29–31]. Moreover, the increase

in free intracellular iron via heme degradation results in an

augmentation of synthesis of ferritin, a protein involved in

iron sequestration [32,33]. Indeed, the binding of free iron

to the cytoplasmic ‘iron-sensing’ RNA-binding proteins,

iron-regulatory protein-1 and -2 (IRP1 and IRP2), causes the

coordination of events to modify mRNA stability, through

binding to iron-regulatory elements of proteins such as

H- and L-ferritin, transferrin receptor 1, and ferroportin1, all

of which are critical for iron processing and trafficking [34,35].

1.1. Heme oxygenase in disease: important, yetambiguous and conflicting, roles

The protective role of the HO/CO system has been reported

in several disease conditions, including diabetes, heart

disease, hypertension, neurological disorders (Alzheimer’s

disease) and endotoxemia as well as organ transplantation,

fibrosis and inflammation [11,36,37]. There have also been

some studies that support a protective role of HO-1 against

the development of some types of cancers, i.e. breast and

lymph node, which may also involve its role in protection

against oxidative stress [38,39]. By contrast, the observation

of substantial HO-1 activity in a variety cancers, including

pancreatic and prostate, is consistent with the idea that the

cytoprotective properties of HO can be disadvantageous for

the host through conferring an advantage to the tumour by

contributing to cellular resistance to standard cancer treat-

ments. In this type of situation, HO activity becomes a

potential therapeutic target for selective HO inhibitors.

Indeed, HO-1 activity has been found to be upregulated in

response to several therapeutic treatments and implicated

in promoting tumour growth; its inhibition has been found

to increase responsiveness of pancreatic cancer cells to

chemo- and radiotherapy [40–43]. Further implications can

be made regarding tumour angiogenesis and subsequent

tumour growth as HO-1-derived CO has also been associated

with angiogenesis, inducing vascular endothelial growth

factor (VEGF) synthesis [44,45], and stimulating the prolifer-

ation of endothelial cells [46,47], as well as being implicated

in the promotion of angiogenesis by stromal-cell-derived

factor 1 [48]. There are also conflicting reports regarding the

role HO-1 may have in neuronal cells; its overexpression

has been implicated in protection from oxidative injury,

while lack of or inhibition of HO-1 activity may attenuate

neuronal death (reviewed in [37]). Furthermore, studies

have shown that gene deletion of HO-2, which predominates

in the brain, is cytoprotective. Thus, the precise role of the

HO/CO system in neuronal complications, particularly that

of HO-1, though important, is still not clear.

1.2. Heme oxygenase inhibitorsThe development of isozyme-selective HO inhibitors would

serve as powerful pharmacological tools to dissect the HO/

CO system, especially in clarifying situations in which

conflicting observations have been reported such as in neur-

onal and cancer cells, as well as the mechanisms underlying

its physiological effects in conjunction with specific roles

for each isozyme. Moreover, this avenue may provide novel

therapeutic strategies in dealing with various disease states.

For example, in the treatment of hemolytic disease such

as hyperbilirubinemia, in which the Hmox1 gene is signifi-

cantly induced, it may be beneficial to inhibit the inducible

HO-1 isozyme selectively without any deleterious effect

to the ‘housekeeping’ isozyme, HO-2 [49]. Inhibition of

HO-1, using pegylated zinc protoporphyrin, has already

been associated with anti-tumour activity [50] and has been

shown to enhance the effects of contemporary chemo-

therapeutic agents, particularly those that generate ROS

[51]. Traditionally, the metalloporphyrins (e.g. tin/zinc/

chromium protoporphyrin) have been used as inhibitors

in this field, on the basis of their structure being similar to

the heme moiety, which makes for useful competitive inhibi-

tors for HOs. Interestingly, a recent comprehensive study

looked at the ability of a variety of metalloporphyrins

(i.e. iron/zinc/tin/chromium bis glycol/deuteron/meso/

protoporphyrin) to inhibit the HO isozymes [49]. Tin meso-

porphyrin was found to be the most potent inhibitor for

both isozymes, with HO-2 selectivity being greatest for tin

protoporphyrin while zinc compounds were the least inhibi-

tory towards HO-2. However, none of the metalloporphyrins

tested were selective for HO-1.

(a)

(b) (c)

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

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with r.m.s.d. of the a-carbons ranging from 0.697 to 0.862 A,

for the various conformations determined thus far

(i.e. open and closed conformations of the human HO-1

holoenzyme) [58].

