The human carbonic anhydrase isoenzymes I and II inhibitory effects of some hydroperoxides, alcohols, and acetates

Article abstract of DOI:10.3109/14756366.2015.1120723

The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes, which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K_i s in the range of 24.93–97.99 nM) and hCA II inhibitors (K_i s in the range of 26.04–68.56 nM) compared to acetazolamide as CA inhibitor (K_i : 34.50 nM for hCA I and K_i : 28.93 nM for hCA II).

Full text of DOI:10.3109/14756366.2015.1120723

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
The human carbonic anhydrase isoenzymes I
and II inhibitory effects of some hydroperoxides,
alcohols, and acetates
Leyla Polat Kose, İlhami Gulcin, Alper Yıldırım, Ufuk Atmaca, Murat Çelik,
Saleh H. Alwasel & Claudiu T. Supuran
To cite this article: Leyla Polat Kose, İlhami Gulcin, Alper Yıldırım, Ufuk Atmaca, Murat Çelik,
Saleh H. Alwasel & Claudiu T. Supuran (2015): The human carbonic anhydrase isoenzymes I
and II inhibitory effects of some hydroperoxides, alcohols, and acetates, Journal of Enzyme
Inhibition and Medicinal Chemistry, DOI: 10.3109/14756366.2015.1120723
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1120723
Published online: 18 Dec 2015.
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2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1120723
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RESEARCH ARTICLE
The human carbonic anhydrase isoenzymes I and II inhibitory effects of
some hydroperoxides, alcohols, and acetates
1
Leyla Polat Kose , Ilhami Gulcin , Alper Yıldırım , Ufuk Atmaca , Murat C¸ elik , Saleh H. Alwasel , and
1,2
1
1
1
2
_
3
,4
Claudiu T. Supuran
1
2
Department of Chemistry, Faculty of Sciences, Atat u¨ rk University, Erzurum, Turkey, Department of Zoology, College of Science, King Saud
3
4
University, Saudi Arabia, Dipartimento Di Chimica Ugo Schiff, Universit a` Degli Studi Di Firenze, Firenze, Italy, and Neurofarba Department, Section
of Pharmaceutical and Nutriceutical Sciences, Universit a` Degli Studi Di Firenze, Florence, Italy
Abstract
Keywords
The carbonic anhydrases (CAs, EC 4.2.1.1) represent a superfamily of widespread enzymes,
which catalyze a crucial biochemical reaction, the reversible hydration of carbon dioxide to
bicarbonate and protons. Human CA isoenzymes I and II (hCA I and hCA II) are ubiquitous
cytosolic isoforms. In this study, a series of hydroperoxides, alcohols, and acetates were tested
for the inhibition of the cytosolic hCA I and II isoenzymes. These compounds inhibited both
Acetate, alcohol, carbonic anhydrase, enzyme
inhibition, enzyme purification,
hydroperoxide
History
hCA isozymes in the low nanomolar ranges. These compounds were good hCA I inhibitors (K
in the range of 24.93–97.99 nM) and hCA II inhibitors (K s in the range of 26.04–68.56 nM)
: 34.50 nM for hCA I and K : 28.93 nM for hCA II).
i
s
Received 9 November 2015
Accepted 12 November 2015
Published online 10 December 2015
i
compared to acetazolamide as CA inhibitor (K
i
i
Introduction
the CA-related proteins (CARPs), are devoid of any catalytic
1
4–18
activity
.
The carbonic anhydrases (CAs; EC 4.2.1.1) are a superfamily of
metalloenzymes, which catalyze the interconversion between
The different CA isoforms possess a widely variable kinetic
properties, their pattern of expression in various cellular com-
partments and tissues is diverse, as it is their inhibition profile
ꢀ
carbon dioxide (CO ) and water (H O) to bicarbonate (HCO
+
)
and a proton (H ) by using a metal hydroxide nucleophilic
2
2
3
13,19
1
,2
with various classes of compounds
. An inhibitor is a molecule
mechanism
.
that binds to an enzyme and diminish its activity. Also, it can
hinder a substrate from entering the active site of the enzyme
preventing catalyzing. The inhibition of CA enzymes is very
ꢀ
þ
CO2 þ H2O , H2CO3 , HCO þ H
3
They are virtually ubiquitous in all living systems and
participate in a variety of physiological and pathological
processes such as pH regulation, fluid balance, bone resorption,
glaucoma, calcification, cancer, neurological disorders, osteopor-
osis, tumorigenicity, and biosynthetic reactions (e.g. carboxyl-
11,16
important for living organism
clinically used primarily as anti-glaucoma drugs, diuretics ,
. CA inhibitors (CAIs) were
6
1
9
anticonvulsant agents , and as anti-epileptics, while the novel
generation compounds are undergoing clinical investigation as
20–22
7,11,23
anti-obesity
recent years, CAIs started to be used in the management of
or anti-tumor drugs and diagnostic tools
. In
ations, in which the produced HCO3ꢀ
3–9
is the real substrate ). The
CAs are present in either eukaryotes and prokaryotes, being
encoded by six genetically distinct, non-related gene families: the
a, b, g, d, z, and Z-CAs. Of them, the Z-CA, a novel family of
22
hypoxic tumors .
