Molecular Recognition between Anticancer Drug, Regorafenib and Human Serum Albumin: Interaction Revisited

The wet-lab techniques (fluorimetry and spectrophotometry), along with computational techniques (molecular docking and molecular dynamics (MD) simulation), were applied to re-examine the association of an anticancer drug, regorafenib (REG) with human serum albumin (HSA). The REG-induced protein fluorescence quenching was characterized as static quenching based on a decrement in the KSV (Stern-Volmer constant) with increasing temperature and hyperchromic effect in the absorption spectra. The REG–HSA complex (Ka = 0.63 – 1.17 × 105 M–1) was stabilized by hydrophobic and van der Waals interactions in combination with hydrogen bonds, as revealed by thermodynamic data (ΔrS° = +17.17 J mol–1 K–1 and ΔrH° = –23.00 kJ mol–1), and further supported by molecular docking assessment. Microenvironmental fluctuations around HSA fluorophores and better protein stability against thermal stress were evident due to REG-HSA complexation. Accessibility of both Sudlow\u27s Sites I and II but priority for Site I of the protein for REG was inferred by the competitive ligand displacement and molecular docking assessments. MD simulation results supported the stability of the complex

Probing the interaction of a therapeutic flavonoid, pinostrobin with human serum albumin: multiple spectroscopic and molecular modeling investigations.

Interaction of a pharmacologically important flavonoid, pinostrobin (PS) with the major transport protein of human blood circulation, human serum albumin (HSA) has been examined using a multitude of spectroscopic techniques and molecular docking studies. Analysis of the fluorescence quenching data showed a moderate binding affinity (1.03 × 10(5) M(-1) at 25°C) between PS and HSA with a 1∶1 stoichiometry. Thermodynamic analysis of the binding data (ΔS = +44.06 J mol(-1) K(-1) and ΔH = -15.48 kJ mol(-1)) and molecular simulation results suggested the involvement of hydrophobic and van der Waals forces, as well as hydrogen bonding in the complex formation. Both secondary and tertiary structural perturbations in HSA were observed upon PS binding, as revealed by intrinsic, synchronous, and three-dimensional fluorescence results. Far-UV circular dichroism data revealed increased thermal stability of the protein upon complexation with PS. Competitive drug displacement results suggested the binding site of PS on HSA as Sudlow's site I, located at subdomain IIA, and was well supported by the molecular modelling data

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>_<i>a</i> = 1.35 × 10⁶–5.43 × 10⁵ M⁻¹). The <i>K</i>_<i>SV</i> 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

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 × 10³M⁻¹ 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⁻¹ K⁻¹) and negative Δ<i>H</i> (−6.57 kJ mol⁻¹) 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 Ca²⁺, Zn²⁺, Cu²⁺, Ba²⁺, Mg²⁺, and Mn²⁺ 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

Spectrofluorometric and Molecular Docking Studies on the Binding of Curcumenol and Curcumenone to Human Serum Albumin

Curcumenol and curcumenone are two major constituents of the plants of medicinally important genus of Curcuma, and often govern the pharmacological effect of these plant extracts. These two compounds, isolated from C. zedoaria rhizomes were studied for their binding to human serum albumin (HSA) using the fluorescence quench titration method. Molecular docking was also performed to get a more detailed insight into their interaction with HSA at the binding site. Additions of these sesquiterpenes to HSA produced significant fluorescence quenching and blue shifts in the emission spectra of HSA. Analysis of the fluorescence data pointed toward moderate binding affinity between the ligands and HSA, with curcumenone showing a relatively higher binding constant (2.46 × 105 M−1) in comparison to curcumenol (1.97 × 104 M−1). Cluster analyses revealed that site I is the preferred binding site for both molecules with a minimum binding energy of −6.77 kcal·mol−1. However, binding of these two molecules to site II cannot be ruled out as the binding energies were found to be −5.72 and −5.74 kcal·mol−1 for curcumenol and curcumenone, respectively. The interactions of both ligands with HSA involved hydrophobic interactions as well as hydrogen bonding

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

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_ex = 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_{336 nm}) with increasing PS/HSA molar ratios.</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