The three-dimensional structures of both HOs are predo-

minantly a-helical with the heme sandwiched between the

distal and proximal helices (figure 2b,c) [58–61]. A number

of conserved glycines in the distal helix provide flexibility

to accommodate substrate binding and product release. The

active site of the apoprotein is generally more open than

the holoenzyme. In the holoenzyme, the heme iron is coordi-

nated by His25 in HO-1 (His45 in HO-2) on the proximal side

with a water molecule serving as the sixth ligand on the distal

side, and the distal and proximal helices are positioned closer

together to form a more ‘closed’ conformation. As elucidated

by the crystal structure of the HO-1 holoenzyme, there is still

an inherent flexibility of the distal helix, which allowed it to be

crystallized in both ‘open’ and ‘closed’ conformations. Both

apo- and holoenzymes contain a hydrogen-bond network

involving Asn210, Arg136, as well as a second level of residues,

which includes Tyr58 and Tyr114 to stabilize the position of the

catalytically critical Asp140 residue (Asp160 in HO-2). In the

high-resolution structure of the HO-1 holoenzyme [61], it

was revealed that the distal and proximal helices ‘clamp

down’ to tighten the structure and allow coordination of the

heme moiety with His25 as well as interaction of its propionate

groups with basic side chains without perturbation of the

Asp140 side chain. The tightened structure due to heme conju-

gation also traps critical water molecules in the distal cavity,

which form part of a well-ordered hydrogen-bond network

involving Asp140. This network may serve as a proton shuttle

to anchor the catalytically critical distal water ligand of heme

required for oxygen activation [61]. Although the analogous

network was not fully resolved in the heme-conjugated HO-2

crystal structure, the conformation of Asp160 is similar to

that of Asp140 in HO-1; thus, it is presumed to follow the

same underlying mechanism of catalysis [58].

The major point of sequence divergence between HO-1 and

HO-2 is in the C-terminal region (figure 2a), as well as the pres-

ence of three heme regulatory motifs (HRMs) in HO-2 with a

characteristic Cys-Pro signature; while HRM3 is centred

around Cys127, HRM1 and HRM2 reside in the C-terminus

[58]. There is some controversy with respect to the precise role

of these HRMs. Early studies indicated that these HRMs may

bind heme directly [62,63]. However, recent reports have indi-

cated that the HRMs affect catalytic efficiency without

affecting heme affinity and do not result in additional heme-

binding sites [64,65]. Rather, HRM1 and HRM2 form a thiol/

redox switch that modulates HO-2 affinity, on the basis of the

redox environment. Reducing conditions release the Cys265

thiol group from a disulphide bond with Cys282, allowing it

to form an alternative axial heme ligand with lower affinity

than His45 [66]. Thus, only one heme moiety conjugates with

HO-2, coordinated by either His45 or the lower affinity Cys265.

2. Development of isozyme-selective hemeoxygenase inhibitors: a structure – activityrelationship approach

Our research group is involved in a multi-disciplinary pro-

gramme comprising investigators specializing in medicinal

chemistry, pharmacology and toxicology, and biochemistry/

structural biology, and focuses upon the design and deve-

lopment of novel and potent, isozyme-selective inhibitors

of HO. Our initial investigations were stimulated by reports

of some non-porphyrin-based compounds that were found

to inhibit HO. Novel synthetic peptides were identified as

potent inhibitors of HO-1 activity and shown to have the

ability to prolong heterotopic heart graft survival in rats

[67]. The most notable HO-1 inhibitors included

peptide RESLRNLRGY, having an IC50 of 200 mM, and pep-

tide RNleNleNleRNleNleNleGY-CONH2 (RDP1258), with an

IC50 of 20 mM, against rat spleen HO-1 in vitro. However, this

effect of HO-1 inhibitory activity was compromised by a

secondary effect of inducing HO-1 expression. A series of

imidazopyridines and substituted prolines were also reported

to inhibit both the HO from the parasite Plasmodium yoeliiand the HO of the host mice [68]. Several compounds

were identified in this study, most notably 2-[3-(2-piperidin-

1-yl)ethoxyphenyl]imidazo[1,2-a]pyridine (100% inhibition

of mouse HO at 100 mM) and one stereoisomer of 5-(2-

hydroxyphenyl)-2-methylpyrrolidine-2,3,4-tricarboxylic acid

3,4-diethyl ester (100% inhibition of mouse HO at 100 mM).

Initial work by our group on these compounds did not give

favourable results as potential lead compounds. However, an

imidazole-based compound, namely (2S,4S)-2-[2-(4-chloro-

phenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-4-[((4-aminophe-

nyl)thio)methyl]-1,3-dioxolane (azalanstat, QC-1, figure 3),

originally designed by Syntex as an inhibitor of mammalian

lanosterol 14a-demethylase (14-DM), gave promising results

in its ability to inhibit HO [3]. The non-porphyrin-based

structure of azalanstat was a key initial criterion because

it would minimize cross-reactivity with other heme-binding

proteins. A medicinal chemistry programme was initiated

based on the lead compound, QC-1, and the topological

analysis shown in figure 3. The resultant analogues were

screened by an in vitro CO formation assay, using micro-

somal preparations from rat spleen and brain as

physiological sources of HO-1 and HO-2, respectively [69].

The in vivo efficacies of some select compounds were also

evaluated in mice and rats, as well as in cultured renal

proximal tubule epithelial cells [70]. Initial studies identified

a number of compounds that were selective for the induci-

ble HO-1 isozyme [69]. Indeed, kinetic analyses of QC-1

and its analogues confirmed a non-competitive mode of

inhibition, unlike the metalloporphyrins that compete for

the heme-binding site, thus putatively minimizing

the chances of cross-reactivity with other heme-binding pro-

teins such as NOS and sGC, and increasing selectivity for

the HO isozymes.

