Inhibition of ribonuclease A by nucleoside-dibasic acid conjugates

Article abstract of DOI:10.1016/j.bmc.2009.08.018

We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside-dibasic acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and the pK_a of the carboxylic group

Full text of DOI:10.1016/j.bmc.2009.08.018

Bioorganic & Medicinal Chemistry 17 (2009) 6491–6495
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of ribonuclease A by nucleoside–dibasic acid conjugates
*
*
Joy Debnath, Swagata Dasgupta , Tanmaya Pathak
Department of Chemistry, Indian Institute of Technology, Kharagpur, Kharagpur 721 302, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the inhibition of the ribonucleolytic activity of ribonuclease A (RNase A) by nucleoside–dibasic
acid conjugates for the first time. Agarose gel and precipitation assays show that the spacer length and
the pKa of the carboxylic group have an important role in the inhibitory capacity. Kinetic experiments
Received 17 June 2009
Revised 7 August 2009
Accepted 11 August 2009
Available online 14 August 2009
indicate a competitive mode of inhibition with inhibition constant (Ki) value of 132
3 lM for Oxa-aT.
Docking studies revealed that the carboxylic group of the most active compounds is within hydrogen
bonding distance of His-12, Lys-41 and His-119.
Keywords:
Ribonuclease A
Ó 2009 Elsevier Ltd. All rights reserved.
Nucleoside–dibasic acid conjugates
Inhibition
1. Introduction
Similar inhibitory activities were also observed for nucleobase cat-
echin/epicatechin chimeric molecules.14
Ribonuclease A (RNase A) is one of the most important mem-
bers of the ribonuclease superfamily,1,2 which exhibits a cytotoxic
effect.3 It adsorbs specifically to certain cells, enters the cytosol, de-
grades the RNA and consequently inhibits protein synthesis caus-
ing cell death. Certain pathological conditions like malignancies
and infectious diseases4 are directly linked with the ribonucleolytic
activity5 of specific members of this superfamily. These pathologi-
cal diseases in addition to its cytotoxicity has triggered research in
the area of inhibition of RNase A by low molecular weight com-
pounds.6 The ribonucleolytic center of RNase A is comprised of
multiple subsites which bind the phosphate, nucleobase, and sugar
components of RNA.7,8 The main subsite is the P1 subsite where
cleavage of the phosphodiester bond occurs. His-12, Lys-41 and
His-119 serve as the catalytic residues at the P1 subsite (Fig. 1).
The B1 and B2 sites are recognition sites for the nucleobases of
the corresponding nucleotide residues. In general the B1 and B2
sites show pyrimidine and purine specificity, respectively.9
A large number of phosphate or pyrophosphate based nucleo-
tidic inhibitors8,9 have reported good anti-RNase A activities.6
However, the polyionic nature of these nucleotide-based inhibitors
hinders their transport through the cell membrane.10 In earlier
studies, we have demonstrated that 30-alkylamino-30-deoxy arauri-
dines,11,12 and 50-alkylamino-50-deoxy-uridines and thymidines
efficiently inhibited RNase A,13 where we found that the carboxylic
group directly interacted with the active site histidine residues.
In this study, we have synthesized a series of dibasic acid con-
jugates which are actually ‘monobasic’ compounds because one
of the –COOH groups of the dibasic acid is part of the amide bond
with a free amino group in the molecule. The amide group along
B1
Val 43
Asn 44
Thr 45
Phe 120
Ser 123
Cyt
or
Ura
O
P
O
O
O
R1
OH
O
P
His 12
Lys 41
O
O
Asn 67
His 119
Gln 69
Asn 71
Glu 111
Ade
O
O
P1
R2
OH
O
P
B2
O
O
O
* Corresponding authors. Tel.: +91 3222 283306; fax: +91 3222 255303 (S.D.);
tel.: +91 3222 283342 (T.P.).
E-mail addresses: swagata@chem.iitkgp.ernet.in (S. Dasgupta), tpathak@chem.
iitkgp.ernet.in (T. Pathak).
