Amino and carboxy functionalized modified nucleosides: A potential class of inhibitors for angiogenin

Article abstract of DOI:10.1016/j.bioorg.2013.11.005

The 3′-amino and carboxy functionalize thymidines execute their ribonucleolytic inhibition activity for angiogenin. These modified nucleosidic molecules inhibit the ribonucleolytic activity of angiogenin in a competitive manner like the other conventional nucleotidic inhibitors, which have been confirmed from kinetic experiments. The improved inhibition constant (K _i) values 427 ± 7, 775 ± 6 μM clearly indicate modified nucleosides are an obvious option for the designing of inhibitors of angiogenesis process. The chorioallantoic membrane (CAM) assay qualitatively suggests that amino functionalized nucleosides have an effective potency to inhibited angiogenin-induced angiogenesis. Docking studies further demonstrate the interaction of their polar amino group with the P₁ site residues of angiogenin, i.e., His-13 and His-114 residues.

Full text of DOI:10.1016/j.bioorg.2013.11.005

Bioorganic Chemistry 52 (2014) 56–61
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Amino and carboxy functionalized modified nucleosides: A potential
class of inhibitors for angiogenin
b
b
Joy Debnath a, , Swagata Dasgupta , Tanmaya Pathak
⇑
a School of Chemical and Biotechnology, SASTRA University, Thanjavur, Tamil Nadu 613401, India
b Department of Chemistry, Indian Institute of Technology, Kharagpur, Kharagpur 721302, India
a r t i c l e i n f o
a b s t r a c t
The 30-amino and carboxy functionalize thymidines execute their ribonucleolytic inhibition activity for
angiogenin. These modified nucleosidic molecules inhibit the ribonucleolytic activity of angiogenin in
a competitive manner like the other conventional nucleotidic inhibitors, which have been confirmed from
Article history:
Received 13 July 2013
Available online 26 November 2013
kinetic experiments. The improved inhibition constant (Ki) values 427 7, 775
6 lM clearly indicate
Keywords:
Thymidine
Angiogenesis
Chorioallantoic membrane (CAM) assay
Docking
modified nucleosides are an obvious option for the designing of inhibitors of angiogenesis process. The
chorioallantoic membrane (CAM) assay qualitatively suggests that amino functionalized nucleosides
have an effective potency to inhibited angiogenin-induced angiogenesis. Docking studies further demon-
strate the interaction of their polar amino group with the P1 site residues of angiogenin, i.e., His-13 and
His-114 residues.
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
pyrimidine specificity whereas the B2 site has an affinity for purine
nucleobase [9].
Angiogenin is a member of ribonuclease superfamily protein [1]
and is a potent inducer of angiogenesis [2] (growth of new blood
vessel). The angiogenesis process is found to operate at an alarm-
ing rate during the proliferation of tumor cells for the supply of
nutrients necessary for their growth to solid tumor [3]. Angiogenin
and ribonuclease A (RNase A) being the members of same super-
family they share a structural homology regarding their ribonucle-
olytic site but the ribonucleolytic activity of angiogenin is about
105-106 times weaker than that of RNase A [4]. However, ribonuc-
leolytic activity of angiogenin is crucial for its angiogenic activity
[5]. Therefore the enzymatic site (ribonucleolytic site) of angioge-
nin is an important target from therapeutic point of view. Abolition
of the ribonucleolytic activity by low molecular weight inhibitors
would therefore tantamount to inhibit the angiogenic activity of
angiogenin leading to the inhibition of the undesired neovascular-
ization. The ribonucleolytic site of RNase A and angiogenin is com-
prised of multiple subsites which bind the phosphate, base, and
sugar components of RNA [6–8]. The P1 subsite where cleavage
of the phosphodiester bond occurs is comprised by His-12, Lys-
41 and His-119 for RNase A and His-13, Lys-40 and His-114 for
angiogenin and it serves as the main catalytic site (Fig. 1). There
are two other important B1 and B2 subsites which recognize the
nucleobases of normal RNA. Among these subsites, B1 site shows
Most of the nucleotide-based inhibitors of angiogenin are func-
tionalized with phosphate or pyrophosphate group. These inhibi-
tors inhibit the ribonucleolytic activity of angiogenin by virtue of
their structural similarities with its normal substrate [7,10]. How-
ever, these conventional nucleotidic inhibitors have a limited suc-
cess towards angiogenin compared to RNase A [11], as well as, the
high negative charge on the phosphate groups is a great disadvan-
tage regarding their cell permeability [12]. Because of the limited
success of the nucleotidic inhibitors different non-nucleosidic mol-
ecules are tried on angiogenin [13] and also dinucleosides with
amide linkage [14].