The initial structure–activity relationship (SAR) study [69]

investigated the ortho-, meta- and para-amino derivatives of

each of the four diastereomeric analogues of QC-1. Although

no pattern in potency/selectivity for HO-1 was observed

with respect to the position of the amino functionality, it was

found that the (2S,4S) and the (2R,4S) diastereomeric configur-

ations produced analogues with greater potency and

selectivity for HO-1. In a further study, candidate compounds

lacking the aminothiophenol moiety of azalanstat were syn-

thesized and evaluated; the specific compounds synthesized

were the four diastereomeric analogues having a methyl

group in place of the methylene(aminothiophenol) moiety

attached to the 1,3-dioxolane ring, as well as the compound

(QC-15) in which this moiety was replaced by a hydrogen

O O

O O

N

western region

N

N

NH2

Scentral region

Clazalanstat (QC-1)

N

Cl

NN

O

O

NN

N

N

N

O

4

4

32

15

321

5

O ON

N

CF3

S

N

northeastern region

eastern region

ClQC-15 QC-80

QC-86QC-82

QC-308

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-

dazol-1-yl)methyl]-4-methyl-1,3-dioxolane hydrochloride. QC-

15 exhibited similar potency/selectivity for HO-1, albeit with-

out containing any stereogenic centres, an attractive feature for

synthesis. The results of the study thus far would suggest that

structural differences between HO-1 and HO-2 exist, differ-

ences that may be exploited to introduce selectivity. The

compounds resulting from the second study were also found

to be selective for HO, with little or no effect on neuronal

NOS and sGC, although they still showed potent inhibitory

activity towards cytochromes P450 (i.e. CYP3A1/3A2,

CYP2E1) [71,72].

The structures of the novel imidazole–dioxolane-based

HO inhibitors are similar to the known azole-based anti-

fungal compounds, particularly ketoconazole (KTZ), which

has been shown also to have anti-tumour effects in prostate

cancer [73]. Given the increase in HO-1 protein expression

in tumours, such as hyperplastic and undifferentiated malig-

nant prostate tissue [40], as well as the requirement of HO-1

for many solid tumours [43], it was hypothesized that

perhaps the anti-tumour effect of KTZ may be mediated

by HO-1 inhibition [74]. Testing of a series of antifungal

agents demonstrated that the structures containing diazoles

or triazoles are potent inhibitors of both HO isozymes.

It was hypothesized that the azole moiety of these

compounds may interfere with HO activity by binding the

heme iron and forming a complex that is inaccessible to the

catalytic site. However, it was found that KTZ did not

affect the heme absorbance spectrum at the low concen-

trations resulting in inhibition. Indeed, analyses showed

that KTZ inhibition was non-competitive and that it was

also not due to interference with cytochrome P450 reductase

or destruction of the HO protein. Moreover, KTZ showed

weak inhibition towards NOS, yet no inhibition towards

NADPH cytochrome P450 reductase.

In continuation of our medicinal chemistry programme

based on the azalanstat lead, a series of 2-oxy-substituted

1-(1H-imidazol-1-yl)-4-phenylbutanes were synthesized hav-

ing halogens in the phenyl ring and replacement of the

dioxolane moiety by a hydroxyl or carbonyl functional

group [75]. The entire library of compounds was found to

be highly active, with the bromine- and iodine-substituted

derivatives being the most potent. The imidazole–dioxolanes

were all selective for HO-1, and exhibited substantially lower

activity towards HO-2. The corresponding imidazole–

ketones and imidazole–alcohols showed selectivity towards

HO-1 to a lesser degree than the similarly substituted

imidazole–dioxolanes.

The SAR study has involved the design and synthesis of

many analogues in addition to the types described earlier,

for example: imidazole–dioxolane compounds having a

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variety of substituents at the 4-position of the dioxolane ring,

2-oxy-substituted 1-azolyl-4-phenylbutanes to study the

effect of the variation of the azole moiety, a series of 1-aryl-2-

(1H-imidazol-1-yl/1H-1,2,4-triazol-1-yl)ethanones and their

derivatives, and a series of a-(1H-imidazol-1-yl)-v-phenylalk-

anes to study the effect of introduction of heteroatoms

in the alkyl linker. Reference to these analogues is made

later in the discussions of the X-ray crystallographic

structural analyses.

