Abstact

Transthyretin (TTR) amyloidosis arises from the extracellular aggregation of misfolded TTR monomers into beta-sheet-rich fibrils, leading to progressive tissue damage. To inhibit this process, we designed and synthesized pyrazole-based kinetic stabilizers targeting the thyroxine-binding sites of TTR. Structure-activity relationship studies revealed that derivatives with hydrophobic trans-alkene linkers and 3,5-substituted pyrazole rings showed enhanced stabilizing potency, particularly those bearing carboxylic acid, amide, or sulfonamide groups. A covalent fluorescent probe derived from trans-styrylpyrazole was developed to selectively react with Lys15, enabling fluorescence probe exclusion and native PAGE assays to evaluate stabilizer selectivity in human serum. Among these, 3,5-dichloropyrazole derivatives exhibited efficacy comparable to that of tafamidis and acoramidis. X-ray crystallography of the TTR-17 complex confirmed hydrogen bonding with Ser117/117' and electrostatic interactions with Lys15. Pharmacokinetic studies of compounds 16 and 17 demonstrated favorable exposure, bioavailability, and metabolic stability, supporting their preclinical development for hereditary- and wild-type TTR amyloidosis.