GmbH, Heidesee, Germany) and incubated for approx. 1 min at
RT in the dark. The reaction was stopped with 3 m H2SO4
(50 μL/well), and the absorbance was measured at 450 nm with a
plate reader (infinite M200 Pro, TECAN). The IC50 value (with
95% confidence interval) of each compound resulted from a
sigmoidal fit to 16 data points, obtained from two serial dilution
rows, by using Origin Pro 9.0.0 G software package. All the IC50
values determined were referenced to the affinity of the internal
standard.
Acknowledgements
CLM thanks the University Grant Commission (UGC), New
Delhi, Govt of India, [Award No. F./311/2017/PDFSS-2017-18-
RAJ-14211]. Grand to GJS (SSB-000726) is also gratefully
acknowledged.
References and notes
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(1) 1.0 μg mL-1, human vitronectin; Merck Millipore.
(2) 2.0 μg mL-1, human αvβ3 integrin, R&D.
(3) 2.0 μg mL-1, mouse anti-human CD51/61, BD Biosciences.
(4) 2.0 μg mL-1, anti-mouse IgG-POD, Sigma-Aldrich.
αvβ5
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(1) 5.0 μg mL-1, human vitronectin, Merck Millipore.
(2) 3.0 μg mL-1, human αvβ5 integrin, R&D.
(3) 1:500 dilution, anti-αv mouse anti-human MAB1978,
Merck Millipore.
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(1) 0.4 μg mL-1, LAP (TGF-β), R&D.
(2) 0.5 μg mL-1, human αvβ8 integrin, R&D.
(3) 1:500 dilutions, anti-αv mouse anti-human MAB1978,
Merck Millipore.
(4) 2.0 μg mL-1, anti-mouse IgG-POD, Sigma-Aldrich.
α5β1
(1) 0.5 μg mL-1, human fibronectin, Sigma-Aldrich.
(2) 2.0 μg mL-1, human α5β1integrin, R&D.
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αIIbβ3
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For NMR spectroscopic studies, approximately 5 mg of the
compound was dissolved in 500 l of DMSO-d6. The required
NMR spectra were recorded at 298 K on Bruker 700 MHz
spectrometer equipped with TXI cryo-probe. 1H-1D, DQF-
COSY, TOCSY, ROESY, 1H-13C HSQC, and 1H-13C HMBC
NMR experiments were acquired. To estimate the solvent
shielding or hydrogen bonding strengths of NH protons,
temperature dependency of the NH chemical shifts was studied
by acquiring 1H-1D spectra from 295 K to 325 K at 5 K intervals.
Mixing times of 80 ms and 300 ms were used for TOCSY and
ROESY experiments, respectively. HSQC spectra were recorded
with a direct proton carbon coupling constant of 140 Hz, and
1
HMBC spectra with a long-range H-13C coupling constant of 7
Hz. For HSQC spectra, a 13C composite pulse decoupling was
utilized. 4k (except HSQC and HMBC: 2k) data points in the
direct dimension, 384 (for DQF-COSY and TOCSY), 512 (for
HSQC), 640 (for ROESY) and 1k (for HMBC) increments in the
indirect dimension were recorded. For all spectra, a 1.5 s
relaxation delay was used after every transient. Exponential /
square sine window functions were used for apodization of the
spectra.
5