2.1. Enhancing structural knowledge of inhibition withX-ray crystallography

As the number of HO inhibitors increased, functional analyses

identified a number of potent inhibitors, including several

deemed isozyme-selective for HO-1. With the knowledge

obtained from the three-dimensional structure of a truncated

human HO-1 derivative based on previous X-ray crystallo-

graphy studies (figure 2b) [59–61], a structural study of

complexes formed between recombinant human HO-1 in com-

plex with some of our potent and representative inhibitors was

initiated. While the previous approach gave valuable infor-

mation regarding SARs, and the iterative and systematic

methodology was successful in identifying salient features

for inhibition, what was missing was the ‘why’—i.e. the

underlying mechanism leading to inhibition as well as iso-

zyme selectivity. The inhibitors for which crystal structures

were determined in complex with HO-1 were: 2-[2-(4-chloro-

phenyl)ethyl]-2-[(1H-imidazol-1-yl) methyl]-1,3-dioxolane

(QC-15), 1-(adamantan-1-yl)-2-(1H-imidazol-1-yl)ethanone

(QC-82), 4-phenyl-1-(1,2,4-1H-triazol-1-yl)butan-2-one (QC-

86), (2R,4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-

yl)methyl]-4-[((5-trifluoromethylpyridin-2-yl)thio)methyl]-1,3-

dioxolane (QC-80), and 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-

butanone (QC-308) (figure 3). Structural analyses and com-

parisons of the various complexes enabled us to definitively

identify structure–functional relationships to understand the

mode of binding to HO-1 and the mechanism underlying

the inhibition of heme degradation. Moreover, identification

of essential structural features critical for inhibition, both

within the inhibitor as well as structural changes within the

protein, provided valuable insights that enhanced the design

of more effective HO-1 inhibitors. During the course of our

study, the X-ray crystal structure of a truncated, human HO-

2 derivative containing a Cys127Ala mutation was also deter-

mined [58], which provided more insights regarding potential

structural differences that may be used in inhibitor design to

enhance isozyme selectivity (figure 2c).

2.2. A common binding mode for imidazole-basedheme oxygenase-1 inhibitors

One of the early, potent inhibitors found to be isozyme-

selective for HO-1 was QC-15, with IC50’s of 4 + 2 mM for

HO-1 and greater than 100 mM for HO-2 as determined with

CO-formation assays using rat spleen and brain microsomal

fractions, respectively [72]. The potency of this compound

was very similar to that of QC-1 (6 + 1 mM) for HO-1, but

removal of the bulky 4-aminophenylthiol groups in the north-

eastern region resulted in decreased activity towards HO-2 in

comparison with QC-1 whose IC50 was 28 + 18 mM. This com-

pound was also the first to be crystallized, as reported by

Sugishima et al. [76], in complex with rat HO-1 (rHO-1,

2.70 A). Soon thereafter, we successfully crystallized QC-82,

also an isozyme-selective and potent inhibitor of HO-1 with

an IC50 of 7 + 1 mM, in complex with human HO-1 (hHO-1)

at a resolution of 1.54 A [77]. The similarity in inhibitory

effect was quite interesting, given the structural differences

between these two compounds. While QC-15 is similar to

QC-1 except in the absence of the bulky northeastern sub-

stituent, the structure of QC-82 diverges even further.

The central dioxolane group was replaced by a ketone, the

4-chlorophenyl group in the western region was replaced by

a bulky adamantanyl group and the central linker region

between the two had been shortened by two carbon atoms.

The only commonality was the eastern imidazole moiety. Inter-

estingly, spectral analyses of both inhibitor–enzyme

complexes also showed a red-shift in the Soret peak implying

that, while the heme was still conjugated to the enzyme, its

environment had been modified [76,77].

Comparison of the structures of the two inhibitor–HO-1

complexes revealed a common binding mode for these see-

mingly structurally distinct, imidazole-based compounds

(figure 4a,b) [76,77]. While it appears that the inhibitors

bind within the heme-binding pocket, they do not displace

and rather require the heme moiety for binding, which

explains the non-competitive nature of the inhibition as

determined through kinetic analyses. This observation has

been confirmed recently by Sorrenti et al. [78], who reported

that the substrate was required for binding to membrane-free

full-length hHO-1. The inhibitor binds to the distal side of

heme, with the imidazolyl group serving as an anchor with

the nitrogen at position 3 coordinating with the heme iron

to form a true hexacoordinated heme, replacing the critical

distal water molecule as the sixth coordinating ligand. The

western region extends back into the distal side of the

heme-binding pocket where it fits into a hydrophobic

pocket. Comparison of the structures of inhibitor–HO-1 com-

plexes with the respective holoenzymes revealed that there is

very little change in the overall structure to accommodate the

inhibitors. Alignment of the hHO-1–QC-82 complex with the

‘closed’, active conformation of the native holoenzyme gave a

Ca r.m.s.d. of only 0.74 A. Both the heme and proximal helix

shift back to expand the distal pocket to some extent. In the

structure of rHO-1 with QC-15, the heme and proximal

helix are shifted approximately 0.8 A towards the a-meso

carbon along the a–g axis of heme; in that of hHO-1 with

QC-82, the coordinating nitrogen of His25 and the Fe

atom are shifted by 0.91 and 0.85 A, respectively. However,

it is the inherent flexibility of the distal helix that allows the

heme-binding pocket to open up to allow the inhibitor to

slip in and also expands the distal pocket to accommodate

the inhibitor. Moreover, the extent of expansion is dependent

upon the size of the inhibitor with the helix being shifted to a

greater extent to accommodate the bulkier adamantanyl

group of QC-82, resulting in a distal pocket which is more

open relative to either the native holoenzyme or rHO-1 in

complex with QC-15. Indeed, accommodation of the bulky

adamantanyl group of QC-82 resulted from a significant con-

formational change in the distal helix (Ser142 to Ile150) with a

maximal displacement of 3.92 A corresponding to Gly144

(figure 5a). In the complex of rHO-1 with QC-15, the main-

chain conformation of Ser142 and Gly143 changed along

with the bending angle so as to open up the distal helix to

such an extent that the helical structure at the bending

point (Gly143–Gln143) was disrupted. By contrast, the

(c) (d )

(a)

(c)

(e)

(b)

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

(a) (b)

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

affect its IC50 significantly (IC50 ¼ 4.7 + 0.5 mM), whereas

substitution to a central hydroxyl group increased its potency

by almost 10-fold (IC50 ¼ 0.5 + 0.1 mM). This may be due,

in part, to a greater potential to form hydrogen bonds

with trapped water molecules or neighbouring residues.

