NMR and Docking Calculations Reveal Novel Atomistic Selectivity of a Synthetic High-Affinity Free Fatty Acid vs. Free Fatty Acids in Sudlow's Drug Binding Sites in Human Serum Albumin

Abstract

Saturation transfer difference (STD), inter-ligand NOEs (INPHARMA NMR), and docking calculations are reported for investigating specific binding sites of the high-affinity synthetic 7-nitrobenz-2-oxa-1,3-diazoyl-4-C₁₂ fatty acid (NBD-C₁₂ FA) with non-labeled human serum albumin (HSA) and in competition with the drugs warfarin and ibuprofen. A limited number of negative interligand NOEs between NBD-C₁₂ FA and warfarin were interpreted in terms of a short-range allosteric competitive binding in the wide Sudlow's binding site II (FA7) of NBD-C₁₂ FA with Ser-202, Lys-199, and Trp-214 and warfarin with Arg-218 and Arg-222. In contrast, the significant number of interligand NOEs between NBD-C₁₂ FA and ibuprofen were interpreted in terms of a competitive binding mode in Sudlow's binding site I (FA3 and FA4) with Ser-342, Arg-348, Arg-485, Arg-410, and Tyr-411. NBD-C₁₂ FA has the unique structural properties, compared to short-, medium-, and long-chain saturated and unsaturated natural free fatty acids, of interacting with well-defined structures with amino acids of both the internal and external polar anchor sites in Sudlow's binding site I and with amino acids in both FA3 and FA4 in Sudlow's binding site II. The NBD-C₁₂ FA, therefore, interacts with novel structural characteristics in the drug binding sites I and II and can be regarded as a prototype molecule for drug development.

Keywords: HSA; INPHARMA NMR; NBD-C12 FA; STD NMR; docking calculations.

Figure 1

HSA Sudlow’s binding sites I and II. The fatty acid binding sites FA3/FA4 and FA7 are indicated on the left and right, respectively.

Figure 2

¹H NMR spectra (500 MHz) of (a) warfarin (W) (2 mM) with native HSA (25 μΜ) in 50 mM PBS buffer in D₂O with 20% DMSO-d₆ (T = 323 K); (c) as in (a) after the addition of 2 mM of NBD-C₁₂ FA; (b) STD ¹H NMR spectrum of (a). (d) STD ¹H NMR spectrum of (c). The STD amplification factor of warfarin in the binary HSA warfarin complex is shown in blue color and the % reduction upon addition of NBD-C₁₂ FA is shown in black.

Figure 3

Interligand 2D Tr-NOESY (INPHARMA) NMR spectrum (500 MHz) of NBD-C₁₂ FA (0.8 mM saturated solution) in the presence of warfarin (W) (1.6 mM) with native HSA (25 μΜ) in 50 mM PBS buffer in D₂O with 20% DMSO-d₆, T = 323 K, mixing time = 300 ms. The red cross-peaks denote interligand NOEs between NBD-C₁₂ FA and warfarin.

Figure 4

¹H NMR spectra (500 MHz) of: (a) ibuprofen (I) (2 mM) with native HSA (25 μΜ) in 50 mM PBS buffer in D₂O with 20% DMSO-d₆ (T = 323 K); (c) as in (a) after the addition of 2 mM of NBD-C₁₂ FA; (b) STD ¹H NMR spectrum of (a). (d) STD ¹H NMR spectrum of (c). The STD amplification factor of ibuprofen in the binary HSA ibuprofen complex is shown in blue color and the % reduction upon the addition of NBD-C₁₂ FA is shown in black.

Figure 5

Interligand 2D Tr-NOESY (INPHARMA) NMR spectrum (500 MHz) of NBD-C₁₂ FA (0.8 mM saturated solution) in the presence of ibuprofen (IB) (1.6 mM) with native HSA (25 μΜ) in 50 mM PBS buffer in D₂O with 20% DMSO-d₆, T = 323 K, mixing time = 300 ms. Interligand NOEs between NBD-C₁₂ FA and ibuprofen are denoted with the red cross-peaks (x is a multiplication factor).

Figure 6

(A) Atom-numbering of warfarin molecule. (B–D). Different views of successful docking pose of warfarin in the presence of NBD-C₁₂-FA. (E) Successful docking pose of NBD-C₁₂-FA in the presence of warfarin. (F) Self-docking of NBD-C₁₂-FA and superposition with the crystal structure.

Figure 7

Superposition of NBD-C₁₂ FA and ibuprofen in FA3/FA4 binding sites (crystal structure codes in PBB: 6ezq.pdb and 2bxg.pdb).