jm030347n_si_001.pdf (20.51 MB)
Diflunisal Analogues Stabilize the Native State of Transthyretin. Potent Inhibition of Amyloidogenesis
journal contribution
posted on 2004-01-15, 00:00 authored by Sara L. Adamski-Werner, Satheesh K. Palaninathan, James C. Sacchettini, Jeffery W. KellyAnalogues of diflunisal, an FDA-approved nonsteroidal antiinflammatory drug (NSAID), were
synthesized and evaluated as inhibitors of transthyretin (TTR) aggregation, including amyloid
fibril formation. High inhibitory activity was observed for 26 of the compounds. Of those, eight
exhibited excellent binding selectivity for TTR in human plasma (binding stoichiometry >0.50,
with a theoretical maximum of 2.0 inhibitors bound per TTR tetramer). Biophysical studies
reveal that these eight inhibitors dramatically slow tetramer dissociation (the rate-determining
step of amyloidogenesis) over a duration of 168 h. This appears to be achieved through ground-state stabilization, which raises the kinetic barrier for tetramer dissociation. Kinetic stabilization of WT TTR by these eight inhibitors is further substantiated by the decreasing rate of
amyloid fibril formation as a function of increasing inhibitor concentration (pH 4.4). X-ray
cocrystal structures of the TTR·182 and TTR·202 complexes reveal that 18 and 20 bind in
opposite orientations in the TTR binding site. Moving the fluorines from the meta positions in
18 to the ortho positions in 20 reverses the binding orientation, allowing the hydrophilic
aromatic ring of 20 to orient in the outer binding pocket where the carboxylate engages in
favorable electrostatic interactions with the ε-ammonium groups of Lys 15 and 15‘. The
hydrophilic aryl ring of 18 occupies the inner binding pocket, with the carboxylate positioned
to hydrogen bond to the serine 117 and 117‘ residues. Diflunisal itself appears to occupy both
orientations based on the electron density in the TTR·12 structure. Structure−activity
relationships reveal that para-carboxylate substitution on the hydrophilic ring and dihalogen
substitution on the hydrophobic ring afford the most active TTR amyloid inhibitors.