Potent and selective inhibitors of long chain l-2-hydroxy acid oxidase reduced blood pressure in DOCA salt-treated rats

Article abstract of DOI:10.1021/ml2001938

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao

Full text of DOI:10.1021/ml2001938

Letter
pubs.acs.org/acsmedchemlett
Potent and Selective Inhibitors of Long Chain L-2-Hydroxy Acid
Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats
Dinesh A. Barawkar,*,† Ashwin Meru,† Anish Bandyopadhyay,† Abir Banerjee,† Anil M. Deshpande,†
Chandrashekhar Athare,† Chandrasekhar Koduru,† Goraksha Khose,† Jayasagar Gundu,† Koshu Mahajan,†
Pradeep Patil,† Sachin R. Kandalkar,† Sanjay Niranjan,† Shubhangi Bhosale,† Siddhartha De,†
Sudit Mukhopadhyay,† Sumit Chaudhary,† Summon Koul,† Umesh Singh,† Anita Chugh,†
Venkata P. Palle,† Kasim A. Mookhtiar,† Joseph Vacca,‡ Prasun K. Chakravarty,‡ Ravi P. Nargund,‡
Samuel D. Wright,‡ Sophie Roy,‡ Michael P. Graziano,‡ Sheo B. Singh,‡ Doris Cully,‡
and Tian-Quan Cai‡
†Drug Discovery Facility, Advinus Therapeutics Limited, Quantum Towers, Plot-9, Phase-I, Rajiv Gandhi InfoTech Park, Hinjewadi,
Pune 411 057, India
‡Merck Research Laboratories, Rahway, New Jersey 07065, United States
S
* Supporting Information
ABSTRACT: L-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant
expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood
pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the
management of blood pressure, selective Hao2 inhibitors were developed. Optimization of
screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat
Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats.
Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood
pressure in an established or developing model of hypertension, deoxycorticosterone acetate-
treated rats. This is the first report demonstrating a pharmacological benefit of selective
Hao2 inhibitors in a relevant model of hypertension.
KEYWORDS: Hao2, hypertension, pyrazolecarboxylic acid, DOCA rat
oxidase from Mycobacterium (L-lactate 2-monooxygenase),
uman hypertension is a complex, multifactorial disorder
Hresulting from the interplay of multiple environmental flavocytochrome b2 from yeasts (L-lactate cytochrome c
oxidoreductase), and mandelate dehydrogenase from Pseudo-
monas putida.9 In mammals, this family of enzymes was first
identified as an L-amino acid oxidase in the kidney and the
liver of rats and later found to have activity similar to that of
L-2-hydroxy acid.10,11 Two α-hydroxy acid oxidases were also
reported from hog renal cortex, named long chain L-α-hydroxy
acid oxidase (Hao2) and short chain L-α-hydroxy acid oxidase
(Hao1), because of their substrate specificity toward long and
short carbon chain L-α-hydroxy acids, respectively. In both
prokaryotes and eukaryotes, all the members of the hydroxy
oxidase family are highly conserved in terms of both nucleotide
and amino acid sequences. Human Hao2 has 351 amino acids
with a predicted molecular mass of 39 kDa, while human Hao1
comprises 370 amino acids and has a predicted molecular mass
of 41 kDa. Human Hao2 shares ∼50% identity with human
Hao1 and 72−74% identity with rodent (rat and mouse) Hao2.
Hao2 is predominantly expressed in the liver and kidney, with
greatest potency for long chain 2-hydroxy acid substrates
and genetic factors, and this common disorder can lead to an
increased risk of heart attack, stroke, and renal failure. The
mechanisms underlying the initiation and maintenance of the
hypertensive process remain unclear.1 Almost one-third of the
U.S. adult population has high blood pressure (BP), which
increases the risk of cardiovascular and renal disease and
shortened life expectancy.2−4 Various antihypertensive drugs
have been developed, including diuretics, beta blockers, calcium
channel blockers (CCBs), renin inhibitors,5 angiotensin-
converting enzyme (ACE) inhibitors, and angiotensin II
receptor blockers (ARBs).6,7 However, these drugs either lack
sufficient efficacy or are associated with significant adverse
effects. In a search for a novel antihypertensive target, we have
identified L-2-hydroxy acid oxidase (Hao2)8 as a potential target
for pharmacological intervention.