Figure 1. Active site residues of RNase A.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.018

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J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 6491–6495
with the methylene groups attached to it act as linkers of different
lengths for delivering the –COOH group to the active site. We
understand that other factors including transport across mem-
branes, metabolism etc. will be important in vivo. This is a matter
of further investigation. Thus we have designed nucleoside-based
inhibitors with lower ionic character compared to reported phos-
phate inhibitors15 but with a capacity to ionize in biological sys-
tems. The notion is to deliver a carboxylic group to the P1 site of
the enzyme through spacers of different chain length. Electrostatic
interactions present due to the ionized carboxylic group with the
active site histidine residues of RNase A ought to disrupt the local
acid-base equilibrium by electrostatic interactions.16 The extent of
electrostatic interactions is expected to vary with the changing pKa
values of the residual carboxylic acid moiety. Increase in chain
length between the two carboxylic acid groups of the dibasic acid
would also help in identifying the role of the spacer length in inhi-
bition. Apart from this, an alternation of the hydrophobic character
in the dibasic acid part can help us estimate the hydrophobic tol-
erance in RNase A inhibition.
6 and 7 were unambiguously characterized by spectral and analyt-
ical data.
2.2. Enzymatic activity
The inhibition of the ribonucleolytic activity of RNase A was ini-
tially checked qualitatively by an agarose gel-based assay, where
the degradation of tRNA by RNase A was monitored (Fig. 2). The
most intense band observed in lane 1 for each gel is due to the
presence of the control tRNA. The maximum possible degradation
of tRNA by RNase A results in the faint intensity of the band ob-
served in lane 2. The differential intensity of bands in lanes 3 to
5 indicated the extent of RNase A inhibition by the compounds
at three different concentrations (lower to higher concentrations,
respectively). These results qualitatively show better inhibition po-
tency for Oxa-aT (2), Mal-aT (3), Suc-aT (6), compared to Glu-aT
(7).
The histogram (Fig. 3) of relative ribonucleolytic activities ob-
tained from a precipitation assay at a particular inhibitor concen-
tration (1 mM) gave us a quantitative idea about the comparative
inhibitory efficacy of the nucleoside–dibasic acid conjugates.
It was observed that Oxa-aT showed the most potent inhibition
of RNase A. An increase in the chain length of the dibasic acid part
resulted in a weaker inhibition with that for Glu-aT being quite
low. This suggests that the distance between the ‘free carboxylic
group’ and the nucleoside moiety plays a decisive role in the ribo-
nuclease inhibition process. The comparative bar diagram for the
residual ribonucleolytic activity of RNase A obtained from the aga-
rose gel (quantified by individual band analysis) and precipitation
assays is shown in Figure 4. It showed same trend of inhibitory
activity on RNase A.
2. Results and discussion
2.1. Chemistry
In order to synthesize the targeted conjugates, the known 30-
amino-20,30-dideoxy-50-O-trityl thymidine 117,18 was either reacted
with diesters or anhydrides. Thus compound 1 on reactions with
diethyl oxalate19 and diethyl malonate18 at elevated temperature
followed by hydrolysis of the products under basic conditions
and detritylation afforded compounds 2 and 3, respectively, with
moderate yields. On the other hand, 1 was reacted with succinic
anhydride20 and glutaric anhydride in the presence of Et3N at room
temperature to generate the partially protected conjugates 4 and 5,
respectively. Compounds 4 and 5 were detritylated to afford 6 and
7, respectively, in good overall yields (Scheme 1). Compounds 2, 3,
To determine the nature of inhibition and the corresponding
inhibition constants, kinetic experiments were conducted with
the synthesized compounds. The inhibition constant values(Ki) ob-
tained for Oxa-aT, Mal-aT and Suc-aT were 132 3, 380 2 and
918
2 lM, respectively. The nature of the Dixon plots for Oxa-
aT, Mal-aT, Suc-aT (Fig. 5A–C, respectively) are indicative of a
competitive mode of inhibition. The order of values obtained for
the inhibition constants correlated well with those obtained from
O
NH
T
HO
O
O
TrO
N
(i)
(ii)
O
1
2
3
4
5
1
2
3
4
5
a
c
b
d
NH
O
NH2
n
1
O
OH
2 n = 0 (45%)
3 n = 1 (47%)
(iii)
Figure 2. Agarose gel assay, RNase A and inhibitor concentrations 0.66
l
M and
1.5 mM, respectively. Lane1: tRNA; Lane 2: tRNA and RNase A; Lane 3, 4 and 5:
TrO
T
T
HO
tRNA and RNase with increasing concentration of nucleoside–dibasic acid
A
(iv)
O
O
conjugates; (a) Oxa-aT, (b) Mal-aT, (c) Suc-aT; (d) Glu-aT.
