NMR and computational studies reveal novel aspects in molecular recognition of unsaturated fatty acids with non‐labelled serum albumin

Abstract

An approach based on the combined use of saturation transfer difference (STD), Tr-NOESY and Inter-ligand NOEs for PHArmacophore Mapping (INPHARMA) NMR techniques and docking calculations is reported, for the first time, for mapping interactions and specific binding sites of caproleic acid (10 : 1 cis-9), oleic acid (18 : 1 cis-9), linoleic acid (18 : 2 cis-9,12) and linolenic (18 : 3, cis-9,12,15) free fatty acids (FFAs) with non-labelled serum albumin (BSA/HSA). Significant negative inter-ligand NOEs between the FFAs and the drugs ibuprofen and warfarin, through competition experiments, were observed. The inter-ligand NOEs and docking calculations were interpreted in terms of competitive binding mode, the significant folding of the bis allylic region and the presence of two orientations of the FFAs in the warfarin binding site (FA7), due to two potential distinctive anchoring polar groups of amino acids. This conformational flexibility is the reason that, the location and conformational states of the FFAs in the binding site of warfarin could not be determined accurately, despite numerous available X-ray structural studies. α-Linolenic acid competes favourably with warfarin at the binding site FA7. Isothermal titration calorimetry experiments of the preformed HSA/α-linolenic acid complex upon titration with warfarin show a significant reduction in the binding constant of warfarin, in very good agreement with NMR and computational data. The combined use, therefore, of STD, Tr-NOESY and INPHARMA NMR, ITC and docking calculations may find promising applications in the field of protein-lipid recognition research.

Molecular Basis for the Selectivity of DHA and EPA in Sudlow’s Drug Binding Sites in Human Serum Albumin with the Combined Use of NMR and Docking Calculations

“…Furthermore, although the serum albumin binding assay is the usual procedure for the development of new drugs, the binding modes of the polyunsaturated FFAs in Sudlow's sites I and II have not been elucidated. We report herein structural aspects of the binding modes of the polyunsaturated free fatty acids DHA and EPA to HSA with the combined use of saturation transfer difference (STD) [28][29][30], Tr-NOESY [31][32][33], and Interligand NOEs for Pharmacophore Mapping (INPHARMA) [26,27,[34][35][36] NMR techniques and molecular calculations [27,[34][35][36][37][38][39][40]. Particular emphasis was given to The various fatty acid and drug-binding sites have been investigated extensively over the past 40 years [12][13][14][15][18][19][20].…”

“…Particular emphasis was given to The various fatty acid and drug-binding sites have been investigated extensively over the past 40 years [12][13][14][15][18][19][20]. X-ray crystallographic [16][17][18][21][22][23][24] and NMR [24][25][26][27] studies revealed the presence of seven to nine binding sites that may be occupied by both medium-and long-chain saturated fatty acids, with chain lengths from C10 to C18. FFA binding sites are asymmetrically distributed throughout the protein.…”

“…Interestingly, weak secondary binding of warfarin in subdomain IB was also observed in the absence of fatty acid. Nevertheless, to assess whether the reduction in the linewidths of the STD signals reflected competitive interactions in relation to binding site FA7, rather than allosteric phenomena, the INPHARMA NMR technique was applied [26,27,[34][35][36]42]. This approach relies on the inter-NOE connectivities of two ligands, provided that they share a common binding site with a distance < 5 Å.…”

“…Figure 3 shows a significant number of interligand NOEs shared between DHA and EPA and warfarin (red cross-peaks), demonstrating that they share a common binding site at FA7. Of particular interest are the common inter-NOEs shared between the H2,3 and terminal CH 3 groups of DHA/EPA and the aromatic protons of both the benzopyran and the phenyl butyl rings of warfarin, although the crystallographic distance of the two aromatic systems is ~6.9 Å [16][17][18][19][20][21][22][23][24][25][26][27]. These results can be attributed to two anchor binding sites within the FA7 binding pocket (see the discussion on computations).…”

“…This was attributed to the formation of high-molecular-weight aggregates of the inhibitors in aqueous environments, which results in negative NOEs that are not mediated through specific interactions with the protein. Since FFAs in an aqueous solution show the presence of negative NOEs due to the formation of micellar aggregates [27], it was necessary to perform further 2D Tr NOESY experiments to investigate whether the drug warfarin is incorporated within the micellar system. Positive intra-NOEs (anti-phase with respect to the diagonal) were observed for warfarin between H2′, 6′ and H9 and H10 due to their spatial proximity (Figure S1, Supplementary Materials), without any cross-peaks with DHA.…”