L-2-Hydroxy acid oxidases are flavin mononucleotide
(FMN)-dependent peroxisomal enzymes, which are members
of the flavoenzyme family that are responsible for the oxidation
of a number of L-2-hydroxy acids to ketoacids at the expense
of molecular oxygen, resulting in the formation of hydrogen
peroxide. Several examples of such enzymes have been identified
in different organisms, e.g., glycolate oxidase from plants, lactate
Received: August 17, 2011
Accepted: October 7, 2011
Published: October 7, 2011
© 2011 American Chemical Society
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Figure 1. Structures of screening hits (1 and 2) and optimized leads (3 and 4). (A) Cartoon representation of docked compound 3 in the active site
of rat Hao2 (Protein Data Bank entry 1TB3). The FMN ring is colored light blue. (B) Close-up view of docked compound 3 (yellow) in the active
site of the enzyme. The FMN ring is colored light pink. The putative hydrogen bonds are shown as dashed lines.
a
Table 1. PK Properties of Lead Compounds 3 and 4
a
NA means not available. iv (1 mg/kg), po (10 mg/kg); vehicle: iv [dimethylacetamide (2%), crempohor (2%), PEG400 (2%), and Milli Q water
(94%)], po [Tween 80 (0.5%) and 0.5% CMC (99.5%)].
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(displays the highest activity toward 2-hydroxypalmitic acid).
Hao1 is expressed primarily in liver and pancreas and shows
greatest potency for the two-carbon 2-hydroxy acid substrate
(glycolic acid) but also displays activity on long chain 2-hydroxy
fatty acids. Both Hao2 and Hao1 are capable of oxidizing
2-hydroxy fatty acids, but the endogenous physiological
substrates remain to be identified.
Hao2 has been identified as a candidate gene for the systolic
BP quantitative trait locus (QTL) in rats.12−14 Genome-wide
linkage analysis in humans locates a BP QTL in a defined
region containing Hao2 (located in Ch. 1 at 119.6 cM), thus
supporting a potential link between Hao2 and hypertension in
humans, as well.15
To establish a pharmacological validation of Hao2 in blood
pressure regulation, potent and selective rat Hao2 inhibitors
were developed. Pyrazole carboxylic acids, 1 and 2 (Figure 1),
were first identified as inhibitors of Hao2 by a focused enzyme
screen of our compound collection. Subsequent optimization of
these hits resulted in the discovery of compounds 3 and 4 as
potent inhibitors of rat Hao2, each exhibiting an IC50 value of
0.3 μM.16 These two compounds were further characterized in
a spectrum of assays, including intervention studies in a well-
established deoxycorticosterone acetate (DOCA) salt hyper-
tension model.
Molecular modeling studies using the rat Hao2 crystal
structure (Protein Data Bank entry 1TB3)17 were used to
understand the potential binding mode of the inhibitor (3) in the
enzyme active site. The modeling suggests that the carboxylic
acid moiety binds in the active site by forming salt bridge
interactions with basic residues R250 and R164 (Figure 1). The
interaction of the carboxylic acid moiety with these residues
has also been observed in homologous enzymes of Hao2.18,19
The NH group of the pyrazole moiety forms putative hydrogen
bonds with the catalytic H247 and nearby Y129 residues. The
biphenyl moiety resides in the hydrophobic pocket and is
surrounded by residues such as F79, A185, L172, L174, E188,
F23, and L161.
The model suggests that a one-carbon linker between the
pyrazole and aromatic moiety (3 and 4) is optimal for filling the
available space and placing the aromatic moiety in an
appropriate orientation. A longer and flexible carbon linker
may increase the conformational entropy of these structures
(1 and 2), thereby decreasing their activity. Deletion of the
carbon linker makes the molecules very rigid and leads to poor
occupancy of the phenyl moiety in the active site. Hence,
compound 5 without any linker shows substantially lower
activity (Figure 1).