NH
n
NH
n
30
20
10
O
O
O
O
OH
OH
4 n = 2 (68%)
5 n = 3 (64%)
6 n = 2 (87%)
7 n = 3 (94%)
Abbreviations
:
2 = Oxa-aT; 3 = Mal-aT; 6 = Suc-aT; 7 = Glu-aT
Scheme 1. Reagents and conditions: for compound 2: (i) diethyl oxalate, 110 °C,
3 h; (ii) NaOH 4 N, refluxed for 2 h, 37% aq HCl; for compound 3: (i) diethyl
malonate, 150 °C, 3 h; (ii) NaOH 4 N, refluxed for 2 h, 37% aq HCl; for compound 4:
(iii) succinic anhydride, Et3N, DCM, rt, 12 h; for compound 5: (iii) glutaric
anhydride, Et3N, DCM, rt, 12 h; for compounds 6 and 7; (iv) 20% TFA in DCM, rt, 2 h.
0
Oxa-aT Mal-aT Suc-aT Glu-aT
Figure 3. Ribonucleolytic inhibition of RNase A by nucleoside–dibasic acid conju-
gates. RNase A and compound concentration are 0.2 lM and 1 mM, respectively.

J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 6491–6495
6493
25
0
Oxa-aT
Mal-aT
Suc-aT Glu-aT
Figure 4. Comparative diagram for RNase A inhibition obtained from agarose gel
(gray) and precipitation assay (black).
2300
1700
1100
2300
1700
1100
500
Figure 6. Docked conformations of Oxa-aT (cyan) and Mal-aT (orange) with RNase
A (1FS3).
500
-0.05
-0.15
0.05
0.15
-0.4
-0.2
0
0.2
[Inhibitor] mM
[Inhibitor] mM
2500
1900
1300
2700
2100
1500
0
0.1
700
-0.2
-1
-0.6
[Inhibitor] mM
Figure 5. Dixon plots for inhibition of RNase
A
l
by (A) Oxa-aT: substrate
M (j) and 150 M (N); (B) Mal-aT: substrate
M (j) and 143 M (N); and (C) Suc-aT: substrate
M (j) and 150 lM (N). RNase A concentration:
Figure 7. Docked conformations of Suc-aT (cyan) and Glu-aT (purple) with RNase A
(1FS3).
concentrations: 250
concentrations: 238
concentrations: 250
l
l
l
M (d), 200
M (d), 190
M (d), 200
l
l
l
l
9 lM.
its carboxylic group near the active site residues as observed for
the other compounds but instead interacted with B2 site resi-
dues.21 Moreover, Suc-aT and Glu-aT did not interact with Lys-
41 which forms a single hydrogen bond to stabilize the transition
state during catalysis.22 As a result they showed lower inhibitory
activity compared to Oxa-aT and Mal-aT. These features corrobo-
rate the competitive mode of inhibition by this series of inhibitors.
the agarose gel and precipitation assay. In the kinetics experiment,
Glu-aT did not show any inhibition (i.e., initial velocity in presence
and absence of Glu-aT remain practically unchanged).
Keeping these results in mind, we further proceeded for pro-
tein–ligand docking studies to visualize the possible conformation
of inhibitors in the protein–ligand complexes. From the docking
experiments it was found Oxa-aT is in close proximity to the three
P1 site residues His-12, Lys-41 and His-119 (Fig. 6) and the pyrim-
idine (thymine) group occupied the B1 site. The interaction of the
carboxylic group with both His-12 and His-119 is expected to per-
turb the normal acid–base mechanism of RNase A thereby inhibit-
ing its enzymatic activity. For Mal-aT the carboxylic group appears
to interact with His-12 and His-119 residues in a more or less sim-
ilar manner as for Oxa-aT. However, for this compound the nucle-
obase is far from the pyrimidine base recognition site, which is the
probable reason for its weaker inhibition compared to Oxa-aT.