The increased rotational flexibility of the hydroxyl group,

relative to a carbonyl or dioxolane, may also allow interac-

tion with Asp140 to stabilize binding further and/or

disrupt the critical Asp140 hydrogen-bond network further.

Unfortunately, the increased potency observed for the

alcohol derivative of QC-15 is marred by a loss of selecti-

vity (IC50 ¼ 4.0 + 0.6 mM for HO-2). Thus, culmination of

all the structural and functional data reveals that, while

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hydrogen-bond networks involving trapped water

molecules and neighbouring residues may contribute to the

stabilization of the central region of an inhibitor, they are not

critical for its binding to HO-1.

Recently, to gain more insights into the structural require-

ments for the central region of the QC compounds, a series of

a-(1H-imidazol-1-yl)-v-phenylalkanes was synthesized to

examine the effect of introducing heteroatoms into the central

alkyl linker [82]. Moreover, linkers of various lengths were

also investigated, which provided information regarding

the size limits required for effective binding. Interestingly,

introduction of an oxygen atom decreased inhibitory potency

against HO-1, while derivatives containing a sulphur atom

had increased potency relative to those with a simple alkyl

linker. The presence of polar groups within the distal hydro-

phobic pocket, e.g. Met34 and Met51, may be playing a role

in these stabilizing/destabilizing interactions. Increasing

linker length from one to five carbon atoms resulted in a pro-

gressive increase in inhibitory potency against HO-1, from

44 + 6 to 3.5 + 0.7 mM, respectively; the absence of a

linker resulted in no activity. Presumably, this trend is due

to greater contacts with the distal hydrophobic pocket as

the linker is increased, resulting in greater stabilization of

the western phenyl moiety. A minimal linker is required

for activity, which suggests that stabilization by the distal

hydrophobic pocket is in fact crucial for binding of this

class of compounds. While a five-carbon linker resulted in

the most potent compounds, the most HO-1 selective com-

pounds contained a four-atom linker between the phenyl

and imidazolyl moieties. Alignment of the crystal structures

of the hHO-1 complex with QC-82 (PDB code 3CZY, i.e. our

largest inhibitor binding site) with that of hHO-2 (PDB code

2QPP) shows that a potential difference in the size of the

western hydrophobic pocket may account for this selectivity.

The HO-2 pocket is larger partly due to substitutions of

shorter amino acids; for example, the side chains of

Met51 and Val50 in HO-1 extend further in towards the

western region of the QC compounds than their Thr71 and

Ala70 counterparts in HO-2. Thus, a longer linker may be

required to allow the proper hydrophobic contacts for

stabilization in HO-2.

The common binding mode of the imidazole-based HO-1

inhibitors involves coordination of the heme iron through an

eastern imidazolyl anchor and stabilization of the western

region by a distal, hydrophobic pocket through hydrophobic

interactions and potentially p–p stacking interactions.

Movement of the distal helix allows expansion of the distal

pocket to accommodate bulky groups on the western end.

The central region is not critical for binding although

water-mediated hydrogen-bond networks may contribute to

its stabilization and an optimal linker length of four carbons

may allow isozyme selectivity towards HO-1 without bind-

ing HO-2. It is possible that this central region’s main

contribution is to the proper spacing between the eastern

and western regions, i.e. the ‘anchor’ and the hydrophobic

moiety, so that they may make optimal contacts of heme

coordination and within the distal hydrophobic pocket,

respectively. Interestingly, binding of inhibitor does not per-

turb the critical Asp140 residue, nor any of the surrounding

resides involved in its critical hydrogen-bond network.

Rather, the underlying mechanism by which the imidazole-

based compounds inhibit HO is by disrupting the ordered

solvent structure and, thus, the hydrogen-bonded network,

and ultimately displacing the catalytically critical distal

water ligand to inhibit heme oxidation.

2.3. Anchoring inhibitionThe elegance of the structure and mechanism underlying heme

oxidation has allowed its inhibition by this diverse class of non-

porphyrin-based compounds. The pentacoordinate nature of

the HO-1 heme, with His25 serving as a proximal coordinating

ligand, as well as the immutable positioning of the critical

Asp140 and its hydrogen-bond network, allows the precise

positioning of the catalytically critical water molecule to

serve as a sixth ligand on the distal side upon heme conjugation

[60,61,83,84]. It also makes the distal side of heme a prime

target in designing inhibitors with a coordinating functional

group to serve as an anchor for binding to displace the critical

water ligand. Indeed, the main feature for binding of our QC

compounds is coordination of the heme iron through the N-3

nitrogen of an unsubstituted imidazolyl moiety. Thus, a poten-

tial strategy for improving inhibitor potency is to build a

stronger ‘anchor’ by modifying this imidazolyl group.

