Conformational analysis of pharmaceutical suramin free and in the presence of its target receptors by solution NMR: What we learned from and about transfered NOESY

The concept of structure-based design of protein inhibitors has become increasingly popular during the past decade. Traditional techniques for structure determination such as X-ray crystallography or solution NMR require large quantities and/or isotopic labeling of the receptors, and quite often sample preparation is either impossible or associated with major difficulties. Transferred NOESY was originally developed as a technique that would circumvent the problems associated with preparing large amounts and isotopically labeling the receptors. The information about the bound conformation of the ligands can in principle be obtained from transferred NOESY--derived interproton distances of the bound ligand. Small amounts of the protein, and possibility to study ligands complexed with very large proteins, made the technique very attractive.; Suramin is a pharmaceutical used for treating sleeping sickness caused by Trypanosoma brucei, and a lead compound against cancer and AIDS. Target receptors of suramin have been identified or proposed in each case. We analyzed the conformations of suramin free in solution and complexed with the target receptors, phosphoglycerate kinases from T. brucei and yeast (an analog of the corresponding mammalian enzyme), and ribonuclease H from Murine Moloney virus by transferred NOESY/ROESY methods. Suramin free in solution has multiple conformers, and only weak conformational preferences exist, as is evident from the NOE data. At concentrations higher than 0.5 mM the compound aggregates, as is evident from NOESY spectra and light-scattering measurements.; The transferred NOESY buildup curves for suramin complexed with PGK and RNaseH cannot be treated assuming fast-exchange kinetics implied from the equilibrium dissociation constants. Lag periods are observed in the buildup curves and suggest intermediate exchange regime. A kinetic scheme involving formation of an intermediate encounter complex has been proposed and is the only scheme consistent with the experimental transferred NOESY buildup curves. The transferred NOESY data indicate difference in conformational preferences of suramin bound to PGK and RNaseH.; In the process of these studies it became necessary to address a more general question of relevance and limitations of transferred NOE technique for conformational analysis of bound molecules, which is a major subject of this thesis, and has also been discussed in recent literature by several other groups.