The docked conformation of compounds Suc-aT and Glu-aT
(Fig. 7) showed that though the carboxylic group of Suc-aT was
within hydrogen bonding distance with both His-12 and His-119
for Glu-aT only His-119 is within hydrogen bonding distance. Thus
it was expected that Suc-aT perturbed the enzymatic activity more
efficiently than Glu-aT. This may be attributed to the compara-
tively longer chain length of Glu-aT, that makes it unable to place
3. Conclusion
Nucleoside–dibasic acid conjugates are shown to inhibit the
ribonucleolytic activity of RNase A for the first time. The carboxylic
group is expected to alter the microenvironment of the active site
residues like their phosphate analogue. Although all compounds of
this series contain one carboxylic group (exists in carboxylate form
at the physiological pH), their inhibitory activity was found to be
dependent on the length of dibasic acid part. With the increase
of spacer length hydrophobicity of the molecules get increased as
a result the binding of the molecules with the ionic active site be-
comes unfavorable which reflected from their Ki values. Thus the
inhibitory activity for Oxa-aT was three fold greater than Glu-aT.
Docking studies further substantiated these experimental observa-
tions. The major advantage of these compounds with substantial
inhibitory properties is their low ionic character which is expected
to facilitate transportation through biological membranes more

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J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 6491–6495
easily unlike nucleosides functionalized with phosphate or
pyrophosphate.
The inhibitory properties in combination with their low ionic
characters make these compounds suitable candidates for studies
on their transportation through biological membranes in future.
evaporated under reduced pressure. Crude residues thus obtained
were purified over silica gel using 5% methanol in chloroform as
eluent to afford compound 4 (0.4 g, 68%); white solid; mp 82 °C
(crystalized from DCM and MeOH). 1H NMR: (DMSO-d6): d 1.43
(s. 3H), 2.12 (m, 1H), 2.26–2.39 (m, 5H), 3.15 (m, 1H), 3.26 (m,
1H), 3.86 (br s, 1H), 4.42–4.49 (m, 1H), 6.20 (t, J = 6.8 Hz, 1H),
7.23–7.38 (m, 15H), 7.52 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 11.34 (s,
1H). 13C NMR (DMSO-d6): d 12.2, 29.4 (CH2), 30.4 (CH2), 37.2
(CH2), 49.5, 64.1 (CH2), 83.5, 84.0, 86.8 (C), 110.0 (C), 127.6,
128.4, 128.7, 136.1, 143.9 (C), 150.8 (C), 164.1 (C), 171.4 (C),
174.3 (C). HRMS (ESI+): m/z calcd for C33H33N3O7Na [M+Na]+:
606.2217; found: 606.2221.
4. Experimental
Bovine pancreatic RNase A, yeast tRNA, 20,30-cCMP, human ser-
um albumin (HSA) were from Sigma–Aldrich. All other reagents
were from SRL India. Column chromatographic separations were
performed using silica gel (60–120 and 230–400 mesh). Solvents
were dried and distilled following standard procedures. TLC was
carried out on pre-coated plates (Merck silica gel 60, f254), and
the spots visualized with UV light or by charring the plates dipped
in 5% H2SO4–MeOH solution or 5% H2SO4/vanillin/EtOH or 5% nin-
hydrin in MeOH solution. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a Bruker NMR spectrometer
(d scale). UV–vis measurements were made using a Perkin Elmer
UV–vis spectrophotometer (Model Lambda 25). Concentrations of
the solutions were determined spectrophotometrically using the
following data: e278.5 (RNase A),23 e268 (20,30-cCMP)24 are 9800
and 8500 Mꢀ1 cmꢀ1, respectively.
