Selective binding of coumarin enantiomers to human α1-acid glycoprotein genetic variants
Graphical abstract
Introduction
Human α1-acid glycoprotein (AGP, orosomucoid (ORM)), a member of the lipocalin family, is one of the most important glycoprotein components of blood plasma that binds both endogenous and exogenous ligands with a chemically diverse structure.1, 2 Although its precise biological function is yet unclear, AGP is suggested to play a role in immunomodulation.3, 4 Binding of drugs to AGP may have clinical importance as it influences both the pharmacokinetics and pharmacodynamics5 of a drug. Moreover, under various pathological and physiological conditions the plasma concentration of AGP may increase up to three- or fourfold which results in an alteration in the binding of drugs and other ligands.2, 4
The AGP molecule consists of a single polypeptide chain of 183 amino acids and of five asparaginyl linked glycans. Besides the high heterogeneity of glycans, the protein part has also been found to show polymorphism.6 The variants are encoded by two different genes: The F1 and S variants are encoded by the alleles of the same gene, while the A variant is encoded by a different gene.6 There is a difference of at least 22 amino acid residues between the F1–S (ORM 1) and A (ORM 2) variants, while F1 and S forms differ only in a few residues.7 ORM 1 and ORM 2 variants were shown to possess different binding properties.8, 9, 10, 11 Warfarin is a selective ligand of ORM 1 variant of human AGP9 and the possibility of high binding stereoselectivity was raised.9 Nakagawa et al.12 determined preference in binding of (S)-warfarin to ORM 1 variant. Furthermore, structurally related acenocoumarol binding on native AGP was found to have a stereoselectivity factor (KS/KR ratio) of 3 in favour of the (S)-enantiomer.13, 14
The three-dimensional X-ray structure of AGP is unknown, therefore, little information is available about the binding mechanism. A three-dimensional model of AGP based on other members of the lipocalin family was presented in 199315 and refined in 2003.16 Former docking calculations of some of the present authors based on the latter model yielded the unlikely result where warfarin was bound at the surface of AGP,17 indicating that the model underestimates the size of the binding cavity.
In the current study, stereoselective binding of warfarin and two more structurally related anticoagulants, phenprocoumon and acenocoumarol (Fig. 1), to F1–S (ORM 1) and A (ORM 2) variants of AGP was investigated. Direct binding measurements with racemates and displacement study using pure enantiomers were carried out. It was anticipated that examination of the binding of these three compounds helps in revealing which functional group of the chiral molecule is responsible for interactions with AGP. Furthermore, a new homology model of ORM 1 and ORM 2 was constructed based on published X-ray structure of members of the lipocalin protein family in order to identify the interactions influencing the ligand binding properties of the genetic variants.
Section snippets
Chemicals
rac-Warfarin, human AGP and quinaldine red (QR) was purchased from Sigma–Aldrich and Co. (St. Louis, MO). Sulfopropylated β-cyclodextrin, α-cyclodextrin and random methylated β-cyclodextrin were kindly donated by Cyclolab (Budapest, Hungary). rac-Acenocoumarol was obtained from Alkaloida Chemical Factory (Tiszavasvári, Hungary). Phenprocoumon enantiomers were generous gift of Hoffmann-LaRoche (Basel). All other chemicals were from Reanal, Hungary. Warfarin enantiomers were obtained using the
Ligand binding measurements by stereoselective analysis of the ultrafiltrates
Binding of racemates to AGP genetic variants was investigated by the ultrafiltration method. The determination of unbound ligand concentration and resolution of the free enantiomers in the ultrafiltrates was carried out by capillary electrophoresis method. Figure 1 shows the electropherograms of the coumarin enantiomers demonstrating that baseline separation was achieved for each investigated enantiomer pair. In Table 1, the free fractions of coumarin enantiomers in the ultrafiltrates are
Discussion
The binding of drugs to plasma proteins, especially to albumin and AGP, is an important factor in drug distribution and disposition, especially since the relative concentrations of each genetic variant of AGP may change under various physiological and pathological conditions.4 The genetic variants of AGP were shown to have altered ligand binding properties.9 The purpose of this study was to characterize the binding affinity and selectivity of coumarins to AGP genetic variants and explain the
Acknowledgments
This research was supported by research grants of OTKA T049721 and NKFP 1/A/005/04 (Medichem 2) project. Skillful assistance of Mrs. Ilona Kawka is acknowledged. For the chromatographic separation of AGP variants, we are grateful to Dr. György Mády.
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