Many classes of CAIs bind to the catalytic zinc ion (Zn
2+
)
within the enzyme active site and prevent its activity.
Acetazolamide (AZA) is the first clinically used sulfonamides
23
as CAI .
1
0–13
CA, was discovered quite recently
.
All human CAs (hCAs) belong to the a-CAs. Until now, 16
isozymes have been recognized in mammals where they play
crucial physiological roles. Among these, only 13 isoforms are
catalytically active (CAs I-IV, CAs VA, VB, CAs VI, VII, CA IX,
and CAs XII-XV). Some of them are cytosolic ones (CA I, II, III,
VII, and XIII), others are membrane associated (CA IV, IX, XII,
XIV, and XV), mitochondrial (CA VA and VB), and there is a
secreted one (CA VI) too. On the other hand CA VIII, X and XI,
The catalytic mechanism involves the presence of a hydroxide
ion coordinated to the zinc within the CA active site (i.e. the basic
form of the enzyme). During the catalytic cycle, the acidic form is
generated, with water coordinated to zinc. For regeneration of the
basic form of CA, a proton is transferred from the zinc
+
coordinated water molecule to the solvent. This H transfer may
be assisted by active site residues or by buffers present in the
1
4,24,25
medium
.
Molecular oxygen is the most plentiful and accessible oxidant.
Oxidations of many organic compounds (e.g. alkenes) are
important in the synthesis of many widely used chemicals.
Singlet oxygen is an electronically excited state of molecular
_
Address for correspondence: Prof. Dr. Ilhami Gulcin, Faculty of Sciences,
Department of Chemistry, Atat u¨ rk University, TR-25240, Erzurum,
Turkey. Tel: +90 442 2314375. Fax: +90 442 2314109. E-mail:
igulcin@atauni.edu.tr; gulcin@ksu.edu.sa

2
L. P. Kose et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
3
oxygen, which is generated by reaction of triplet oxygen ( O ) Results and discussion
2
with photoexcited sensitizer. Singlet oxygen is produced
by irradiation of ground state triplet oxygen with light in the
presence of triplet sensitizers such as meso-tetraphenyl porphyrin
(TPP), Rose Bengal or Methylene Blue. The olefins with allylic
Clinical use of the CAIs proved to be a reliable therapeutic
method for a number of human diseases and for several decades
such compounds were a major component of the therapy for high
64,65
blood pressure, glaucoma, etc.
are involved in crucial physiological processes connected with
It was well known that CAs
hydrogen atoms undergo ene-type reactions with singlet oxygen to
form allylic hydroperoxides, which are useful intermediates
–
2
6–29
CO /HCO transport and homeostasis, electrolyte secretion in a
2 3
in many synthetic reactions
involved in the development of rancidity in fat, the disruption of
. Allylic hydroperoxides are
variety of tissues and organs, biosynthetic reactions including
ureagenesis, gluconeogenesis, and lipogenesis, respiration,
3
0
lipid membranes, but also in the biosynthesis of prostaglandins .
As hydroperoxides were not yet investigated for their inter-
action with the CAs, in this study, we studied the potential
inhibition effect of some allylic hydroperoxides (1–5), alcohols
32,66,67
tumorigenicity, bone resorption, and calcification
.
In this study, we report the inhibition profiles of allylic
hydroperoxides, alcohols, and acetates (1–15) against the ubiqui-
tous cytosolic isoform (hCA I) and the more rapid cytosolic
isoenzyme (hCA II). Hydroperoxides are strong oxidants like
ozone and have some toxic effects on living organisms by
inactivation of enzymes and impairing metabolic processes. They
(5–10), and acetates (10–15) against hCA I and II.
