Synthesis and pharmacological activity of metabolites of the 5-HT(4) receptor antagonist SB-207266.

Three metabolites of N-[(1-butyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]-oxazino[3,2_a]indole-10-carboxamide (SB-207266) (1) were synthesised and their pharmacological activity determined.

A proton nuclear magnetic resonance and molecular modeling study of calmidazolium (R24571) binding to calmodulin and skeletal muscle troponin C.

1H NMR spectroscopy at 360 MHz has been used to study the interactions between the calmodulin function inhibitor calmidazolium (R24571) and (i) calmodulin (CaM) and (ii) skeletal muscle troponin C (sTnC). One equivalent of racemic calmidazolium binds tightly to CaM and perturbs a number of protein signals, corresponding to residues in both dicalcium-binding domains, in a manner characteristic of slow exchange. Calmidazolium binds with lower affinity to sTnC but still induces widespread perturbations in both domains. Extensive spectral overlap precludes definite assignment of intermolecular nuclear Overhauser effect (NOEs) although intraprotein NOEs do indicate the nature of some drug-induced conformational changes. Relaxation enhancements induced by two spin-labeled calmidazolium analogues demonstrate that several methionine residues of CaM, significantly immobilized by calmidazolium binding, are in fact located at or near its binding sites. These and other residue-specific broadening effects have enabled low resolution models to be constructed of the predominantly hydrophobic drug-binding sites on each domain of CaM. The hydrophobic portions of calmidazolium itself, and its analogues, contact side chains of Ala-15, Leu-18, Phe-19, Val-35, Met-36, Leu-37, Leu-39, Met-51, Met-71, Met-72, and Met-76 in the N-terminal domain of calmodulin, and Ala-88, Val-91, Phe-92, Val-108, Met-109, Leu-112, Phe-141, and Met-145 in its C-terminal domain. The model, and an analogous one of sTnC, can be used to rationalize drug-induced changes in intraprotein NOEs. Issues pertaining to the possible simultaneous binding of calmidazolium to both globular domains of the proteins are discussed in terms of the experimental results and the overall structures of each protein.

A proton and fluorine-19 nuclear magnetic resonance and fluorescence study of the binding of some natural and synthetic thyromimetics to prealbumin (transthyretin).

Interactions between prealbumin and several thyromimetic compounds have been examined by proton magnetic resonance spectroscopy. One equivalent of thyroxine (T4) or reverse triiodothyronine (rT3) selectively broadens a number of protein signals, while addition of a second equivalent induces much less widespread changes. One equivalent of triiodothyronine (T3), however, produces much less dramatic changes, and effects comparable with T4 and rT3 are only apparent when a second equivalent binds. The broadening is ascribed to immobilization of flexible residues. The non-halogenated analogue 3,5-dimethyl-3'-isopropylthyronine induces qualitatively different changes suggesting incomplete entry into the thyromimetic binding channel. The fluorinated analogue SK&F 95049 (3,5-bis-thiotrifluoromethyl-3'-isopropylthyronine) induces very similar changes to T3. A fluorine-19 NMR signal with a half-height line width of approximately 150 Hz can be observed from the bound ligand. Finally, a spin-labeled T4 analogue, with a nitroxyl on the alanyl moiety, induces changes identical to those induced by T4 itself, and additionally broadens some signals corresponding to residues at the opening of the ligand binding channel. The natural tryptophan fluorescence of the protein is shown to be a sensitive indicator of binding. The possible influence of the dynamic restrictions induced by binding the first molecule of T4 or rT3 on the protein's affinity for a second hormone is discussed. It is suggested that the first interaction confers rigidity on the second site and reduces its ability to flex open and accommodate a second thyromimetic, which results in the marked negative co-operativity associated with the occupancy of this site.