Differential Binding Affinities and Kinetics of Transthyretin Stabilizers

Transthyretin amyloid cardiomyopathy (ATTR-CM) is a progressive, fatal disease. Dissociation of tetrameric transthyretin (TTR) is the triggering event in the pathogenic mechanism; destabilizing TTR mutations accelerate the process. The TTR stabilizers, tafamidis and acoramidis, are the only FDA approved treatments for patients with ATTR-CM. By mimicking the stabilizing characteristics of the super-stabilizing, disease-protecting variant T119M, we hypothesize that acoramidis displays differential TTR binding, kinetic stability, and tetramer stabilization compared with other TTR stabilizers, such as tafamidis and diflunisal. The TTR binding affinity and thermodynamic stability of TTR interaction of acoramidis and tafamidis were assessed by surface plasmon resonance (SPR) and microscale thermophoresis (MST). Tetrameric TTR stabilization by acoramidis, tafamidis, and diflunisal in the presence of plasma proteins against acidic denaturation was measured by immune blots. In kinetic studies, SPR demonstrated 4 times longer residence time for acoramidis bound to TTR compared with tafamidis. The dissociation constants were consistent with those determined by equilibrium measurements in MST. The affinity of acoramidis for purified TTR, as measured by MST, was 4 times higher than that of tafamidis. When tested at clinically relevant plasma concentrations, acoramidis stabilized TTR against acidic denaturation to a much higher extent (≥90%) than tafamidis or diflunisal. Of note, both tafamidis and diflunisal demonstrated partial stabilization of tetrameric TTR. Relative to other stabilizers, acoramidis is more potent as independently assessed by TTR binding affinity, kinetic stability, and acid-mediated denaturation. These properties may contribute to the ability of acoramidis to achieve near-complete stabilization of TTR in plasma samples.