Experimental
The hCA I and II isoenzymes were purified by Sepharose-4B-L occur as end products of polyunsaturated fatty acids biosynthetic
3
1–36
68–78
tyrosine-sulphanilamide affinity chromatography
7
as pub- pathways/degradation, including linoleic acid
. They can give
3
lished in previous studies . Sodium dodecyl sulphate-polyacryl- rise to secondary oxidative damage, which can happen in two
amide gel electrophoresis (SDS-PAGE) was used for checking ways. The first is via one electron (free radical) reaction to get
3
7–42
enzymes purity
each isoenzyme
, and a single band was observed for secondary radicals, whereas the second via two electron (molecu-
79
. For this purpose acrylamide in the running lar) reactions with suitable nucleophiles .
4
3,44
(10%) and the stacking gel (3%), with SDS (0.1%) were
employees
The allylic hydroperoxides (1–5) were prepared by the Schenk
method. The chemical structures of allylic hydroperoxides,
4
5–48
.
CA isoenzyme activities were determined according to alcohols and acetates (1–15) are shown in Figure 1. The reduction
4
9–51
Verpoorte et al.
One unit of CA activity was expressed as of the hydroperoxides with dimetilsulfide in the presence of
ꢁ
52
mmol/L of released p-nitrophenol (NP) per minute at 25 C . catalytic amount of Ti(O-iPr) produced the corresponding allylic
1
The quantity of protein during enzyme purification procedure alcohols (6–10) in high yield. Allylic alcohols represent an
4
was spectrophotometrically determined at 595 nm according interesting building block in organic synthesis. All allylic alcohols
5
to the Bradford method . Bovine serum albumin was used as (6–10) were converted into the corresponding monoacetates with
3
5
4–56
30
the standard protein
The inhibition effects of allylic hydroperoxides, alcohols, and synthesis of these allylic hydroperoxides, alcohols, and acetates
.
NaOAc/Ac O at room temperature in excellent yields . The
2
3
acetates (1–15) on both CA isoenzymes was measured by using (1–15) was performed as described previously . The synthesis
p-nitrophenyl acetate (NPA) hydrolysis to NP
catalysed reaction of CO hydration was first observed in the (1–15) is shown in Figure 2.
0
5
7,58
. The CA- route of some allylic hydroperoxides, alcohols, and acetates
2
absence of allylic hydroperoxides, alcohols, and acetates (1–15)
and used as a control for both CA isoenzymes.
For evaluation of the biological activity of hydroperoxides,
alcohols, and acetates 1–15, the physiologically relevant human
Activity (%)-[allylic hydroperoxide, alcohol, or acetate] isoforms hCA I and II have been included in the study. Allylic
graphs were drawn and the half maximal inhibitory concentration hydroperoxides, alcohols, and acetates 1–15 demonstrated effect-
(IC ) values of each allylic hydroperoxides, alcohols, and ive inhibitory effects against both CA I and II isoforms (Table 1).
acetates (1–15) exhibiting more than 50% inhibition of CA The following structure–activity relationship could be drawn from
50
were calculated. In addition, the K values were also determined. data of Table 1:
i
Five different concentrations of substrate were used and
5
(i) The CA I isoenzyme is found in many tissues and is
Lineweaver–Burk curves were drawn in order to determine involved in retinal and cerebral edema. Its inhibition may be a
9
6
0–63
21
valuable tool for fighting these conditions . For hCA I, the
the Kis
.
Figure 1. The chemical structures of allylic
hydroperoxides, alcohols, and acetates (1–15)
used for carbonic anhydrase isoenzymes
OOH
OOH
OOH
OOH
OOH
(
hCA I and II) inhibition effects.
1
2
3
4
5
OH
OH
OH
OH
OH
6
7
8
9
10
OAc
OAc
OAc
OAc
OAc
1
1
12
13
14
15

DOI: 10.3109/14756366.2015.1120723
CA inhibitory effects of hydroperoxides
3
hν/TPP
Figure 2. The synthesis route of some allylic hydroperoxides, alcohols, and acetates (1–15).
7
8
Table 1. Human carbonic anhydrase isoenzymes (hCA I and II) inhibition benzylamine derivatives , melatonin , sulfonamide derivatives of
9
values with some hydroperoxides, alcohols, and acetates by an esterase aminoindanes and aminotetralins , dimethoxy-bromophenol
1
0
assay with NPA.