To assess selectivity and to demonstrate compounds 3 and 4
are selective inhibitors of rat Hao2, we profiled these two
Figure 2. (A and B) Lowering of SBP and (C and D) change in SBP from baseline by Hao2 inhibitors 3 and 4, respectively, in a DOCA model (n =
6−8). Compared to the control group on each day, *P < 0.05, **P < 0.01, and ***P < 0.001. Two-way analysis of variance (ANOVA) followed by a
Bonferroni post test.
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Table 2. Drug Levels of Compounds 3 and 4 in Plasma and Kidney
concn 1 h postdose (n = 3)
concn 12−15 h postdose (n = 3)
compd
dose (mg/kg)
plasma (μM)
kidney (μmol/g)
plasma (μM)
kidney (μmol/g)
ΔSBP (mmHg) on day 15
3
3
30
3
2.2 ± 0.5
8 ± 2
48 ± 11
157 ± 9
10 ± 1
24 ± 3
34 ± 5
0.2 ± 0.1
1.9 ± 0.8
12 ± 2
3 ± 1
7 ± 2
32 ± 7
31 ± 4
27 ± 8
33 ± 6
4
13 ± 12
a
30
113 ± 36
86 ± 13
NA
a
Not available.
compounds in a range of in vitro assays. First, we tested their
potential cross reactivity against a closely related enzyme,
Hao1.16 Compound 3 inhibited rat Hao1 with an IC50 of 45.7
μM, while compound 4 was completely inactive at 10 μM,
indicating that compounds 3 and 4 have a minimum 150-fold
selectivity against rat Hao1. Compounds 3 and 4 were further
profiled against a panel of 125 targets (MDS PanLab Drug Matrix
Screen), which includes most targets known to regulate blood
pressure, and were shown to be completely inactive at 10 μM.
Additionally, compounds 3 and 4 were screened against the
GPR109a20 receptor agonist assay, as they are structurally similar
to the known high-affinity agonists 3-methylpyrazole-5-carboxylic
acid and 3-n-butylpyrazole-5-carboxylic acid, and were found to be
completely inactive at 10 μM.
The in vitro pharmacokinetic (PK) properties of compounds 3
and 4 are summarized in Table 1. Both leads showed low rates
of oxidative metabolism in rat liver microsomes (RLM) and no
cytotoxicity in the HepG2 cell line. They neither inhibit nor
induce cytochrome P450 at 10 μM. Both were highly (>99%)
bound to rat plasma protein.
The in vivo PK properties of 3 and 4 were examined in rats
(Table 1). Both compounds exhibited low systemic plasma
clearance (8 mL/min/kg for 3 and 0.52 mL/min/kg for 4) and
elimination half-lives (1.7 h for 3 and 3.7 h for 4). At an oral
dose of 10 mg/kg, compound 4 showed much higher plasma
exposure (Cmax = 134 μM) than compound 3 (Cmax = 24 μM)
with an AUC0−8 h values of 517 and 27 μM h−1 for compounds
4 and 3, respectively. Compounds 3 and 4 exhibited oral
bioavailability of 38 and 53%, respectively. Thus, both leads
were selected for investigation of their antihypertensive effect in
the well-studied DOCA salt model of hypertension in
treatment as well as prevention of hypertension mode.
pressure at a dose of 3 mg/kg, but the scale of blood pressure
reduction by compound 4 was lower at a dose of 3 mg/kg.
To demonstrate the role of Hao2 inhibition in the observed
lowering of SBP, the degree of inhibition of enzyme activity in
vivo (target engagement) was assessed. Rats were sacrificed at
the end of the treatment period, and their plasma and kidneys
were collected 1 and 12−15 h postdosing for estimation of drug
concentration and ex vivo inhibition of Hao2 activity.
Appropriate drug concentrations for Hao2 inhibition were
observed (Table 2). The ex vivo Hao2 activity was studied by
monitoring the conversion of [3H]-2-hydroxyoctanoic acid into
2-ketooctanoic acid with the release of [3H]H2O using kidney
slices.16 Both compounds (3 and 4) showed target engagement
1 h postdosing at 3 and 30 mg/kg, whereas 12−15 h postdosing,
target engagement was observed at only the 30 mg/kg dose
(Table 3). Demonstration of appropriate drug concentration
(compound 3 or 4) and target engagement data clearly indicate
the role of Hao2 inhibition in the observed lowering of SBP.
