Structure-Based Design of N-Phenyl Phenoxazine Transthyretin Amyloid Fibril Inhibitors

Starting with the published 2.0 A X-ray crystal structure of the transthyretin·(flufenamic acid)2 complex, a simple structure-based ligand design strategy was employed to conceive of N-phenyl phenoxazine transthyretin (TTR) amyloid fibril inhibitors. Fifteen N-phenyl phenoxazines were chemically synthesized and evaluated using a quantitative amyloid fibril assay in vitro. The structure of one of the two most active phenoxazines, 4, bound to TTR was solved to a resolution of 1.9 A to understand the structural basis of its efficacy. N-phenyl phenoxazine 4 binds similar to the orientation anticipated, although not as deeply into the channel as expected. Like flufenamic acid, 4 mediates binding-induced conformational changes that enable intersubunit H-bonding in tetrameric TTR which may be important for preventing fibril formation. Analytical ultracentrifugation analysis demonstrates that 4 blocks the first step of TTR amyloid fibril formation, that is, tetramer dissociation to the alternatively folded amyloi...