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paring working solutions which were also filtered through Sartorius
Stedim Biotech filters of 0.2 mm.
290 nm, temperature slope 908ChÀ1, bandwidth 0.5 nm, data pitch
0.28C, delay time 0 s, time constant 1 s.
Crystallization
UV/Vis spectroscopic titration
A search for crystallization conditions was planned based on the
work of Campbell and Parkinson using the sitting drop vapor diffu-
sion method.[37] Crystallization experiments were performed for the
adducts formed by each semisynthetic derivative of berberine and
the human telomeric sequences Tel23 and Tel12, however, only
crystals for NAX053-Tel12 were obtained by mixing 1 mL of
NAX053 and Tel12 solution in 1:2 stoichiometric ratio at concentra-
tion of 1 mm with 1 mL of 1m (NH4)2SO4, 0.05m Li2SO4 and 0.05m
sodium cacodylate at pH 6.5 and equilibration against 100 mL of
2.4m (NH4)2SO4 solution at T=238C. Yellow prismatic crystals were
observed after two weeks upon drops deposition.
Absorbance spectral studies were performed on a Thermo Scientif-
ic Evolution 220 UV-vis spectrophotometer (Waltham, MA, USA) at
258C. Every experiment was conducted in 150 mm KCl and 10 mm
potassium phosphate buffer at pH 7. Matched quartz cells (Hellma,
Germany) of 1 cm path length were used. The Tel24 concentration
was determined by using e260nm =244600mÀ1 cmÀ1; for berberine
and its semisynthetic derivatives, e345nm =22500mÀ1 cmÀ1 and
e341nm =21500mÀ1 cmÀ1, respectively, were used. These values were
determined under our experimental conditions and are in agree-
ment with previously published data.[16,17,20,22]
Small aliquots of Tel24 solution were added to a solution of ligand
at known concentration (~5 mm) until saturation was reached.
Equal additions were made to a reference cell during each titration.
Readings were noted 5 min after each addition and subsequent
mixing in order to guarantee the homogeneous adduct formation.
The parameters used for recording spectra are listed as follows:
spectral range 400–240 nm, scan speed 100 nmminÀ1, bandwidth
1 nm, integration time 0.6 s, data interval 1 nm. The variation in
peak intensity at about 340 nm for the ligands was used to con-
struct the binding isotherm following the method reported by
Ghosh and co-workers.[20–22] Briefly, the isotherms were constructed
by plotting DA/DAmax at the chosen wavelength as a function of in-
creasing quadruplex concentration. The experimental points were
fitted by using Equation (1):
Data collection and structure refinement
Diffraction experiments on the crystals were performed at 100 K,
using as cryoprotectant the crystallization condition supplemented
with 30% v/v glycerol. Data were collected at the ID23-1 beam-line
(ESRF, Grenoble) up to a maximum resolution of 1.70 , using
1.000 wavelength X-ray. Data were integrated and scaled using
the program XDS.[38] The structure of the adduct was solved by
molecular replacement using the program Phaser.[39] The crystallo-
graphic coordinates of the Tel12-coptisine adduct were used, as
a search model, after deleting atoms belonging to ligand and sol-
vent molecules (PDB accession number 4P1D). The FO-FC electron
density maps showed a clear density for the drug molecule located
on a 4-fold screw axis. The T18 and T24 thymine residues are disor-
dered and they have been only partially localized in the crystal
structure. The model was refined using the program Refmac5 from
the CCP4 package.[40] Thermal factors were treated as isotropic. The
NAX053 molecule was built by using the program JLigand and
added manually into the electronic density maps.[41] Manual re-
building of the model was performed using the program Coot.[42]
The crystal packing analysis was made by means of the Mercury
program.[43] Final coordinates and structure factors have been de-
posited with the Protein Data Bank (PDB accession number 5CDB).
Statistics of the data collection and refinement are reported in
Table S1 in the Supporting Information.
2
C0ðDA=DAmaxÞ ÀðC0 þ Cp þ KdÞðDA=DAmaxÞ þ Cp ¼ 0
ð1Þ
where C0 is the concentration of ligand, DA is the variation in ab-
sorbance at the chosen wavelength after each quadruplex addi-
tion, DAmax is the DA value at ligand saturation, and Cp is the quad-
ruplex concentration. The intercept on the abscissa at DA/DAmax
=
0.5 gives the dissociation constant Kd. DAmax was obtained from
the plot 1/DA vs. 1/(Cp-C0) using Equation (2):
1=DA ¼ 1=DAmax þ 1=ðCp-C0Þ
ð2Þ
The binding stoichiometry was estimated from the interception of
straight lines resulting from fitting of the experimental points be-
longing to the initial and the saturation regions of the isotherm.
The quadruplex concentration at the interception was thus divided
by the concentration of the ligand to yield the binding stoichiome-
try.
Evaluation of cytotoxic activity on cancer cells
Human HeLa (uterine cervix carcinoma), A375 (melanoma), SW613-
B3 and HCT116 (colon carcinoma), and MCF-7 (breast carcinoma)
cell lines were grown at 378C and 5% CO2 atmosphere, in Dulbec-
co’s modified Eagle’s medium (DMEM) supplemented with 10%
FBS, 0.1 mgmLÀ1 penicillin, 100 UmLÀ1 streptomycin, 2 mm gluta-
mine and 2% sodium pyruvate (all reagents were from Celbio,
Milano, Italy). Twenty-four hours after seeding, cells were treated
for 24 h with the BER derivative NAX042 or NAX053 (stock solu-
tions: 10 mm in DMSO) at different concentrations. Cell viability
was analyzed by the MTT metabolic assay, which measures mito-
chondrial activity, by applying a previously reported procedure.[33]
Briefly, 2103 cells were seeded in 96-multiwell plates and, 24 h
later, incubated for 24 h with increasing concentrations (1–10 mm)
of NAX053. At the end of the incubation, 20 mL of Cell Titer 96
AQueous One Solution Cell Proliferation Reagent (Promega Italia,
Milano, Italy) were added to each well. The plates were then main-
tained for 4 h at 378C; the absorbance of each sample was mea-
sured with a microplate reader (EZ Red 400, Biochrom, Cambridge,
UK) at 492 nm. Experiments were performed in quadruplicate and
Circular Dichroism Spectroscopy
CD spectra were recorded on a Jasco J-810 Spectropolarimeter
(Jasco Cooperation, Tokyo, Japan) equipped with a Peltier tempera-
ture controller (model JWJTC-484). Matched quartz cells (Hellma,
Germany) of 0.1 cm path length were used. Solutions at known
concentration of Tel24 (56.3 mm) were prepared increasing the
ligand concentration up to ligand:quadruplex ratio of 4:1. Solu-
tions were prepared in 90 mm LiCl, 10 mm KCl and 10 mm lithium
cacodylate at pH 7. For each solution, CD spectra at 258C and ther-
mal melting curves were recorded. The technical parameters of the
experiments are listed as follows. CD spectra: spectral range 400–
230 nm, scan speed 100 nmminÀ1, bandwidth 0.5 nm, data pitch
0.5 nm, time constant 1 s, 6 accumulations. Melting curves: l=
Chem. Asian J. 2016, 11, 1107 – 1115
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