A series of compounds was designed in which different

azole groups were substituted for imidazole in the eastern

region [85]. Functional analyses determined that 1,2,4-tria-

zole- and 1H-tetrazole-based inhibitors are potent inhibitors

of HO-1, with an improved selectivity over HO-2. A crystal

structure for hHO-1 in complex with a compound containing

a 1,2,4-triazolyl substituent, namely 4-phenyl-1-(1,2,4-1H-

triazol-1-yl)butan-2-one (QC-86, IC50 ¼ 2.5 + 0.4 mM), was

determined to a resolution of 2.20 A. Analysis of the

enzyme–inhibitor interaction revealed that the triazolyl

moiety is able to provide an additional point of anchor stabil-

ization as the presence and positioning of the N-2 nitrogen

atom is close enough to form a potential hydrogen bond

with the amide group of Gly143 (figure 4c). This additional

hydrogen bond may explain the slightly increased potency

of this compound relative to its imidazolyl counterpart

(cf. IC50 ¼ 4 + 2 mM). Interestingly, shifting the position of

the additional nitrogen atom to form the 1,2,3-triazolyl

variant significantly decreases potency (IC50 ¼ 89 + 1 mM).

Because there was no obvious structural basis for the

decrease, this phenomenon was investigated further by calcu-

lating the Mulliken charges for the coordinating nitrogens of

each of these azole variants. Similar values were determined

for both the 1,2,4-triazole ring (20.464) and the imidazole

ring (20.446), while the resultant delocalized electron

system of the 1,2,3-triazole ring decreased the electronegativ-

ity of the ‘coordinating’ nitrogen (20.281) to potentially

weaken its coordination with the heme iron. Further destabi-

lization of the anchor may be a result of repulsive forces

contributed by the ketone group of Gly139 as the N-2 nitro-

gen of the 1,2,3-triazole would be located 3.05 A away from

this functional group.

Recent studies have shown that replacing the imidazole

with either a 1,2,4-triazole or a tetrazole significantly dimini-

shes the inhibitory effects on the cytochrome P450 family of

drug metabolizing enzymes [86]. Thus, strengthening the

QC-anchor in this manner would be an efficient strategy to

design potent and selective HO-1 inhibitors. It may also be

worthwhile to explore other strategies to strengthen the

anchor by replacing the coordinating atom itself. It was puz-

zling, however, to observe that the azole–dioxolanes and the

azole–alcohols derived from the active 1,2,4-triazole- and

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1H-tetrazole-based ketones were generally less active than

their imidazole-based derivatives [81].

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2.4. A novel, inducible binding modeWhile initial work had concluded that the aminothiophenol

moiety in the northeastern region of azalanstat is not essential

for HO inhibition, it had been noticed that general HO

potency was dependent upon the position of the nitrogen

atom and that, more importantly, its removal altogether

enhanced HO-1 selectivity [69,71,72]. Thus, further investi-

gation of this region was postulated to be important in

determining selectivity for HO-2 using thiophenol and sub-

stituted phenol derivatives as well as small functionalized

derivatives at the 4-position of the dioxolane ring, i.e. the

northeastern region [87]. Unfortunately, most of the com-

pounds synthesized were highly potent inhibitors of both

HOs with only moderate selectivity for HO-1. The addition

of a second aromatic ring system was well tolerated by

HO-1 and enhanced selectivity, although an upper limit to

this region was found based on the loss of inhibitory activity

when this was extended to a bulky adamantanyl group. Inter-

estingly, within the thiophenol/phenol series, four of the six

most selective compounds contained hydrocarbon moieties

in the northeastern region, suggesting the importance of

non-polar functionality in this region. Replacement of the

4-aminothiophenol moiety with smaller, polar, functional

groups also resulted in potent inhibitors but with no to

modest selectivity for HO-1. Thus, it was concluded that modi-

fications solely in this northeastern region would not result in

HO-2 selectivity and, moreover, may not be an efficient

avenue in the development of highly selective HO-1 inhibitors.

From a structural point of view, however, it was not

directly obvious as to how these bulky northeastern groups

would be accommodated to allow binding to HO-1. None

of the previous structures indicated much flexibility within

the proximal region, with most of the movement occurring

distally. A putative proximal hydrophobic pocket had been

observed in the structure of hHO-1 in complex with the

bulky adamantanyl derivative, QC-82, which had not been

apparent in previous structures (figure 4b). Computational

analyses were successful in virtually docking both QC-1

[77] as well as (2R, 4S)-2-[2-(4-chlorophenyl)ethyl]-2-[(1H-

imidazol-1-yl)methyl]-4-[(phenylsulphanyl)methyl]-1,3-dioxo-

lane [87] such that the bulky northeastern groups fit into this

potential pocket. However, the crystal structure of QC-80

(IC50 ¼ 2.1 + 0.6 mM) in complex with hHO-1 revealed that

the binding of this class of compounds is, in fact, a result of

a novel, inducible binding mode, which could not be antici-

pated from any of the structural data obtained previously

[80]. The bulky northeastern region is accommodated by

shifting residues in the proximal helix to induce the creation

of a secondary hydrophobic pocket distinct from that

suggested from previous structures (figure 4d ).