4.4. N-[5-(5-Methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
2-trityloxymethyl-tetrahydro-furan-3-ylcarbamoyl]-butyric
acid (5)
Compound 1 (0.5 g, 1 mmol) was reacted with glutaric anhy-
dride (0.1 g, 1.0 mmol) following the procedure described for 4 to
afford compound 5 (0.4 g, 64%); white solid; mp 196 °C (crystal-
lized from DCM and MeOH). 1H NMR: (DMSO-d6): d 1.45 (s, 3H),
1.65 (m, 2H), 2.05–2.18 (m, 5H), 2.31 (m, 1H), 3.15–3.25 (m, 2H),
3.84 (br s, 1H), 4.47 (m, 1H), 6.17 (t, J = 6.4 Hz, 1H), 7.23–7.38
(m, 15H), 7.53 (s, 1H), 8.21 (d, J = 7.2 Hz, 1H), 11.33 (b, 1H). 13C
NMR (DMSO-d6): d 12.2, 21.0 (CH2), 33.4 (CH2), 34.8 (CH2), 37.3
(CH2), 49.1, 64.0 (CH2), 83.5, 84.0, 86.8 (C), 110.0 (C), 127.5,
128.3, 128.7, 136.1, 143.9 (C), 150.7 (C), 164.1 (C), 171.9 (C),
174.6 (C). HRMS (ESI+): m/z calcd for C34H35N3O7Na [M+Na]+:
620.2373; found: 620.2371.
4.1. N-[2-Hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-
2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl]-oxalamic acid (2)
A
mixture of 30-amino-20,30-dideoxy thymidine (1) (0.6 g,
1.2 mmol) and diethyl oxalate (0.5 g, 3.6 mmol) was stirred at
110 °C for 3 h. The excess diethyl oxalate was then removed by dis-
tillation. The oily residue thus obtained was treated with 4 N NaOH
solutions and refluxed for 2 h. After cooling the reaction mixture it
was filtered and the filtrate was acidified with 37% HCl and kept at
2 °C for 16 h. The aqueous layer was then evaporated under re-
duced pressure. Crude residues thus obtained were purified over
silica gel using 30% methanol in chloroform as eluent to afford
compound 2 (0.17 g, 45%); white hygroscopic solid. 1H NMR:
(DMSO-d6): d 1.75 (s, 3H), 2.13–2.30 (m, 2H), 3.59–3.75 (m, 3H),
3.88 (br s, 1H), 6.21 (t, J = 6.4 Hz, 1H), 7.75 (s, 1H). 13C NMR
(DMSO-d6): d 12.6, 37.4 (CH2), 50.6, 61.2 (CH2), 83.8, 84.7, 109.8
(C), 136.6, 150.8 (C), 164.2 (C), 171.9 (C), 174.6 (C). HRMS (ESI+):
m/z calcd for C12H15N3O7Na [M+Na]+: 336.0809; found: 336.0808.
4.5. N-[2-Hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-
2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl]-succinamic acid (6)
Compound 2 (0.4 g, 0.7 mmol) was stirred with 50% TFA in DCM
(7 ml) for 5 h at room temperature. The acid was then removed un-
der reduce pressure. Crude residues thus obtained were purified
over silica gel using 20% methanol in chloroform as eluent to afford
compound 6 (0.2 g, 87%); white hygroscopic solid. 1H NMR:
(DMSO-d6): d 1.75 (s, 3H), 2.06–2.21 (m, 2H), 2.33 (m, 4H), 3.50–
3.61 (m, 2H), 3.75 (s, 1H), 4.27 (br s, 1H), 5.16 (br s, 1H), 6.15 (t,
J = 6.4 Hz, 1H), 7.78 (s, 1H), 8.44 (br s, 1H), 11.28 (br s, 1H). 13C
NMR (DMSO-d6): d 12.6, 29.5 (CH2), 30.3 (CH2), 37.4 (CH2), 49.5,
61.6 (CH2), 84.0, 85.5, 109.7 (C), 136.7, 150.8 (C), 164.2 (C), 171.6
(C), 174.2 (C). HRMS (ESI+): m/z calculated for C14H19N3O7Na[M+-
Na]+: 364.1121; found: 364.1100.