In literature it has been reported that for RNase A the pKa values
of His-12 and His-119 changes from ꢀ5.22/6.78 to ꢀ6.30/8.10 dur-
ing its binding with the substrate [15]. Keeping this point in mind
we employed a series of 30-modified nucleosides functionalized
with amino and carboxy groups for angiogenin inhibition study.
At the physiological pH these polar groups are likely to remain in
their conjugate form. Now if these molecules are recognized at
the ribonucleolytic site of angiogenin because of the presence of
nucleodase and ribose moieties then amino and carboxylic groups
have a possibility to interact with the P1 site residues. However,
replacement of the phosphate or pyrophosphate group by one
amino or carboxylic group lowers the net negative charge of these
molecules compared to their nucleotide analogs.
In this particular report we have selected 20-deoxynucleoside to
protect the amide bond towards the hydrolysis so that they can de-
liver the free amino and carboxylic groups at the catalytic site.
⇑
Corresponding author. Fax: +91 4362 264120.
E-mail address: joydebnath@scbt.sastra.edu (J. Debnath).
0045-2068/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2013.11.005

J. Debnath et al. / Bioorganic Chemistry 52 (2014) 56–61
57
B1
B1
Ile42
B
A
Val 43
Asn 43
Asn 44
Thr 45
Phe 120
Ser 123
Cyt
or
Ura
Thr 44
Thr 80
Leu 115
Gln 117
O
P
O
Cyt
or
Ura
O
P
O
O
O
O
R1
O
R1
OH
OH
O
P
His 12
Lys 41
His 119
O
P
His 13
Lys 40
His 114
Asn 67
O
O
O
O
Asn 68
Leu 69
Glu 108
Gln 69
Asn 71
Glu 111
Ade
O
Ade
O
O
O
P
1
P1
R2
R2
B2
OH
B2
OH
O
P
O
P
O
O
O
O
O
O
Fig. 1. Key residues of ribonucleolytic site of (A) Human angiogenin and (B) RNase A.
Otherwise in presence of the 20-OH, the inhibitor molecules can
undergo easy hydrolysis of its amide bond as found in case of
angiogenin’s normal substrate (RNA).
2. Results and discussion
Amino and carboxy functionalize modified nucleosides*
showed their inhibitory activity on RNase A [16,17] and it encour-
ages us to extend the inhibition study for angiogenin, a homolo-
gous protein of RNase A (structures of the inhibitor molecules of
these series is given in Fig. 2). Therefore, the most potential com-
pounds of those series were selected for this study. However, com-
parison of the inhibitory potential of these two series can also
produce valuable information for the further modification of low
molecular weight inhibitor of angiogenin.
Fig. 3. Percentage inhibition of ribonucleolytic activity of angiogenin by compounds 1,
2, 3, 4 (black) and 5, 6, 7, 8 (gray).
the dibasic acid part played the determining role in inhibition
process.
