Human factor Xa bound amidine inhibitor conformation by double rotational-echo double resonance nuclear magnetic resonance and molecular dynamics simulations.

Double rotational-echo double resonance (double REDOR) NMR was used to investigate the conformation of a 13 C-, 15 N-, and 19 F-labeled inhibitor (Berlex Biosciences compound no. ZK-806299) bound to human factor Xa. Conformationally dependent carbon-fluorine dipolar couplings were measured by 13 C{ 19 F} REDOR. Natural abundance carbon signals in the full-echo spectra were removed by 13 C{ 15 N} REDOR. Major and minor binding modes were suggested by the NMR data, but only the former had adequate signal to noise for distance determinations. Molecular dynamics simulations restrained by double-REDOR-determined intramolecular 13 C- 19 F distances revealed two models for the dominant binding mode that are consistent with the NMR data. We conclude that ZK-806299 binds similarly to both FXa. Moreover, it appears to bind to FXa in a fashion previously demonstrated for ZK-807834, a more selective FXa inhibitor.