Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 10: Identification of inhibitors for the liver microsomal acetoxycoumarin: Protein transacetylase

Article abstract of DOI:10.1016/S0968-0896(02)00515-1

The quantitative structure-activity relationship (QSAR) studies conducted by us earlier revealed the cardinal role of the pyran ring carbonyl group in the acetoxy polyphenolic compounds for the acetoxy polyphenol:protein transacetylase (TAase) activity. H

Full text of DOI:10.1016/S0968-0896(02)00515-1

Bioorganic & Medicinal Chemistry 11 (2003) 1015–1019
Mechanism of Biochemical Action of Substituted
4-Methylbenzopyran-2-ones. Part 10: Identification of
Inhibitors for the Liver Microsomal Acetoxycoumarin:
Protein Transacetylase
Hanumantharao G. Raj,a Ishwar Singh,b Ekta Kohli,a Ranju Kumari,a Garima Gupta,a
Yogesh K. Tyagi,a Ajit Kumar,a Ashok K. Prasad,b Narendra K. Kaushik,b
Carl E. Olsen,c Arthur C. Wattersond and Virinder S. Parmarb,d,
*
aDepartment of Biochemistry, V P Chest Institute, University of Delhi, Delhi-110 007, India
bBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi-110 007, India
cChemistry Department, Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark
dInstitute of Nano Science Engineering and Technology, Department of Chemistry, University of Massachusetts,
One University Avenue, Lowell, MA 01854, USA
Received 18 June 2002; accepted 7 October 2002
Abstract—The quantitative structure–activity relationship (QSAR) studies conducted by us earlier revealed the cardinal role of the
pyran ring carbonyl group in the acetoxy polyphenolic compounds for the acetoxy polyphenol:protein transacetylase (TAase)
activity. Hence, an attempt was made to examine whether such substrate analogues of benzopyran acetates which lack in the pyran
ring carbonyl group, such as 7-acetoxy-2,3-dihydro-2,2-dimethylbenzopyran (BPA), cetachin pentaacetate (CPA) and hematoxylin
pentaacetate (HPA) could inhibit the 7,8-diacetoxy-4-methylcoumarin (DAMC):protein (glutathione-S-transferase) transacetylase
activity. These compounds were indeed found to remarkably inhibit the TAase activity in a concentration dependent manner and
exerted their inhibitory action very rapidly. Further BPA, CPA and HPA were found to abolish the TAase mediated activation of
NADPH cytochrome C reductase as well as the inhibition of liver microsome catalyzed aflatoxin B1 (AFB1)-DNA binding by
DAMC very effectively. These results strongly suggest that the acetoxybenzopyrans merit as potent inhibitors of TAase.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
the assay represented TAase activity.3 TAase was puri-
fied from mammalian liver microsomes to a reasonable
degree of purity, nevertheless need was felt to devise an
affinity chromatographic procedure for the purification of
TAase. In order to accomplish this task, we undertook
QSAR studies4 which gave the hint for the possible inhi-
bitors of TAase. We have in this communication identified
several benzopyran acetates as inhibitors of TAase.
Our earlier investigations in this series1À3 conclusively
evidenced the presence of a unique enzyme in liver and
other tissues possibly catalyzing the transfer of acetyl
groups from a model acetoxycoumarin (DAMC) to
certain receptor proteins, such as glutathione S-trans-
ferase (GST), cytochrome P-450-linked mixed function
oxidases and NADPH cytochrome C reductase result-
ing in the modulation of their catalytic activities. The
enzyme was termed as DAMC: Protein Transacetylase
(TAase) which was assayed3 using GST as the second
substrate, the inhibition of GST under the conditions of
Materials and Methods
Chemicals
NADPH, cytochrome C, reduced glutathione (GSH),
AFB1, 1-chloro-2,4-dinitrobenzene (CDNB), (+)-cat-
echin, hematoxylin and calf thymus DNA were purchased
*Corresponding author. Tel.: +91-11-766-6555; fax: +91-11-766-
7206; e-mail: virparmar@yahoo.co.in
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00515-1