The ribonucleolytic inhibitory activity of these compounds on
angiogenin was quantitatively estimated by precipitation assay
[18]. We observed the similar trend of inhibition in case of angio-
genin as for RNase A for both class of compounds. Compound 2 and
5 from amino and carboxy functionalized nucleosides respectively
showed the best inhibitory activity on angiogenin (22.00% and
23.42% respectively) (Fig. 3). It suggested both amino and carboxy
functionalizations have an equal importance in angiogenin inhibi-
tion. The bar diagram obtained from this assay (Fig. 3) indicated
that for amino functionalize nucleosides inhibitory activity de-
pends upon the nature of the side chain part for respective amino
acids. However, for the other set of compounds the chain length of
The mode of ribonucleolytic inhibition of angiogenin was fur-
ther confirmed with the help of kinetic experiment where 6-
FAM-dArUdAdA-6-TAMRA was used as the substrate of angiogenin
protein [19]. This study was based on the fluorescence quenching
of the fluorescein moiety attached to rhodamine by a phosphodies-
ter bond. The enhanced fluorescence of fluorescein moiety was
measured during the cleavage of facile phosphate bond by angio-
genin. The inhibition constant value (Ki) for compounds 2, 3, 5
and 6 was found 427 7, 1251 4, 775 6 and 1,114
3 lM
respectively and the nature of the Lineweaver-Burk plots con-
firmed their competitive mode of inhibition (Fig. 4). The Ki values
of these four compounds are much better than 30-CMP
(12,703 0.03 lM), a nucleotidic inhibitor of angiogenin (deter-
mined using the same substrate). However, the Ki values obtained
from this experiment clearly show the preference of amino group
over the carboxy one. The comparative Ki values of these inhibitors
for RNase A and angiogenin are given in Table 1.
In view of the results obtained from inhibition of the ribonucle-
olytic activity of angiogenin based on the fluorimetric assay, it ap-
peared pertinent to observe the effect of the amino functionalized
modified nucleosides on the angiogenin induced angiogenesis. The
chorioallantoic membrane (CAM) assay [1] was therefore con-
ducted to verify the inhibition of angiogenin-induced angiogenesis
by compounds 2 and 3. As expected, vascularization was enhanced
in presence of angiogenin (Fig. 5A). Vascular growth was compara-
ble to the blank (Fig. 5B) for the cases when compounds mixed
with angiogenin. It suggested that both 2 (Fig. 5C) and 3 (Fig. 5E)
suppress the growth of blood vessels induced by angiogenin. This
Fig. 2. Structures of amino and carboxy functionalize modified nucleosides. *Note:
Synthesis of these inhibitor molecules has been reported in Refs. [16,17].

58
J. Debnath et al. / Bioorganic Chemistry 52 (2014) 56–61
Fig. 4. Lineweaver-Burk plots for the inhibition of angiogenin by (A) 2: 0 mM (ꢀ), 0.2 mM (j) and 0.8 mM (N); (B) 3: 0 mM (ꢀ), 0.4 mM (j) and 1.2 mM (N); (C) 5: 0 mM (ꢀ),
0.3 mM (N) and 1.2 mM (j); (D) 6: 0 mM (ꢀ), 0.7 mM (N) and 2.5 mM (j). Angiogenin concentration: 0.36 lM.
Table 1
Docking studies were then performed with the help of FlexX to
get an insight about the possible interactions involved between the
protein residues and ligand molecules. In the most preferred dock-
ing poses for 2 and 3 with human angiogenin (1B1I), we found that
the amino groups were within hydrogen bonding distance of His-
114 (Fig. 6A and B). Along with this possible interaction compound
2 also formed a hydrogen bonding network with His-13 with its 50-
OH and 30-amide oxygen. However, the thymine nucleobase of 2
was found to interact with Lys-40 and Asp-41 residues of angioge-
nin. In case of compound 3 only the 30-amide oxygen atom was
within hydrogen bonding distance of His-13 and its nucleobase
moiety was found to interact only with Asp-41 which is a residue
of angiogenin near the B1 subsite. The interactions with the ribo-
nucleolytic site residues corroborated competitive nature of the
synthesized inhibitors as observed in the Lineweaver-Burk plots
for compounds 2 and 3. The hydrogen bonding distance between
the amino acid residues of angiogenin and compounds 2 and 3
are given in Table 2.
Comparative Ki values for RNase A and angiogenin with compounds 2, 3, 5 and 6.
