Category: Preclinical Development
Purpose: Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a fatal disease with no available disease-modifying therapies. While destabilizing TTR mutations increase the risk of developing ATTR-CM, the naturally occurring Thr119Met (T119M) variant stabilizes TTR compared to wild-type and can prevent disease in compound heterozygous carriers of a destabilizing TTR mutation. The two S117 side chain hydroxyl groups of monomers A and B in T119M variant TTR form direct hydrogen bonds with a distance of 2.8 Å, which are not observed in TTRwt (distance between the two S117 residues ~6.0 Å). Genetic and pharmacologic data have shown that kinetic stabilization of tetrameric TTR can slow/halt disease onset and/or progression. Ligands that bind tightly to the T4 binding site of TTR reduce tetramer dissociation and subsequent assembly of misfolded monomers into toxic aggregates1. Previously our lab reported the first high-throughput screen for TTR kinetic stabilizers1. We used these compounds as a starting point for structure-activity relationship (SAR) studies and synthesized a series of analogues of a lead compound which resulted in the discovery of AG102. AG10 is an orally available, small molecule TTR stabilizer that just completed successful Phase 2 clinical trials for ATTR-CM3. Structural features that infer high potency of AG10 for stabilizing TTR remains incompletely characterized. Our studies provide mechanistic insights into the unique binding mode of AG10, which could be attributed to mimicking the stabilizing T119M variant. Our objective is to study the structure activity relationship of AG10 and several designed analogs in order to evaluate the structural similarities between AG10 binding and T119M variant in stabilizing TTR. Our objective is to study the structure activity relationship of AG10 and several designed analogs in order to evaluate the structural similarities between AG10 binding and T119M variant in stabilizing TTR.
Methods: In order to investigate the contribution of hydrogen bonds between the pyrazole ring of AG10 and the two S117/S117’ on the stabilization of TTR, we synthesized two AG10 analogues (compound 1 and 2). The analogues were purified by preparative HPLC and their chemical identity was confirmed by NMR spectroscopy and mass spectrometry. Isothermal titration calorimetry (ITC) was used to determine the binding affinities (Kd) of ligands to TTR. Biochemical studies were used to compare the potency of AG10 relative to other ligands in stabilizing TTR in buffer and human serum.
Results: As predicted by modeling, the addition of a methyl group on the pyrazole ring of compound 1 would restrict the pyrazole ring to form only one hydrogen bond with Ser117 on one of the adjacent TTR subunits. The bulky diethyl groups of analogue 2 would prevent the molecules for reaching deep in the T4-binding site thereby decreasing its ability to potentially form any hydrogen bonds with S117/S117′. Our data showed that the contribution of the enthalpy on binding of compound 1 (ΔH = -4.73 kcal/mol) and 2 (ΔH = -2.1 kcal/mol) was much lower than AG10 (ΔH = -13.6 kcal/mol, Kd = 4.8 ± 1.9 nM). This was translated in lower binding affinity for compounds 1 and 2 for TTR in buffer (Kd = 251 ± 12 nM and 1253 ± 79 nM, respectively).
This reduced affinity was translated into a significant decrease in potency for TTR in buffer and human serum. The order potency for stabilizing TTR was similar in both buffer and serum (AG10 > 1 > 2).
Conclusion: These results highlight the crucial role played by the pyrazole ring of AG10 and the importance of the hydrogen bonds it forms with the two TTR dimers, mimicking the interactions in the protective T119M-TTR mutation and enhancing the kinetic stability of the TTR tetramer.