¹9F nuclear magnetic resonance screening of glucokinase activators.

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R2 relaxation of the (19)F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the (19)F signal intensity after an 80-ms spin echo correlates nicely with the EC50, opening a route for NMR screening of GK activators.

Detection of natural abundance 1H-13C correlations of cholesterol in its membrane environment using a gradient enhanced HSQC experiment under high resolution magic angle spinning.

The quality and signal to noise ratio of a J-based HETCOR performed on a standard MAS probe have been compared with a gradient enhanced HSQC performed on a HR-MAS probe at 500 MHz. The sample selected was cholesterol, inserted at 30 mol% in acyl chain deuterated phospholipids (DMPC-d54), at a temperature where the bilayer is in a liquid crystalline phase (310 K). It is representative of any rigid molecule undergoing fast axial diffusion in a bilayer as the main movement. After optimization of the spinning rate and carbon decoupling conditions, it is shown that the ge-HSQC/MAS approach is far superior to the more conventional J-HETCOR/MAS in terms of signal to noise ratio, and that it allows the detection of all the natural abundance cross peaks of cholesterol in a membrane environment. Clear differences between the 1H and 13C chemical shifts of cholesterol in a membrane and in chloroform solution were thus revealed.