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 13C-, 15N-, and 19F-labeled inhibitor (Berlex Biosciences compound no. ZK-806299) bound to human factor Xa. Conformationally dependent carbon−fluorine dipolar couplings were measured by 13C{19F} REDOR. Natural abundance carbon signals in the full-echo spectra were removed by 13C{15N} 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 13C−19F 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.