12 research outputs found
Interactive association between RhoA transcriptional signaling inhibitor, CCG1423 and human serum albumin: Biophysical and <i>in silico</i> studies
<p>Multiple spectroscopic techniques, such as fluorescence, absorption, and circular dichroism along with <i>in silico</i> studies were used to characterize the binding of a potent inhibitor molecule, CCG1423 to the major transport protein, human serum albumin (HSA). Fluorescence and absorption spectroscopic results confirmed CCG1423–HSA complex formation. A strong binding affinity stabilized the CCG1423–HSA complex, as evident from the values of the binding constant (<i>K</i><sub><i>a</i></sub> = 1.35 × 10<sup>6</sup>–5.43 × 10<sup>5</sup> M<sup>−1</sup>). The <i>K</i><sub><i>SV</i></sub> values for CCG1423–HSA system were inversely correlated with temperature, suggesting the involvement of static quenching mechanism. Thermodynamic data anticipated that CCG1423–HSA complexation was mainly driven by hydrophobic and van der Waals forces as well as hydrogen bonds. <i>In silico</i> analysis also supported these results. Three-dimensional fluorescence and circular dichroism spectral analysis suggested microenvironmental perturbations around protein fluorophores and structural (secondary and tertiary) changes in the protein upon CCG1423 binding. CCG1423 binding to HSA also showed some protection against thermal denaturation. Site-specific marker-induced displacement results revealed CCG1423 binding to Sudlow’s site I of HSA, which was also confirmed by the computational results. A few common ions were also found to interfere with the CCG1423–HSA interaction.</p
Binding and thermodynamic parameters for the interaction between PS and HSA, studied at different temperatures, pH
<p>Binding and thermodynamic parameters for the interaction between PS and HSA, studied at different temperatures, pH</p
Fluorescence quench titration of HSA with increasing PS concentrations.
<p>[HSA] = 3 µM, [PS] = 0–22.5 µM with 1.5 µM intervals (1–16), λ<sub>ex</sub> = 280 nm studied in 10 mM Tris-HCl buffer, pH 7.4, 25°C. Arrow depicts the blue shift in the emission maximum of HSA with increasing PS concentrations. Inset shows the decrease in the relative fluorescence intensity of HSA at 336 nm (FI<sub>336 nm</sub>) with increasing PS/HSA molar ratios.</p
Analysis of fluorescence quenching data.
<p>(A) Stern–Volmer and (B) against plots of PS–HSA system at different temperatures. Inset of (B) shows the van’t Hoff plot for PS–HSA interaction.</p
Characteristics of three-dimensional fluorescence spectra of native HSA and its complexes with PS at pH 7.4, 25°C.
<p>Characteristics of three-dimensional fluorescence spectra of native HSA and its complexes with PS at pH 7.4, 25°C.</p
Distance of the predicted hydrogen bonds formed between interacting residues of HSA and PS.
<p>Distance of the predicted hydrogen bonds formed between interacting residues of HSA and PS.</p
Interaction of a tyrosine kinase inhibitor, vandetanib with human serum albumin as studied by fluorescence quenching and molecular docking
<p>Interaction of a tyrosine kinase inhibitor, vandetanib (VDB), with the major transport protein in the human blood circulation, human serum albumin (HSA), was investigated using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking analysis. The binding constant of the VDB–HSA system, as determined by fluorescence quenching titration method was found in the range, 8.92–6.89 × 10<sup>3 </sup>M<sup>−1</sup> at three different temperatures, suggesting moderate binding affinity. Furthermore, decrease in the binding constant with increasing temperature revealed involvement of static quenching mechanism, thus affirming the formation of the VDB–HSA complex. Thermodynamic analysis of the binding reaction between VDB and HSA yielded positive Δ<i>S</i> (52.76 J mol<sup>−1</sup> K<sup>−1</sup>) and negative Δ<i>H</i> (−6.57 kJ mol<sup>−1</sup>) values, which suggested involvement of hydrophobic interactions and hydrogen bonding in stabilizing the VDB–HSA complex. Far-UV and near-UV CD spectral results suggested alterations in both secondary and tertiary structures of HSA upon VDB-binding. Three-dimensional fluorescence spectral results also showed significant microenvironmental changes around the Trp residue of HSA consequent to the complex formation. Use of site-specific marker ligands, such as phenylbutazone (site I marker) and diazepam (site II marker) in competitive ligand displacement experiments indicated location of the VDB binding site on HSA as Sudlow’s site I (subdomain IIA), which was further established by molecular docking results. Presence of some common metal ions, such as Ca<sup>2+</sup>, Zn<sup>2+</sup>, Cu<sup>2+</sup>, Ba<sup>2+</sup>, Mg<sup>2+</sup>, and Mn<sup>2+</sup> in the reaction mixture produced smaller but significant alterations in the binding affinity of VDB to HSA.</p> <p>Binding orientation of the VDB in the Sudlow’s binding site I (subdomain IIA) of HSA.</p
Synchronous fluorescence spectra of HSA obtained in the absence and presence of increasing PS concentrations.
<p>[HSA] = 3 µM, [PS] = 0–22.5 µM with 1.5 µM intervals (1–16) studied in 10 mM Tris-HCl buffer, pH 7.4, 25°C. The difference between excitation and emission wavelengths (Δλ) was (A) 15 nm and (B) 60 nm. Arrows depict the position of the emission maximum of HSA in presence of increasing PS concentrations.</p
3-D fluorescence spectral projections and corresponding contour maps of HSA and various PS–HSA complexes.
<p>(A and A′) Free HSA, (B and B′) 1∶1 PS–HSA, (C and C′) 2∶1 PS–HSA and (D and D′) 3∶1 PS–HSA. The spectra were recorded in 10 mM Tris-HCl buffer, pH 7.4, 25°C using a protein concentration of 3 µM.</p
Cluster analysis of PS docking to binding sites I and II of HSA.
<p>The different crystal structures of HSA used in the analysis were (A) 1BM0, (B) 2BXD and (C) 2BXF. A total of 100 runs were performed for each binding locus.</p