Compound
Ki value for RNase A (
l
M)
Ki value for angiogenin (lM)
2
3
5
6
80
451
132
380
3
2
2
2
427
1251
775
7
4
6
3
1114
On the other hand, for carboxy functionalized nucleoside 5 it
was found that the carboxylic group was in a position to form a
hydrogen bonding network with His-13 and His-114, like the phos-
phate groups of nucleotidic inhibitors. This also corroborated the
competitive nature of 5 as obtained the nature of Lineweaver-Burk
plot for 5. The thymine moiety of 5 was found within the hydrogen
bonding distance of Asp-41 and Arg-121. From the docked confor-
mation of 6, however, we found the free carboxylic group was pro-
jected in a different orientation than for 5 and formed a possible
hydrogen bonding network with Ser-118 and Asp-116 (Fig. 7A
and B). The ribonucleolytic site residues His-13 and His-114 were
found within the hydrogen bonding distance of thymine nucleo-
base as well as with the carbonyl oxygen of the amide bond at
the 30-position. The most important thing found from docking
experiment was that for amino functionalized modified nucleo-
sides alternation of the amino acid side chain did not changed
the binding patterns of the ligands with angiogenin. However, for
carboxy functionalized modified nucleosides incorporation of addi-
tional CH2 group drastically changed their binding pattern. The
Fig. 5. CAM assay (A) 10 ng angiogenin; (B) control; (C) 0.1
angiogenin; (D) 0.1
(F) 0.1 M compound 3.
l
M compound 2 + 10 ng
l
M compound 2; (E) 10 ng angiogenin + 0.1
l
M compound 3;
l
is more apparent for 2 than 3 indicating, albeit qualitatively, but it
indicate compound 2 acts as a better inhibitor than compound 3.
On the other hand the compounds themselves inhibit vascular
growth to a negligible extent (Fig. 5D and F).

J. Debnath et al. / Bioorganic Chemistry 52 (2014) 56–61
59
Fig. 6. Docking poses (A) compound 2 and (B) compound 3 with human angiogenin (1B1I).
Table 2
Hydrogen bonding distance between the amino acid residues of angiogenin and compounds 2 (A), 3 (A), 5 (B) and 6 (C).
Protein 1B1I
Compound 2
Compound 3
Amino acid residues of 1B1I
(A)
g
1
g
Arg-5 [N
Gln-12 [O
Gln-12 [N
]
]
2.82 [O
]
e
1
2.54 [O amide of amino acid]
3.28 [O amide of amino acid]
3.21 [O amide of amino acid],
3.42 [O50]
3.28 [O2], 3.20 [N1], 3.02 [O(sugar)]
2.51 [O4], 2.64 [N3]
3.37 [O50], 3.10 [NH2] amino acid
e
2
]
e
2
His-13 [N
]
3.12 [O amide of amino acid]
Lys-40 [Nf]
Asp-41 [Od1
]
2.57 [O4], 2.42 [N3]
3.15 [NH2] amino acid
His-114 [Nd1
]
Angiogenin (1B1I)
Amino acid residue of 1B1I
(B)
Compound 5
e2
His-13 [N
]
2.92 [O (COOH)]
2.87 [O (amide bond)]
3.33 [N (3)]
Lys-40 [Nn]
Asp-41 [Od1
]
3.13 [O (4)]
3.08 [O (2)]
2.73 [N (3)]
2.66 [OH (COOH)]
3.23 [O (COOH)]
3.44 [O (COOH)]
2.96 [O(4)]
Asp-41 [Od2
]
His-114 [Nd1
Leu-115 [N]
Leu-115 [O]
]
g
1
2
Arg-121 [N
Arg-121 [N
]
]
g
2.43 [O(4)]
Angiogenin (1B1I)
Amino acid residue of 1B1I
(C)
Compound 6
e2
His-13 [N
His-114 [N
Leu-115 [O]
]
3.10 [O(4)]
2.82 [O (amide bond)]
2.87 [O(2)]
e
2
]
2.58 [N(3)]
Asp-116 [Od1
Asp-116 [Od2
]
]
2.99 [OH (COOH)]
3.44 [N (amide bond)]
3.30 [O (amide bond)]
2.37 [OH (COOH)]
2.81 [O(2)]
Gln-117 [N (back bone)]
Ser-118 [N]
3.09 [OH (50)]
c
Ser-118 [O ]
2.56 [OH (COOH)]
g
2
Arg-121 [N
]
2.81 [OH (50)]
e
Arg-121 [N ]
2.87 [OH (50)]
hydrogen bonding distance between the amino acid residues of
angiogenin and compounds 5 and 6 are given in Table 2.
Theoretical calculations of binding energies along with docking
studies had been performed for the better understanding of inhibi-
tion by compounds 2 and 3 compared to that of 30-CMP as
observed experimentally. These two compounds were selected
for their more or less similar binding pattern with angiogenin
and to find out the reason for better inhibitory activity of amino
functionalize modified nucleosides over their phosphate analogs.