Despite the bulky northeastern region, QC-80 follows the

typical binding mode, interacting with hHO-1 in a manner

analogous to the azole-based compounds that lack substitu-

ents in this region, with one exception. Previous structures

of hHO-1, including the apo- and holoenzymes, as well as

in complex with the other QC-inhibitors lacking a north-

eastern region showed a sharp bend of approximately 608in the proximal helix (Asn30-Ala31) that precedes another

helical segment (Glu32-Lys39). Interaction of the bulky

5-trifluoromethylpyridin-2-yl moiety of QC-80 appeared to

push back residues in this region to ‘unkink’ and extend

the proximal helix up to Gln38 and induce the formation of

a secondary peripheral hydrophobic pocket to accommodate

the northeastern substituent. Residues linking this newly

formed pocket that may contribute hydrophobic interactions

to stabilize the 5-trifluoromethylpyridin-2-yl group include

Ala28, Ala31, Arg35, Phe214 and Glu215. This conformation-

al change expands the binding pocket to a greater extent than

observed with any of the other inhibitors, shifting Gln38 as

much as 11 A from its original position in the native holoen-

zyme. Moreover, there is a rearrangement of residues

between the interior and exterior of the pocket. Binding of

the northeastern region of the inhibitor displaces the

thiol group of Met34, which shifts to the exterior of the

newly formed hydrophobic pocket along with the functio-

nal groups of Phe33, Phe37 and Gln38, while the functional

groups of both Arg35 and Glu32 shift into the interior

where they may be stabilized by electrostatic interactions

between the two side chains (figure 5b).

Thus, the crystal structure of the QC-80 complex revealed

that the proximal helix of hHO-1 also contains a degree of

intrinsic flexibility, although not to the same extent as the

distal helix, which allows binding of inhibitors with a bulky

northeastern region, such as QC-1, by inducing the formation

of a secondary hydrophobic pocket to stabilize this region.

While binding by an ‘induced fit’ mechanism would be more

energetically costly than binding to a preformed binding site,

it has provided further insights into the structural properties

of hHO-1 that were not apparent previously, as well as a

rationale underlying the potency of inhibitors such as azalan-

stat which contain bulky northeastern regions. This new

information may be helpful in future inhibitor design.

2.5. Structure-aided drug design: a novel,‘double-clamp’ binding mode

The discovery of the novel, induced formation of the secondary

hydrophobic pocket to bind the northeastern region of QC-80

shed new light on the observation of the putative proximal, sec-

ondary hydrophobic pocket seen in the structure of hHO-1 with

QC-82 [77]. While this proximal pocket is not involved in bind-

ing the northeastern region as previously postulated, there was

potential in its exploitation to improve inhibitor potency by

creating compounds that could occupy both preformed hydro-

phobic pockets simultaneously. The synthesis and

characterization of 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-buta-

none (QC-308, IC50¼ 0.27 + 0.07 mM), which contains an

additional phenyl moiety in the western region, in contrast to

the usual single hydrophobic group in the QC compounds,

demonstrated a approximately 15-fold increase in potency rela-

tive to its monophenyl analogue, 4-phenyl-1-(1H-imidazol-1-

yl)-2-butanone (QC-65; IC50¼ 4.06 + 1.8 mM) [75]. This was

quite remarkable given that QC-65 was already quite a potent

inhibitor. Determination of the crystal structure of the hHO-1

complex with QC-308 revealed the common elements required

for binding the majority of the QC compounds and, further,

confirmed the presence of a definitive secondary hydrophobic

pocket to accommodate and stabilize the second phenyl

moiety in the western region of QC-308 (figure 4e) [88].

Thus, each of the two phenyl moieties of the diphenyl analogue

fit into separate hydrophobic pockets, resulting in a ‘double-

clamp’ binding mode to provide additional stabilization,

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which likely accounts for the increased potency relative to

the monophenyl variant. Unfortunately, neither QC-65

nor QC-308 are isozyme-selective, being potent inhibitors

of both HO isozymes (IC50¼ 11.3 + 4.7 mM [75] and 0.46 +0.15 mM [88], respectively, for HO-2 in rat brain microsomes).

Replacement of the central ketone moiety of QC-65 with a diox-

olane group had conferred isozyme selectivity (QC-57; 2-[2-

phenylethyl]-2-[(1H-imidazol-1-yl)methyl]1,3-dioxolane) with

IC50’s of 0.76 + 0.4 and greater than 100 mM for HO-1 and

HO-2, respectively [75]. Thus, using the ‘double-clamp’ strategy

to increase the potency of an HO-1 isozyme-selective derivative

such as this would be an avenue worth pursuing.

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3. Looking forwardOver the past decade, since the discovery of azalanstat as a

specific HO inhibitor, we have amassed a large group of

azole-based derivatives that exhibit potent inhibitory activity

specific for HO, with several showing isozyme selectivity.

