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. 2013 Jun 11;110(24):9992-7.
doi: 10.1073/pnas.1300761110. Epub 2013 May 28.

AG10 Inhibits Amyloidogenesis and Cellular Toxicity of the Familial Amyloid Cardiomyopathy-Associated V122I Transthyretin

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Free PMC article

AG10 Inhibits Amyloidogenesis and Cellular Toxicity of the Familial Amyloid Cardiomyopathy-Associated V122I Transthyretin

Sravan C Penchala et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Ig light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African American subjects, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 y causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the development of AG10, a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10, combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy.

Keywords: crystal structure; drug design.

Conflict of interest statement

Conflict of interest statement: E.T.P. and J.W.K. have a financial interest in the regulatory agency-approved drug tafamidis.

Figures

Fig. 1.
Fig. 1.
The TTR amyloidogenesis cascade and a table summarizing TTR-mediated amyloidoses. TTR amyloidoses require rate-limiting tetramer dissociation to dimers, followed by dissociation into monomers before partial unfolding of monomers yields the aggregation-prone amyloidogenic intermediate. The amyloidogenic intermediate can misassemble to form a variety of toxic aggregates, including amyloid fibrils. Disease-associated destabilizing mutations can kinetically or thermodynamically destabilize TTR. Kinetic stabilization can be achieved through trans allelic suppression with the kinetically stabilized T119M-TTR, binding to T4 or other small molecules (Lower Left).
Fig. 2.
Fig. 2.
AG10 binds to TTR and stabilizes it in buffer. (A) Chemical structures of TTR kinetic stabilizers. (B) Evaluation of ligand binding to TTR in buffer by FP. Competition of FP-probe 2 from TTR by increasing concentrations (0.006–12.5 µM) of AG10 (Kapp = 193 nM; R2 = 0.994) and tafamidis (Kapp = 247 nM; R2 = 0.990). Each point shows the mean (SD) of three replicates. (C) Assessment of the binding affinity of AG10 to TTR by ITC. Calorimetric titration of AG10 against TTR (Kd1 = 4.8 nM, Kd2 = 314 nM). Raw data (Upper) and integrated heats (Lower) from the titration of TTR (2 µM) with AG10 (25 µM). (D) Inhibition of WT- and V122I-TTR (4 µM) fibril formation by test compounds (4 and 2 µM) under acidic conditions after 24 h. Aggregation in the presence of solvent alone (DMSO) was defined as 100% fibril formation. Each bar shows the mean ± SD of three technical replicates.
Fig. 3.
Fig. 3.
AG10 binds selectively to TTR in human serum. (A) Fluorescence change caused by modification of TTR in human serum by covalent probe 3 monitored for 6 h in the presence of probe alone (black circles) or probe and TTR ligands (colors). (B) Percentage of covalent probe binding to TTR in the presence of ligands measured after 6 h of incubation relative to probe alone. (The lower the binding of the probe, the higher the binding selectivity of the ligand to TTR.) Each bar shows the mean (SD) of three replicates. (C and D) Stabilization of WT-TTR in serum against acid-mediated denaturation in the presence of AG10 and tafamidis. Serum samples were incubated (with DMSO, AG10, or tafamidis) in acetate buffer (pH 4.0) for the desired time period (0 and 72 h) before cross-linking and immunoblotting. The intensity of TTR bands (TTR tetramer, arrowhead; TTR bound to RBP, solid arrow) was quantified by using an Odyssey IR imaging system (LI-COR Bioscience) and reported as percentage of TTR tetramer, calculated as 100 × [(tetramer and tetramer + RBP density, 72 h)/(tetramer and tetramer + RBP density of DMSO, 0 h)]. (C) The shown blot is representative of four independent blots. (D) Error bars indicate SEM (n = 4).
Fig. 4.
Fig. 4.
Stabilization of V122I-TTR in serum from two patients with FAC against acid-mediated denaturation in the presence of AG10 and tafamidis. Stabilization effect of tafamidis and AG10 on serum from a heterozygous patient with FAC (heterozygous V122I/WT-TTR, African American, male, age 56 y) at 0 h (A and B) and after 72 h (C and D). (E and F) Stabilization effect of tafamidis and AG10 on serum from a homozygous patient with FAC (homozygous V122I-TTR, Caucasian, female, age 62 y). Sample processing and quantification was performed as described in Fig. 3. IgG was analyzed by using IRdye800 goat anti-human IgG. Error bars represent SEM of three replicates.
Fig. 5.
Fig. 5.
Crystal structures of V122I-TTR ligand complexes. (A) Quaternary structure of AG10 bound to V122I-TTR shown as a ribbon representation with monomers colored individually and positions of each of the V122I mutations shown as black spheres located on the H β-strand, which interacts with the adjacent AB-loop on the AC/BD interface. (B) AG10 in complex with V122I-TTR. (C) Tafamidis in complex with V122I-TTR. Close-up views of one of the two identical T4 binding sites with different colored ribbons for the two monomers of the tetramer composing the binding site. A Connolly molecular surface (40) was applied to residues within 10 Å of ligand in the T4 binding pocket and colored gray for hydrophobic and purple for polar residues.

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