1016
Hanumantharao G. Raj et al. / Bioorg. Med. Chem. 11 (2003) 1015–1019
from Sigma Chemical Company, St. Louis, MO, USA.
[3H] AFB1 was the product of Moravek Biochemicals,
Brea, CA, USA.
0.25 M sucrose. Protein content of microsomes and
cytosol were assayed by the method of Lowry et al.8
Assay of TAase
Synthesis of 7,8-diacetoxy-4-methylcoumarin (DAMC)
and inhibitors of TAase (Fig. 1)
TAase was assayed using DAMC and cytosolic GST as
the substrates as per the details given in our earlier
reports.3 The assay mixture consisted of 0.25 M phos-
phate buffer (pH 6.5), liver microsomes (25 mg protein),
DAMC (100 mM) added in 50 mL DMSO, liver cytosol
(10–15 mg protein) and water to make up 0.5 mL. The
contents of the tube were scaled up as per requirement
and preincubated at 37 ꢀC for various periods. The ali-
(a) 7,8-Dihydroxy-4-methylcoumarin (DHMC) was
synthesized by the well-known Pechmann con-
densation of pyrogallol with ethyl acetoacetate,
its diacetyl derivative (DAMC) was prepared by
acetylation of DHMC as described by us earlier.5
(b) 7-Acetoxy-3,4-dihydro-2,2-dimethylbenzopyran
(BPA) was synthesized according to the pro-
cedure of Levai and Timar.6
(c) Catechin pentaacetate (CPA) and hematoxylin
pentaacetate (HPA) were prepared by the
acetylation of (+)-catechin and hematoxylin,
respectively by acetic anhydride/pyridine/DMAP
method and were fully characterized from their
1H and 13C NMR spectral data and comparison
with the data in literature.4,7
quots were removed periodically into
a spectro-
photometer cuvette containing CDNB and GSH to
make up their concentration 1 mM in a total volume of
1 mL and the progress of the GST activity was followed
at 340 nm using a Cary spectrophotometer (Cary
Bio100).9 In control samples, DAMC was replaced by
DMSO. The unit of TAase was expressed in terms of %
inhibition of GST under the conditions of the assay and
ensured that the reaction was linear with respect to
enzyme concentration and incubation time.
Inhibition of TAase activity
Animals
Male albino rats of Wistar strain weighing around 150–
200 g fed on rat chow supplied by Hindustan Lever
Ltd., Mumbai (India) were used.
(a) The inhibitors BPA, CPA and HPA were sepa-
rately preincubated with microsomes and cyto-
solic GST, followed by the assay of GST as
described above in order to quantify the activity
of TAase (Table 1).
Preparation of liver microsomes and cytosol
(b) The inhibitors CPA, BPA and HPA were sepa-
rately included in the reaction mixture for TAase
as described above at a concentration of 100 mM
and preincubated along with DAMC (25 mM),
liver microsomes (25 mg protein) and liver cytosol
(15 mg protein) for various periods, followed by
the assay of GST in order to quantify the TAase
activity. Unit of TAase activity was plotted
against the time of incubation (Fig. 2).
(c) The inhibitors CPA, BPA and HPA were sepa-
rately included in the reaction mixture of TAase
at concentrations varying from 10 to 150 mM and
preincubated for 10 min along with DAMC
Rats were sacrificed by decapitation, liver was removed
and a 30% homogenate was prepared in 10 mM phos-
phate buffer containing 0.25 M sucrose and 1.4 mM
b-mercaptoethanol and pH adjusted to 7.0. The homo-
genate was centrifuged at 10,000g for 30 min in a Sorvall
superspeed centrifuge and the supernatant was spun at
100,000g in a Beckman ultracentrifuge (Model L-7).
The cytosolic fraction was set aside at À20 ꢀC. The
microsomal pellet was resedimented and suspended in
Table 1. Specificity of BPA, CPA and HPA for acetoxy drug protein:
transacetylase
Time of preincubationa
(min)
TAase unitsb
DAMC
BPA
CPA
HPA
(25 mM) (100 mM) (100 mM) (100 mM)
10
20
30
40
8.20
15.80
24.40
30.20
5.50
6.30
7.80
8.40
6.40
9.60
10.90
12.70
3.90
5.30
7.00
8.10
aBPA, CPA and HPA were preincubated with liver microsomes, fol-
lowed by the assay of GST as described under ‘Materials and Meth-
ods’.
bTAase unit is expressed in terms of percent inhibition of GST under
the assay conditions. The values are the average of five separate
observations with error <5%.
Figure 1. Structures of the substrate and the inhibitors of TAase.