4.2. N-[2-Hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-
2H-pyrimidin-1-yl)-tetrahydro-furan-3-yl]-malonamic acid (3)
4.6. 4-[2-Hydroxymethyl-5-(5-methyl-2,4-dioxo-3,4-dihydro-
2H-pyrimidin-1-yl)-tetrahydro-furan-3-ylcarbamoyl]-butyric
acid (7)
A mixture of 1 (0.4 g, 0.8 mmol) and diethyl malonate (0.6 g,
3.5 mmol) was stirred at 150 °C for 3 h. The reaction mixture was
worked up and hydrolyzed following the procedure described for
2 to afford compound 3 (0.13 g, 47%); white hygroscopic solid. 1H
NMR: (DMSO-d6): d 1.75 (s, 3H), 2.25–2.36 (m, 2H), 2.74 (br s,
1H), 3.14 (s, 1H), 3.66 (m, 2H), 3.80 (br s, 1H), 4.02 (br s, 1H),
6.27 (t, J = 6.4 Hz, 1H), 7.73 (s, 1H). 13C NMR (DMSO-d6): d 12.6,
36.0 (CH2), 40.0 (CH2), 50.5, 61.3 (CH2), 83.4, 83.8, 110.0 (C),
136.5, 150.8 (C), 164.2 (C), 172.0 (C), 186.7 (C). HRMS (ESI+): m/z
calcd for C1H17N3O7Na [M+Na]+: 350.0965; found: 350.0965.
Compound 3 (0.3 g, 0.5 mmol) was deprotected following the
procedure described for 6 to afford compound 7 (0.18 g, 94%);
white hygroscopic solid. 1H NMR: (DMSO-d6): d 1.76–1.77 (m,
5H), 2.09–2.26 (m, 6H), 3.51–3.62 (m, 2H), 3.73 (br s, 1H), 4.29
(br s, 1H), 6.15 (t, J = 6 Hz, 1H), 7.75 (s, 1H), 8.30 (s, 1H), 11.26
(br s, 1H). 13C NMR (DMSO-d6): d 12.2, 21.0 (CH2), 33.4 (CH2),
34.8 (CH2), 37.3 (CH2), 49.1, 64.0 (CH2), 84.0, 86.8 (C), 110.0 (C),
136.1, 150.7 (C), 164.1 (C), 171.9 (C), 174.6 (C). HRMS (ESI+): m/z
calcd for C15H21N3O7Na [M+Na]+: 378.1277; found: 378.1250.
4.3. N-[5-(5-Methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
2-trityloxymethyl-tetrahydro-furan-3-yl]-succinamic acid (4)
4.7. Agarose gel-based assay
Compound 1 (0.5 g, 1 mmol) was dissolved in dry DCM (5 ml),
to this solution succinic anhydride (0.1 g, 1.0 mmol) and dry Et3N
(0.2 ml, 2.2 mmol) were added and stirred for overnight at room
temperature. It was then diluted with DCM and extracted with
5% citric acid and then with brine.The organic layer was then
Inhibition of RNase A by all nucleoside–dibasic acid conjugates
was checked qualitatively by the degradation of tRNA in an agarose
gel. In this method, 20
l
l of RNase A (0.66
lM) (in TAE buffer) was
mixed with 10, 15 and 20
ll of the compounds (1.5 mM) to a final

J. Debnath et al. / Bioorg. Med. Chem. 17 (2009) 6491–6495
6495
volume of 50
at 37 °C. 20
with 20 l of tRNA solution (5.0 mg/ml) and 10
(containing 10% glycerol and 0.025% bromophenol blue). The mix-
ture was then incubated for another 30 min. A volume of 15
from each solution were extracted and loaded onto a 1.1% agarose
gel. The gel was run in 0.04 M Tris–acetic acid–EDTA (TAE) buffer
(pH 8.0). The undegraded tRNA was visualized by ethidium bro-
mide staining under UV light. Gel images were captured on a KO-
DAK Gel Logic 200 imaging system and relative intensities of the
bands were estimated using Kodak Molecular imaging software.
l
l and the resulting solutions were incubated for 6 h
l aliquots from incubated mixtures were then mixed
l of sample buffer
nucleoside–dibasic acid conjugates were generated by Sybyl6.92
(Tripos Inc., St. Louis, USA) and their energy-minimized conforma-
tions were obtained with the help of the TRIPOS force field using
Gasteiger-Hückel charges with a gradient of 0.005 kcal/mole. The
FlexX software as part of the Sybyl suite was used for docking of
the nucleoside–dibasic acid conjugates with RNase A. PyMol28
was used for visualization of the docked conformations.
l
l
l
l
l
Acknowledgments
J.D. thanks CSIR, India for a fellowship. S.D. and T.P. are grateful
to the Department of Science and Technology (DST), New Delhi, In-
dia, for financial support.