derivatives incorporating cyclopropane moieties (3,4-dihydrox-
yphenyl) (2,3,4-trihydroxyphenyl), methanone and its deriva-
1
tives , new ureido-substituted sulfonamides incorporating a
2
IC50 (nM)
2
i
K (nM)
1
3
14
GABA moiety , new benzotropone derivatives , guaiacol and
2
r
Compounds hCA I
r
hCA II
hCA I
hCA II
1
15
16
17
catechol derivatives , pyrimidines , capsaicin , hydroquinone ,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
74.17 0.9911 46.63 0.9901 40.46 ± 7.01 47.29 ± 5.20
77.02 0.9930 59.62 0.9972 71.16 ± 36.50 36.66 ± 6.69
91.78 0.9960 52.19 0.9926 60.12 ± 11.36 32.84 ± 1.84
66.71 0.9880 48.05 0.9933 24.93 ± 5.39 26.04 ± 6.51
59.76 0.9954 37.57 0.9877 44.62 ± 17.60 34.60 ± 7.58
89.49 0.9964 68.22 0.9984 82.44 ± 1.94 68.29 ± 6.65
97.59 0.9921 83.01 0.9990 97.99 ± 20.63 68.56 ± 11.10
91.23 0.9831 73.36 0.9971 85.87 ± 4.63 61.19 ± 10.36
67.73 0.9916 66.76 0.9955 51.40 ± 4.86 45.74 ± 9.10
63.38 0.9967 53.52 0.9858 64.15 ± 17.11 49.79 ± 2.01
64.47 0.9987 53.62 0.9990 49.58 ± 7.39 41.11 ± 9.39
59.72 0.9985 61.20 0.9909 80.26 ± 14.01 58.91 ± 4.99
59.26 0.9963 53.83 0.9983 55.78 ± 10.24 51.85 ± 4.54
53.85 0.9946 45.09 0.9985 45.88 ± 8.55 47.06 ± 3.58
52.95 0.9983 44.71 0.9966 53.93 ± 15.60 36.08 ± 8.12
40.46 0.9996 24.16 0.9833 34.50 ± 4.09 28.93 ± 14.77
30
novel sulfamides derived from 1-aminoindanes and anilines ,
32
32
34
34
benzylsulfamides , rosmarinic acid , dantrolene , morphine ,
3
tocopherol , phenolic sulfonamides , N-acylsulfonamides ,
5
36
43
4
3
novel phenolic sulfamides , antioxidant phenols , brominated
49
5
diphenylmethanone and its derivatives , natural phenolic com-
6
6
1,62,84
85
, phenolic acids , antioxidant polyphenol prod-
pounds
81,86
ucts
86
, natural product polyphenols and phenolic acids ,
82
caffeic acid phenethyl ester , carbamates and sulfamoylcarba-
0
1
2
3
4
5
8
mates , natural and synthetic bromophenols , norbornene-fused
7
88
8
pyridazines , (Z)-4-Oxo-4-(arylamino)but-2-enoic acids deriva-
9
9
tives , avermectins , spirobisnaphthalenes , 4-(2-substitutedhy-
0
91
92
9
3
94
drazinyl)benzenesulfonamides , and taxifolin . All of these
studies demonstrate the importance of CA isoenzymes in
95–100
AZA*
biochemical and pharmaceutical application
.
*
Acetazolamide (AZA) was used as a standard inhibitor for both CA
isoenzymes.
Conclusions
The allylic hydroperoxides, the corresponding alcohols and
acetates 1–15 demonstrated effective inhibition profiles against
compounds 1–15 showed K values ranging between 24.93 ± 5.39 hCA I and II. The similar inhibition profiles of these compounds
i
and 97.99 ± 20.63 nM (Table 1). The best inhibition was observed for the two CA isoforms can be due to the high homology between
with hydroperoxide 4 (3-hydroperoxycyclohept-1-ene) with a Ki hCA I and II. Allylic hydroperoxides, alcohols, and acetates
value of 24.93 ± 5.39 nM. On the other hand, AZA, used a CAI for (1–15) were identified as potent low nanomolar CAIs.
the medical treatment of idiopathic intracranial hypertension,
glaucoma, epileptic seizure, altitude sickness, cystinuria, periodic
paralysis, central sleep apnea, and dural ectasia, showed a K value
of 34.50 ± 4.09 nM. hCA I is highly abundant in red blood cells
Acknowledgements
i
I.G. and S.H.A. would like to extend his sincere appreciation to the
Research Chairs Program at King Saud University for funding this
and is found in many tissues but its precise physiological function
2
2,67
research.
is unknown
(
edema, epilepsy, and altitude sickness
.
ii) CA II is involved in several diseases, such as glaucoma,
. For hCA II, com-
pounds 1–15 showed K values ranging between 26.04 ± 6.51 and
2
2,80
Declaration of interest
i
The authors declare no conflict of interest.
6
hydroperoxide 4 (3-hydroperoxycyclohept-1-ene) with a K value
8.56 ± 11.10 nM. As for hCA I, the best hCA II inhibitor was
i
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