Through a combination of a medicinal chemistry SAR

approach and X-ray crystallographic structural analyses, we

have gained valuable insights into the common binding

mode of this class of compounds, as well as information

regarding the enzyme responsible for its inherent flexibility,

which also allows for an ‘induced’ fit to accommodate prox-

imal structural motifs. Each series of derivatives has allowed

us to amass more information, which can be used to develop

the next generation of inhibitors. As such, we have concluded

that this class of azole-based HO-1 inhibitors, originally

derived from azalanstat, inhibit heme oxidation through a

non-competitive, yet heme-dependent, mechanism by dis-

rupting the ordered solvent structure integral to the critical

hydrogen-bond network involving Asp140 and displacing

the catalytically critical distal water ligand.

Moreover, from a physiological point of view, this inhibi-

tory mechanism has already demonstrated potential

therapeutic applications as some of our imidazole-based

HO-1 inhibitors have shown the ability to attenuate the

growth of prostate [89] and breast cancer cells in vivo. The rel-

evance of HO in various experimental situations has been

explored by other laboratories through administration of

these novel imidazole-based drugs to both cultured cells

and intact animals. Accordingly, Di Francesco et al. [90]

tested the role of HO-1 in the response of human umbilical

vein endothelial cells in culture to laminar shear stress.

They observed that TNF-a biosynthesis was reduced by

shear stress, and that this reduction was reversed in the pres-

ence of the selective HO-1 inhibitor QC-15. Similarly,

Csongradi et al. [91] used selective HO-1 inhibitors to study

the effects of renal HO inhibition on blood pressure in

mice. Angiotensin II induced hypertension was exacerbated

in the presence of this (QC-13) selective HO-1 inhibitor, as

well as the first-generation HO inhibitor tin mesoporphyrin

(SnMP). SnMP also induced renal medullary HO-1 protein,

while QC-13 was without effect on HO protein levels,

which obviated the complication of increasing quantities of

the enzyme targeted for inhibition.

The salient features of this class of inhibitors include,

first and foremost, an anchor in the eastern region to coor-

dinate with the heme iron atom, usually an imidazole

although a 1,2,4-triazole moiety may strengthen the inter-

action with the added benefit of increasing specificity by

decreasing activity against the cytochrome P450 family of

enzymes. The anchor is connected by a linker region to a

hydrophobic group in the western region, which fits into a

distal hydrophobic pocket and is stabilized by both hydro-

phobic and aromatic stacking interactions. A four-carbon

linker is the optimal length to maximize potency without

compromising isozyme selectivity for HO-1. A five-carbon

linker results in a more potent compound but one that is

also inhibitory against HO-2, possibly due to the latter’s

larger hydrophobic pocket. The presence of polar residues

in the pocket (e.g. Met34) may also accommodate halogena-

tion of the phenyl group as well as the presence of

heteroatoms nearby in the linker region, although an

alternative explanation may be that the larger halogens pro-

vide more points and better contact in the pocket. The distal

helix of hHO-1 has a high inherent flexibility, which gives

the enzyme the capacity to bind inhibitors with a range of

‘bulkiness’ in the western region, while still maintaining

its heme moiety. The maximal limit may be the size of an

adamantanyl group, as its binding caused disruption in

the helical structure at one point of the distal helix. More-

over, the presence of a putative peripheral secondary

hydrophobic pocket can also be exploited to increase potency

by introducing diphenyl variants to the western region,

which can be stabilized by a ‘double-clamp’ binding mode.

A lesser degree of flexibility is associated with the proximal

helix, which results in the accommodation of compounds

with large substituents in the northeastern region. Interaction

of this series of compounds induces a conformational change,

essentially resulting in an ‘unkinking and extension’ of the

helix and rearrangement of resides from the interior and

exterior of the protein, to form a peripheral hydrophobic

pocket to accommodate these groups.

It should be noted that all of the structural analyses of

HO-1 in complex with the QC-inhibitors to date have been

conducted using soluble, truncated derivatives of the protein

[76,77,80,81,88]. By contrast, functional characterization of

inhibitor potency has been performed using the full-length

protein as found in microsomal preparations. A recent com-

parison of native and recombinant truncated proteins

demonstrated differential sensitivity of recombinant and

microsomal HO-1 towards inhibitors [92]. Also, this has led

to some controversy regarding data interpretation, especially

regarding the issue of isozyme selectivity, as some isozyme-

selective compounds were found to be equipotent against

both isozymes when tested against the recombinant, trun-

cated versions [76,92]. Given the structural and sequence

conservation of the HOs within the catalytic core [58], the

lack of selectivity found with the truncated purified protein

was not surprising. However, the recent characterization of

a recombinant full-length HO-1 had indicated that the full-

length form exhibits a 2- to 3-fold greater activity relative to

that of the truncated, soluble form, which was increased

even further in the presence of lipid. Moreover, the C-terminal

hydrophobic tail has an essential role with respect to mem-

brane incorporation as well as in formation of a high-affinity

complex with cytochrome P450 reductase, deemed essential

for maximal catalytic activity [93–95]. The C-terminal

domain is also the major point of sequence divergence between

the two isozymes, especially in the presence of the HRMs in

HO-2 [58]. While current structural information does not

imply a role for this hydrophobic tail in the binding of this

class of inhibitors, its potential role in influencing conformation

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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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