Probing the conformation of the sugar transport inhibitor phlorizin by 2D-NMR, molecular dynamics studies, and pharmacophore analysis.

Sodium/D-glucose cotransport, one of the prototypes for sodium gradient-driven symport systems in kidney and intestine, is known to be inhibited by aromatic and aliphatic glucosides (Diedrich, D. F. Biochim. Biophys. Acta 1963, 71, 688-700; Diedrich, D. F. Arch. Biochem. Biophys. 1966, 117, 248-256; Kipp, H.; et al. Biochim. Biophys. Acta 1996, 1282, 124-130; Ramaswamy, K.; et al. Biochim. Biophys. Acta 1976, 433, 32-38). The conformation in which the most potent inhibitor, phlorizin, interacts with the transport protein was investigated with different approaches. Phlorizin consists of the glucose moiety and two aromatic rings (A and B) joined by an alkyl spacer. First the interaction of these various parts of the molecule was determined by two-dimensional (2D) solution NMR. From the 2D-NOESY (nuclear Overhauser effect) measurements spatial distances (up to 5 A) between various interacting H atoms could be detected. Using these values as distance constraints, conformations of phlorizin were calculated and analyzed by the valence force-field method. As a result, a set of conformations could be obtained. The most probable phlorizin conformation shows a nearly perpendicular arrangement of the two aromatic rings (A and B) with the ring B situated above the sugar ring. A very similar conformation could be found by using molecular dynamics simulations when water was chosen as the solvent. This phlorizin conformation in aqueous solution then served as a template for conformational analysis of various phlorizin derivatives. The resulting conformations of derivatives were taken as input to establish a pharmacophore model using the DISCO calculation. As a result, the essential elements of phlorizin for interaction with its binding pocket could be deduced: namely hydrogen bonding via hydroxyl groups of the pyranoside at C(2), C(3), C(4), and C(6) and at C(4) and C(6) of aromatic ring A and hydrophobic interactions via the pyranoside ring and aromatic ring A. Finally, from these conformational features of the pharmacophore the dimension of the phlorizin binding site on the sodium/D-glucose cotransporter was estimated to be 17 x 10 x 7 A(3).