| Abstract
| - Protein native state stabilization imposed by small molecule binding is an attractive strategy toprevent the misfolding and misassembly processes associated with amyloid diseases. Transthyretin (TTR)amyloidogenesis requires rate-limiting tetramer dissociation before misassembly of a partially denaturedmonomer ensues. Selective stabilization of the native TTR tetramer over the dissociative transition stateby small molecule binding to both thyroxine binding sites raises the kinetic barrier of tetramer dissociation,preventing amyloidogenesis. Assessing the amyloidogenicity of a TTR tetramer having only one amyloidogenesis inhibitor (I) bound is challenging because the two small molecule binding constants are generallynot distinct enough to allow for the exclusive formation of TTR·I in solution to the exclusion of TTR·I2 andunliganded TTR. Herein, we report a method to tether one fibril formation inhibitor to TTR by disulfidebond formation. Occupancy of only one of the two thyroxine binding sites is sufficient to inhibit tetramerdissociation in 6.0 M urea and amyloidogenesis under acidic conditions by imposing kinetic stabilizationon the entire tetramer. The sufficiency of single occupancy for stabilizing the native state of TTR providesthe incentive to search for compounds displaying striking negative binding cooperativity (e.g., Kd1 innanomolar range and Kd2 in the micromolar to millimolar range), enabling lower doses of inhibitor to beemployed in the clinic, mitigating potential side effects.
|