4.8. Precipitation assay
Inhibition of the ribonucleolytic activity of RNase A was quanti-
fied by the precipitation assay as described by Bond.25 In this
Supplementary data
method 10
nucleoside–dibasic acid conjugate (2 mM) to a final volume of
100 l and incubated for 2 h at 37 °C. A 20 l aliquot of the result-
ing solutions from the incubated mixtures were then mixed with
40 l of tRNA (5 mg/ml), 40 l of Tris–HCl buffer of pH 7.5 contain-
ing 5 mM EDTA and 0.5 mg/ml HSA. After incubation of the reac-
tion mixture at 25 °C for 30 min, 200 l of ice–cold 1.14 (N)
perchloric acid containing 6 mM uranyl acetate was added to
quench the reaction. The solution was then kept in ice for another
30 min and centrifuged at 4 °C at 12,000 rpm for 5 min. A 100 ll
ll of RNase A (2 lM) was mixed with 50 ll of each
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.08.018.
l
l
References and notes
l
l
1. Kurachi, K.; Davie, E. W.; Strydom, D. J.; Riordan, J. F.; Vallee, B. L. Biochemistry
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l
aliquot of the supernatant was taken and diluted to 1 ml. The
change in absorbance at 260 nm was measured and compared to
a control set.
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´
8. Nogues, M. V.; Vilanova, M.; Cuchillo, C. M. Biochim. Biophys. Acta 1995, 1253,
16.
4.9. Inhibition kinetics
9. Witzel, H.; Barnard, E. A. Biochem. Biophys. Res. Commun. 1962, 7, 295.
10. Yakovlev, G. I.; Mitkevich, V. A.; Makarov, A. A. Mol. Biol. 2006, 40, 867.
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2008, 18, 5411.
The inhibition of RNase A by Oxa-aT, Mal-aT, Suc-aT and Glu-aT
was assessed individually by a spectrophotometric method as de-
scribed by Anderson et al.24The assay was performed in oligovinyl-
sulfonic acid free26 0.1 M Mes–NaOH buffer, pH 6.0 containing
0.1 M NaCl using 20,30-cCMP as the substrate. The inhibitor concen-
tration was ranged from 0 to 100
tion was used from 150 to 250 M. The RNase A concentration was
M. The inhibition constants (Ki) were determined from initial
lM and the substrate concentra-
15. Wrobel, J.; Green, D.; Jetter, J.; Kao, W.; Rogers, J.; Perez, M. C.; Hardenburg, J.;
Deecher, D. C.; Lopez, F. J.; Arey, B. J.; Shen, E. S. Bioorg. Med. Chem. 2002, 10,
639.
l
9
l
16. Leitner, N. K. V.; Berger, P.; Legube, B. Environ. Sci. Technol. 2002, 36, 3083.
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De Clercq, E. J. Med. Chem. 1988, 31, 2040.
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Tetrahedron: Asymmetry 2001, 12, 1511.
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velocity data. The reciprocal of initial velocity was plotted against
the inhibitor concentration (Dixon Plot) according to the equation:
ꢀ
ꢁ
1
m
Km
Vmax½SꢁKi
1
Vmax
Km
½Sꢁ
¼
½Iꢁ þ
1 þ
ð1Þ
where
v is the initial velocity, [S] the substrate concentration, [I] the
inhibitor concentration, Km the Michaelis constant, Ki the inhibition
constant and Vmax the maximum velocity.
26. Smith, B. D.; Soellner, M. B.; Raines, R. T. J. Biol. Chem. 2003, 278, 20934.
27. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
28. DeLano, W. L. The PyMOL Molecular Graphics System, DeLano Scientific, San
Carlos, CA, 2006. USA. http://pymol.sourceforge.net/.
4.10. Docking studies of the compounds with RNase A (1FS3)
The crystal structure of RNase A (PDB entry 1FS3) was down-
loaded from the Protein Data Bank.27 The 3D structures of the