The effect of Hao2 inhibitors in a DOCA salt rat model of
hypertension (prevention mode) was examined. Compound 3
was further tested in a hypertension prevention model along
with atenolol. In this study, DOCA salt (25 mg/kg, twice a
week) was administered in uninephrectomized male Wistar
The effect of Hao2 inhibitors in a DOCA salt rat model of
hypertension (treatment mode) was examined. To investigate
the potential effect of Hao2 inhibitors on blood pressure regula-
tion, compounds 3 and 4 along with atenolol (as a positive
control) were studied in a well-established rat model of
hypertension, DOCA salt-treated Wistar rats.16 Treatment with
atenolol resulted in a significant reduction in systolic blood
pressure (SBP) (Figure 2). Treatment with compound 3 or 4
reduced ∼30 mmHg of SBP from the baseline at a dose of
30 mg/kg. Compound 3 exhibited a similar reduction in blood
Figure 3. Prevention of DOCA-induced hypertension (n = 6).
Compared to the control group on each day, *P < 0.05 and ***P <
0.001. Two-way analysis of variance (ANOVA) followed by a
Bonferroni post test.
rats continuously for 35 days.21,22 A sham control group
(surgery with no nephrectomy and without DOCA
administration) was also included in this study. Compound
treatment23 was initiated on day 14 of DOCA administration
and continued for the next 21 days. Blood pressure was
measured in conscious rats on days 0, 7, 14, 28, 30, and 35 of
DOCA administration by tail-cuff plethysmography 12−15 h
after the last dose of compound had been administered.
Treatment with compound 3 at 30 mg/kg significantly
attenuated DOCA salt-induced BP elevation (Figure 3) from
Table 3. Target Engagement Study
Conversion of [3H]-2-Hydroxyoctanoate to [3H]H2O (% inhibition vs
vehicle)
3
4
dose (mg/
kg)
1 h
postdose
12−15 h
1 h
postdose
12−15 h
postdose
postdose
3
29 ± 15
38 ± 13
0 ± 12
28 ± 16
75 ± 5
93 ± 3
0 ± 55
42 ± 9
30
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Letter
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damage.
In summary, we have successfully identified pyrazole-3-
carboxylic acids, lead compounds 3 and 4, as potent and
selective inhibitors of rat Hao2. These compounds are
metabolically stable, with good PK profiles, and demonstrate
in vivo efficacy in the DOCA model of hypertension and for the
first time validated that inhibition of Hao2 leads to lowering of
blood pressure in a well-established rat hypertension model.
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ASSOCIATED CONTENT
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Experimental procedures, analytical data for compounds 3−5,
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screening protocol, protocol for the DOCA model, blood
pressure measurement, and target engagement assay. This
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pubs.acs.org.
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AUTHOR INFORMATION
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P. J.; Tamura, S. Y.; Zou, N.; Rodriguez, N.; Boatman, P. D.; Saga,
C. R.; Lindstrom, A.; Xu, J.; Schrader, T. O.; Smith, B. M.; Chen, R.;
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(23) Compound 3 was orally dosed with the same vehicle (0.5%
Tween 80 and 0.5% sodium methylcellulose) at 3, 10, and 30 mg/kg,
twice a day. Atenonol was used as the positive control in this study,
with a dose of 10 mg/kg, twice a day.
■
Corresponding Author
*Telephone: 91-20-66539630. Fax: 91-20-6653 9620. E-mail:
dinesh.barawkar@advinus.com.
ACKNOWLEDGMENTS
■
This research was part of collaborative program between
Advinus Therapeutics and Merck Research Laboratories. We
thank Dr. Mahesh Mone for analytical support and Dr. Anup
Ranade for managing intellectual property. We thank all the
members of the team, business alliance leaders, and senior
management from both organizations. Advinus Publication
ADV-A-015.
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