Fig 1.
UV-vis absorption spectra of HSA in the presence of NSC48693 (A) and NSC290956 (B).
The inserted figures represent the difference spectra i.e. complex spectrum minus small molecule spectrum. The colored lines represent the absorption spectra of individual component as indicated.
Fig 2.
Steady-state fluorescence spectra of HSA in the presence of NSC48693 (left lane) and NSC290956 (right lane).
Fluorescence quenching spectra of HSA in the presence of NSC48693 (A) and NSC290956 (B). A represents a fluorescence quenching difference spectra: HSA-NSC48693 complex spectra subtracting alone NSC48693 spectra. Synchronous fluorescence quenching spectra of HSA in the presence of NSC48693 (C) and NSC290956 (D) at Δλ = 15 nm. Synchronous fluorescence quenching spectra of HSA in the presence of NSC48693 (E) and NSC290956 (F) at Δλ = 60 nm. Calculated peak positions are shown with dashed lines, and labeled arrows indicate the concentration increasing of anticancer leads.
Fig 3.
Binding constants and binding mechanisms.
Stern-Volmer curves for quenching various concentrations of NSC48693 (A) and NSC290956 (B) with HSA at 293 K (black dots) and 298 K (red dots). Modified Stern-Volmer curves for quenching various concentrations of NSC48693 (C) and NSC290956 (D) with HSA at 293 K. Nonlinear regression fitting curves for quenching various concentrations of NSC48693 (E) and NSC290956 (F) with HSA at 293 K. The inserted pictures show the Scatchard plots of each binding data.
Table 1.
Thermodynamic parameters of binding of anticancer leads to HSA.
Fig 4.
Binding thermodynamic measurements of NSC48693 (A, C) and NSC290956 (B, D) interacting with HSA measured by ITC.
(A) and (B): raw heat released belonging to each injection (dilution spike of blank titration shown in dark blue lines). (C) and (D): integrated heat corresponding to each injection. The solid line shows the best fitting of integrated heat to a 1:1 isothermal binding model. Experiments were performed at 310 K as described in Materials and Methods.
Fig 5.
Overlapping of the fluorescence emission spectrum of HSA with UV-vis absorption spectra of NSC48693 (A) and NSC290956 (B) at corresponding wavelength.
Overlapping area is marked with shadow lines.
Fig 6.
Effect of anticancer leads-bound on HSA conformation.
Far-UV CD spectra of NSC48693 (A) and NSC290956 (B) binding to HSA. [HSA] = 2 μM. Concentration ratio of [small molecule]: [HSA] is shown in colored lines.
Table 2.
Kinetic parameters by fitting SPR data to 1:1 model.
Fig 7.
Kinetic characterization of anticancer leads binding with HSA.
Kinetic signal response determined by SPR is recorded as the difference between the index of refraction of the sample and the index of refraction with buffer. The micro refractive index units (μRIU) is represented by green lines. The best fitting line of 1:1 binding model is shown for NSC48693 (A) and NSC290956 (B) with black solid line. The saturated signals for NSC48693 (C) and NSC290956 (D) are fitted with one step equilibrium binding model.
Fig 8.
Molecular modeling of NSC48693 and NSC290956 to HSA at the respective binding site.
Docking molecules are emphasized with dashed frame, and the detailed binding site structures and surrounding hydrophobic residues are linked by dashed blank lines. In site structure view, possible hydrogen bonds are shown in dashed yellow lines. A: HSA structure and elucidation of drug binding site; B and C: detailed structure and surrounding hydrophobic residues of NSC48693 binding site; D and E: detailed structure and surrounding hydrophobic residues of NSC48693 binding site.