Exploring Ligand Affinities for Proteins by NMR of Long-Lived States

PAR Roberto BURATTO " Twenty years from now you will be more disappointed by the things that you didn't do than by the ones you did do. So throw off the bowlines. Sail away from the safe harbor. Catch the trade winds in your sails. Abstract i Abstract The detection of molecules that can bind to active sites of protein targets and the measurement of their affinities is a promising application of NMR. Nowadays, the screening of drug candidates is routinely done by NMR in pharmaceutical industry. We have proposed to use the relaxation of Long-Lived States (LLS) for drug screening by NMR. Long-lived states are nuclear spin states whose decay time constant í µí±‡ í µí°¿í µí°¿í µí±† can be much longer than the longitudinal relaxation time í µí±‡ 1. LLS can be used to screen and determine the dissociation constant í µí°¾ í µí°· of molecular fragments that bind weakly to protein targets. The use of LLS for fragment screening leads to a spectacular increase in contrast between free and bound ligands, and thus allows one to characterize binding of fragments with very weak affinities, with í µí°¾ í µí°· in the millimolar range, which is difficult to achieve by other methods such as ITC. By exploiting the LLS behavior of a spy molecule, we experimentally demonstrate that it is possible to measure dissociation constants KD as large as 12 mM, corresponding to very weak binding, where most other biophysical techniques fail, including other NMR methods based on the observation of ligands. Furthermore, we have combined LLS for screening for improved contrast with 1 H dissolution-DNP to enhance the sensitivity. DNP-enhanced screening for measuring LLS signals of a weak ligand allows one to use very low concentrations of ligands and proteins. We observed dramatic differences between the spectra of the ligand in the presence or absence of a protein, or in the presence of the protein combined with a stronger ligand. Moreover, we have explored LLS involving pairs of 19 F nuclei to study binding phenomena. Indeed, fluorine detection is quite interesting because it offers the possibility to perform screening experiments without any problems due to overlapping signals. In a custom-designed fluorinated ligand that binds trypsin, we have observed a promising ratio í µí±‡ í µí°¿í µí°¿í µí±† / í µí±‡ 1 > 4. This fluorinated ligand has been used as spy molecule in competition experiments, which allowed …