Background: Metoprolol (MTP) is a cardio-selective β1-blocker used in hypertension, angina pectoris
and chronic heart failure therapies. Serum albumin is the most frequently occurring protein in blood plasma.
The binding of ligands to human serum albumin (HSA) has an important effect on pharmacokinetics and final
Objective: The objectives of this study included a detailed analysis of metoprolol – serum albumin interactions
in low affinity binding sites, on the surface or within the hydrophobic subdomain of a macromolecule, as well as
an analysis of the competition between MTP and fatty acids in binding with protein.
Methods: The analysis of the drug-albumin interaction was based on the observed chemical shifts in combination
with correlation Times (T1
-1 = τ) [1/s], 2D NOESY 1H NMR spectra and association constants Ka [M-1]. For
the determination of chemical shifts σ [ppm], relaxation times T1 [s] and for the NOESY experiment, the final
concentrations of MTP and albumins (in the presence (HSA) and absence of fatty acids (dHSA)) were 5 x 10-3
M and 2 x 10-5 M - 4.55 x 10-4 M, respectively. In order to calculate the association constants, the final concentrations
of MTP and both HSA and dHSA were 2.75 x 10-3 M - 6.25 x 10-2 M and 2.5 x 10-4 M, respectively. For
the analysis, the MTP proton resonances of aliphatic H17, aromatic (H2/H6 and H3/H5) and the methoxy group
H14 were chosen.
Results: Changes in the values of the 1H NMR chemical shift [ppm] are evidence of interaction between MTP,
fatted (HSA) and defatted (dHSA) human serum albumin. With an increase of albumin concentration, changes
in the chemical shift values were observed for the aromatic protons H2/H6 (Δσ = 0.013 ppm and 0.018 ppm)
and H3/H5 (Δσ = 0.015 ppm and 0.019 ppm), the aliphatic proton H17 (Δσ = 0.018 ppm and 0.022 ppm) and
the aliphatic protons of the methoxy group H14 (Δσ = 0.019 ppm and 0.022 ppm) for dHSA and HSA, respectively.
Greater changes in chemical shifts in the presence of fatty acids (FA) were observed. Changes in the
correlation times of MTP aromatic H2/H6 (Δτc = 0.224 1/s and 0.189 1/s) and H3/H5 (Δτc = 0.269 1/s and
0.210 1/s), aliphatic from the methoxy group H14 (Δτc = 0.472 1/s and 0.271 1/s) and aliphatic H17 protons (Δτc = 0.178 1/s and 0.137 1/s) for dHSA and HSA systems, respectively. It confirms the interaction between the
drug and albumin are evidence for the dynamics of the process. In the presence of FA the relaxation time of all
analyzed MTP proton resonance signals significantly increases (due to the decrease of correlation time). This
phenomenon is due to the increase of electron density in the MTP protons' surroundings. Association constants
for the MTP-dHSA complex in the low affinity site range between 0.29 x 102 M-1 and 0.47 x 102 M-1. The presence
of FA results in a two to three-fold increase of the Ka values of protons from aromatic (H2/H6 and H3/H5),
aliphatic H17 and methoxy (H14) groups. In 2D NOESY spectra proton magnetization transfer was observed
between cysteine (Cys-34) and aromatic H3/H5 and H2/H6 protons. Cross-peaks were also observed between
cysteine and aliphatic protons from the methoxy group.
Conclusion: The selective changes in σ [ppm] and τc [1/s] values indicated the unequal participation of chemical
groups of MTP in the interaction with HSA and dHSA. The data obtained suggest that the presence of fatty
acids increases the accessibility of low affinity sites of serum albumin to MTP, which results in the higher affinity
of albumin towards the drug. The results showed that the main binding site of MTP and fatty acid is probably
a low affinity site in subdomain IB, where Cys-34 can be located.