The total ligand-receptor interaction energy values obtained from

60
J. Debnath et al. / Bioorganic Chemistry 52 (2014) 56–61
Fig. 7. Docking poses (A) compound 5 and (B) compound 6 with human angiogenin (1B1I).
PEARLS [20] were found to be -8.75, -6.25, -2.13 kcal/mole for com-
pounds 2, 3 and 30-CMP respectively. This implies that 2 and 3 bind
more strongly with angiogenin than 30-CMP which was in agree-
ment with the experimental observations. Gln-117 of angiogenin
produced a hindrance during pyrimidine binding at the B1 site,
which resulted in the relatively lower ribonucleolytic activity of
the protein [21]. In the docked conformation of 30-CMP with angio-
genin the phosphate group was within hydrogen bonding distance
of His-13 (2.76 Å) and His-114 (3.00 Å). The presence of the sugar
moiety along with phosphate anchoring renders the compound
less effective as an inhibitor compared to 2 and 3 (Fig. 8). For the
compounds 2 and 3, the longer chain and inherent flexibility pro-
vides a positional advantage of the thymine nucleobase to avoid
the hindrance offered by Gln-117 during binding at the B1 site. This
is most likely the cause for the better inhibition of 2 and 3.
From the accessible surface area (ASA) calculation between
angiogenin (uncomplexed) and the respective docked complexes
with compounds 2, 3, 5, 6 it is clearly evident that these ligands
binds with the active site residues and make them less accessible
towards the solvent molecules (Table 3). From the table it is well
documented that compounds 2 and 5 preferentially interact with
His-13 and Lys-41 compared with His-114.
Amino and carboxy functionalized nucleosides showed to inhi-
bit the ribonucleolytic activity of angiogenin. The inhibitory activ-
ity of these two set of compounds was found to be dependent on
the polarity of the amino acid side chain part and on the hydropho-
bicity of the dibasic acid part. Inhibition constant values of 2, 3, 5
and 6 are 427 7, 1251 4, 775 6 and 1,114
which are much lower than 20-CMP, 30-CMP, 20-UMP and 50-AMP
(Ki values are 8700, 13000, 13100 and 7200 M respectively) [8].
3 lM respectively,
l
CAM assay explored that angiogenin-induced angiogenesis was
also inhibited by amino functionalize nucleosides 2 and 3. Along
with the lower Ki values, these compounds are less polar compared
with their nucleotidic analogs. Therefore, these synthesized com-
pounds can be used in vivo more effectively as a potential antian-
giogenic drugs.
3. Materials and methods
3.1. Materials
Recombinant human angiogenin, human serum albumin (HSA),
were from Sigma-Aldrich. 6-FAM-dArUdAdA-6-TAMRA was pur-
chased from Integrated DNA Technologies (Coralville, IA). All other
reagents were from SRL India. UV-Vis measurements were made
Fig. 8. Combined docking poses of 2, 3 and 30-CMP with human angiogenin (1B1I).
Table 3
Change in accessible surface area (
D
ASA) in Å2 of interacting residues of angiogenin (Uncomplexed), and its complexes with compounds 2, 3, 5 and 6.
Amino acid residues
Compound 2
Compound 3
Compound 5
Compound 6
Arg-5
Gln-12
His-13
00
22.25
00
17.31
00
9.36
15.28
00
00
00
00
00
00
00
00
00
6.23
00
00
16.51
28.74
13.65
6.06
10.92
6.85
88.96
34.36
39.62
17.25
21.33
16.49
00
00
00
00
00
32.63
18.32
21.59
7.61
8.48
00
00
00
11.24
99.87
Lys-40
Asp-41
His-114
Leu-115
Asp-116
Gln-117
Ser-118
Arg-121
R
D
ACS
129.05
64.2

J. Debnath et al. / Bioorganic Chemistry 52 (2014) 56–61
61
using a Perkin Elmer UV–Vis spectrophotometer (Model Lambda
25). Fluorescence measurements were recorded on a Spex Fluoro-
log-3 machine. Concentrations of the solutions were determined
of the nucleoside-amino acid conjugates with RNase A. PyMol [27]
was used for visualization of the docked conformations. The total
ligand-receptor interaction energies of the docked structures for
protein-ligand complexes was computed using PEARLS [20].
spectrophotometrically using the following data: e278 (angiogenin)
[22], e260 (6-FAM-dArUdAdA-6-TAMRA) [23] are 12500 and
102,400 Mꢁ1 cmꢁ1 respectively.