Hanumantharao G. Raj et al. / Bioorg. Med. Chem. 11 (2003) 1015–1019
1017
were preincubated at 37 ꢀC for varying periods. The
aliquot (0.5 mL) was removed periodically into a spec-
trophotometer cuvette (1 cm light path) containing
0.1 mM EDTA, 36 mM cytochrome C and 1 mM
NADPH in a total volume of 1 mL. The progress of the
reaction was followed by monitoring absorption at
550 nm.10 In the appropriate blank sample, DAMC/
inhibitor was replaced by DMSO. The percentage acti-
vation of reductase was plotted against time of pre-
incubation (Fig. 4).
Effect of TAase inhibition on the liver microsomes medi-
ated AFB1-DNA binding by DAMC (Table 3). Liver
microsomes (1 mg protein) were pre-incubated sepa-
rately with 100 mM phosphate buffer (pH 7.4), 100 mM
inhibitor (CPA/BPA/HPA) for 30 min at 37 ꢀC followed
by the addition of [3H] AFB1 (250 mCi/mM), 2 mM
NADPH, and 0.1 mg calf thymus DNA in a total
volume of 1 mL and the incubation was continued for
another 30min. After incubation, 2 mL of extraction
mixture (phenol/chloroform/isoamyl alcohol, 50:50:1 v/v)
was added and DNA was isolated by the procedure of
Wang and Cerutti,11 as described in detail in our earlier
publication.12 In the appropriate blank sample, DAMC/
inhibitor was replaced by DMSO.
Figure 2. Inhibition of TAase by BPA,CPA and HPA. The detailed
assay procedure is described under ‘Materials and Methods’. TAase
activity was expressed in terms of percent inhibition of GST under the
conditions of the assay. The values are the average of three values with
error <5%.
(100 mM), liver microsomes (25 mg), liver cytosol
(15 mg protein) and TAase was assayed as
described above. The reaction mixture containing
DAMC devoid of inhibitor served as control.
The inhibitory action was expressed by plotting
TAase activity as percentage of control against
the concentration of the inhibitor (Fig. 3).
Results and Discussion
A novel enzyme termed as acetoxy drug: protein trans-
acetylase (TAase) localized in liver microsomes carrying
out the possible transfer of acetyl group of acetylated
xenobiotics such as acetoxy substituted 4-methyl-
coumarins, flavones, isoflavones, xanthones and chal-
cones to specific enzyme proteins leading to modulation
of their catalytic activities has been characterized in our
previous publications.1À4,13,14 DAMC was used as a model
substrate for the assay of TAase as described above.
TAase was partially purified and characterized from
mammalian liver and human placenta.3,4,15 The QSAR
study of various acetylated polyphenols, that is acetoxy-
coumarins, acetoxyflavones and acetoxyisoflavones
Inhibition of transacetylase mediated biochemical actions
Inhibition of modulation of NADPH cytochrome C re-
ductase by DAMC catalyzed by TAase. The method
consisted of preincubation of DAMC with the inhibitor
and the liver microsomes, followed by the addition of
substrates for the reductase (cytochrome
C and
NADPH) for the assay as described earlier.2 The rat
liver microsomes (40 mg protein) were preincubated with
DAMC (5 mM), 0.05 M phosphate buffer (pH 7.0), the
inhibitor (100 mM) in DMSO and water to make 0.5 mL
volume. The contents, scaled up as per requirement
Figure 3. Effect of the concentration of the inhibitors on the activity
of TAase. The inhibitors BPA/CPA/HPA at the concentration of
10–150 mM were separately included in the reaction mixture of TAase
as described under ‘Materials and Methods’. The values are the aver-
age of three separate observations with error <5%.
Figure 4. Effect of the inhibitors on the liver microsomal TAase cata-
lyzed activation of NADPH cytochrome C reductase by DAMC. The
detailed assay procedure is described under ‘Materials and Methods’.
The values are the average of three separate observation with error <2%.