3.6. Accessible surface area calculation
3.2. Precipitation assay with angiogenin
The accessible surface area (ASA) of angiogenin (uncomplexed)
and their docked complexes with compounds 2, 3, 5, 6 were calcu-
lated using the program NACCESS [28]. The structures correspond-
ing to the minimum score as obtained from the FlexX analysis of
the protein-ligand docked structures were chosen in each case.
Composite coordinates of the angiogenin and inhibitors were gen-
erated to form the docked complex. The change in ASA for residue,
Inhibition of the ribonucleolytic activity of angiogenin was
quantified following the method as described by Bond [18]. In this
method 5
20 M of 1, 2, 3, 4, 5, 6, 7 and 8 individually along with 20
6-FAM-dArUdAdA-6-TAMRA (0.1 mg/ml) and 20 of HSA
(0.02 mg/ml) to a final volume of 55 l and incubated for 5 h at
37 °C. After incubation, the reaction mixture was quenched with
100 l of ice-cold 1.14 N perchloric acid containing 6 mM uranyl
acetate. The solution was then kept in ice for another 30 min and
centrifuged at 4 °C at 12000 rpm for 5 min. 100 l of the superna-
tant was taken and diluted to 550 l. The change in absorbance at
ll of angiogenin (0.2
l
M) was mixed with 10
l
l
l of
l of
l
ll
l
i was calculated using:
D
ASAi ¼ ASAiangiogenin ꢁ ASAai ngiogenin-inhibitor. If
a residue lost more than 10 Å2 ASA when going from the uncom-
plexed to the complexed state it was considered as being involved
in the interaction.
l
l
l
Acknowledgments
260 nm was measured and compared with a control set.
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,
India, for financial support.
3.3. Inhibition kinetics with angiogenin
Purchased recombinant human angiogenin contains a substan-
tial amount of bovine serum albumin (BSA). Pure angiogenin was
obtained after removal of BSA using Amicon Ultra centrifugal filter
devices (Millipore) with a 30kD molecular weight cutoff. Purified
angiogenin was checked to be free of contaminating ribonucleases
by zymogram electrophoresis [24,25]. 6-FAM-dArUdAdA-6-TAM-
RA was used as substrate where 6-FAM is 50,6-carboxyfluorescein
and 6-TAMRA is 30,6-carboxytetramethylrhodamine. Kinetic exper-
iments were carried out in oligovinylsulfonic acid free [21] 0.1 M
Mes-NaOH buffer (pH 6.0) containing 0.1 M NaCl using substrate
concentration from 20 to 122 nM. For 2, 3, 4 and 5 the concentra-
tion ranged from 0 to 0.8, 0 to 1.2, 0 to 1.2 and 0 to 2.5 mM respec-
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2013.11.
005.
References
[1] J.W. Fett, D.J. Strydom, R.R. Lobb, E.M. Alderman, J.L. Bethune, J.F. Riordan, B.L.
Vallee, Biochemistry 24 (1985) 5480–5486.
[2] Guo-Fu Hu, J.F. Riordan, B.L. Vallee, Proc. Natl. Acad. Sci. USA 91 (1994) 12096–
12100.
[3] J. Folkman, M. Klagsbrun, Science 235 (1987) 442–447.
[4] R. Shapiro, J.F. Riordan, B.L. Vallee, Biochemistry 25 (1986) 3527–3532.
[5] R. Shapiro, B.L. Vallee, Biochemistry 28 (1989) 7401–7408.
[6] M.V. Nogue´s, M. Vilanova, C.M. Cuchillo, Biochim. Biophys. Acta 1253 (1996)
16–24.
[7] R.T. Raines, A. Ribonuclease, Chem. Rev. 98 (1998) 1045–1066.
[8] N. Russo, K.R. Acharya, B.L. Vallee, R. Shapiro, Proc. Natl. Acad. Sci. USA 93
(1996) 804–808.
tively. The angiogenin concentration was 0.36 lM. An increase in
fluorescence emission intensity at 515 nm, upon excitation at
490 nm, indicated the progress of the reaction [19]. The inhibition
constants (Ki) were determined from initial velocity data using
Lineweaver-Burk plots.
3.4. Angiogenic activity
[9] H. Witzel, E.A. Barnard, Biochem. Biophys. Res. Commun. 7 (1962) 295–299.
[10] N. Russo, K.R. Acharaya, R. Shapiro, Methods Enzymol. 341 (2001) 629–648.
[11] H. Katoh, M. Yoshinaga, T. Yanagita, K. Ohgi, M. Irie, J.J. Beintema, D. Meinsma,
Biochim. Biophys. Acta 873 (1986) 367–371.
[12] G.I. Yakovlev, V.A. Mitkevich, A.A. Makarov, Mol. Biol. 40 (2006) 867–874.
[13] J.L. Jenkins, R.Y.T. Kao, R. Shapiro, Proteins: Struct. Funct. Genet. 50 (2003) 81–
93.
[14] J. Debnath, S. Dasgupta, T. Pathak, Chem. Eur. J. 18 (2012) 1618–1627.
[15] D.G. Herries, A.P. Mathias, B.R. Rabin, Biochem. J. 85 (1962) 127–134.
[16] J. Debnath, S. Dasgupta, T. Pathak, Bioorg. Med. Chem. 17 (2009) 4921–4927.
[17] J. Debnath, S. Dasgupta, T. Pathak, Bioorg. Med. Chem. 17 (2009) 6491–6495.
[18] M.D. Bond, Anal. Biochem. 173 (1988) 166–173.
The effect of 2 and 3 on angiogenin-induced angiogenesis was
assessed by the chick embryo chorioallantoic membrane (CAM) as-
say as described by Fett et al. [1] The chorioallantoic membrane of
the chicken egg was exposed by carefully drilling a small hole
through the shell on day 2. 10 ll aliquots of angiogenin, angiogenin
mixed with 2 and 3 separately were placed on transparent plastic
disks and dried under laminar flow. The disks were inverted over
the chorioallantoic membrane on day 10 and response was assayed
microscopically after 48 h. The density of blood vessel formation
was compared with a control set.
[19] B.R. Kelemen, T.A. Klink, M.A. Behlke, S.R. Eubanks, P.A. Leland, R.T. Raines,
Nucleic Acids Res. 27 (1999) 3696–3701.
[20] L.Y. Han, H.H. Lin, Z.R. Li, C.J. Zheng, Z.W. Cao, B. Xie, Y.Z. Chen, J. Chem. Inf.
Model. 46 (2006) 445–450.
[21] B.D. Smith, M.B. Soellner, R.T. Raines, J. Biol. Chem. 278 (2003) 20934–20938.
[22] R.T. Raines, M.P. Toscano, D.M. Nierengarten, J.H. Ha, R. Auerbach, J. Biol. Chem.
270 (1995) 17180–17184.
[23] R.B. Wallace, C.G. Miyada, Methods Enzymol. 152 (1987) 432–442.
[24] A. Blank, R.H. Sugiyama, C.A. Dekker, Anal. Biochem. 120 (1982) 267–275.
[25] J.S. Kim, R.T. Raines, Protein Sci. 2 (1993) 348–356.
[26] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N.
Shindyalov, P.E. Bourne, Nucleic Acids Res. 28 (2000) 235–242.
[27] W.L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific, San
Carlos, CA, 2004. USA. <http://pymol.sourceforge.net/>.
[28] S.J. Hubbard, J.M. Thornton, ‘‘NACCESS’’ Computer Program, Department of
Biochemistry and Molecular Biology, University College, London, 1993.
3.5. Docking studies of compounds 2, 3, 5 and 6 with angiogenin and
calculations of interaction energetic
The crystal structure of human angiogenin (PDB entry 1B1I) was
downloaded from the Protein Data Bank [26]. The 3D structures of
the nucleoside-amino acid conjugates were generated by Syb-
yl6.92 (Tripos Inc., St. Louis, USA) and their energy-minimized con-
formations 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