1018
Hanumantharao G. Raj et al. / Bioorg. Med. Chem. 11 (2003) 1015–1019
Table 2. Inhibitory potential of the DAMC: GST transacetylase
inhibitors
inhibitory action of BPA, CPA and HPA was sub-
stantiated by the inhibitor concentration-dependent
diminution of TAase catalytic activity (Fig. 3). The
results shown in Table 2 record considerably close IC50
values for TAase inhibition, thus corroborating the
aforementioned results (Fig. 2). We have established
earlier2 that DAMC through the action of TAase dra-
matically activated microsomal NADPH cytochrome C
reductase activity. TAase catalyzed activation of
NADPH cytochrome C reductase by DAMC was found
to be remarkably abolished by inclusion of the inhibitor
BPA/CPA/HPA in the preincubation mixture (Fig. 4).
The inhibitor(s) alone were not found to have any
influence on the activity of the reductase (data not
shown). Cytosolic P-450-linked mixed function oxidase
proved to be another enzyme protein whose catalytic
activity was shown to be effectively inhibited by the
TAase mediated action of DAMC.1 Accordingly, the
results included in Table 3 demonstrate that liver
microsomal TAase catalyzed inhibition of AFB1 epoxi-
dation (measured as AFB1-DNA binding) by DAMC
was very effectively abolished by TAase inhibitors BPA/
CPA/HPA, while the inhibitors alone were not found to
have any influence on the P-450 catalyzed AFB1-DNA
binding.
Inhibitor
IC50 (mM)
BPA
CPA
HPA
39.00
33.00
41.00
Assay of the transacetylase activity using DAMC and cytosolic GST
as the substrates was carried out as described under ‘Materials and
Methods’. Dose–response plot of enzyme fractional and activity
(vi/Vo) was plotted against inhibitor concentration. The concentration
of the inhibitor required to achieve a half maximal degree of inhibition
(IC50) was determined graphically.16
Table 3. Reversal of transacetylase mediated inhibition of liver
microsome catalysed AFB1–DNA binding by DAMC
Incubation
mixturea
AFB1–DNA binding pmoles
AFB1 bound/30 min/mg protein
DMSO
DAMC
209.2
86.2
BPA
CPA
HPA
BPA+ DAMC
CPA+DAMC
HPA+DAMC
206.7
205.5
204.8
207.1
208.9
208.3
The aforementioned results clearly established the iden-
tification of inhibitors for TAase, the criterion for the
TAase inhibitor being the lack of pyran ring carbonyl
group in a oxygen containing heterocyclic polyphenolic
acetate. There are several motivations for studying the
enzyme inhibition. By the study of relative binding affi-
nity of the inhibitors of varying structures, it is possible
to get insight into the active site of an enzyme in the
absence of a high resolution three-dimensional struc-
ture.14,16 The nature of inhibition of TAase by BPA,
CPA and HPA, and their analogues will be described in
the ensuing publications.
aThe concentration of DAMC, BPA/CPA/HPA was 100 mM. The
values are the average of three separate observations with variation
<5%.
revealed the following features specific for TAase: (a)
the presence of acetoxy groups in proximity to the oxy-
gen heteroatom, and (b) absolute requirement of the
presence of the pyran ring carbonyl group.4 Accord-
ingly, acetoxy groups at the C-7 and C-8 positions on
the benzenoid ring of polyphenols gave highest TAase
activity. The acetylated polyphenols, such as BPA, CPA
and HPA lacking the pyran ring carbonyl group sup-
ported little activity when used as substrates for the
TAase4 (Table 1). It was thought interesting to examine
whether the compounds BPA, CPA and HPA which are
substrate analogues could act as the inhibitors for
TAase. For this purpose, the test compound (BPA/
CPA/HPA) was preincubated with liver microsomes
and DAMC, followed by GST assay for the TAase
activity as described under ‘Materials and Methods’.
The results shown in Table 1 demonstrate TAase activ-
ity when the test compounds were used as substrates. It
is clear from the data that BPA/CPA/HPA are very
poor substrates for the TAase. An effort was made to
examine the outcome of the inclusion of the test com-
pound(s) in the incubation mixture for the assay of
TAase activity. The data shown in Figure 2 highlighted
the remarkable inhibition of TAase activity by BPA,
CPA and as well as HPA. The incubation of BPA/CPA/
HPA for 10 min at 100 mM concentration resulted in
nearly 75–90% reduction of the TAase activity. Also, it
is evident from the data that there is no change in the
extent of inhibition at 40 min of preincubation com-
pared to that at 10 min. Hence, these compounds can be
considered to exert their inhibitory action rapidly. The
Acknowledgements
The financial assistance of the Danish International
Development Agency (DANIDA) and the Department
of Biotechnology (DBT, Government of India) is grate-
fully acknowledged. E.K., I.S., R.K. and Y.K.T. are
Research Fellows of the Council for Scientific and
Industrial Research (CSIR, New Delhi).
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