Inhibiting transthyretin amyloid fibril formation via protein stabilization

Abstract

Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 M) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis-i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.Transthyretin (TTR) is present in human plasma (0.2 mg͞ml; 3.63 M, tetramer) and is composed of four identical ␤-sheetrich subunits that bind and transport thyroxine (T4) and the retinol binding protein (1). In unfortunate individuals, TTR is converted into an insoluble fibrillar structure called amyloid. These fibrils putatively cause senile systemic amyloidosis (wild-type TTR composes the fibrils-late onset) and familial amyloid polyneuropathy (FAP; predominantly variant TTR composing the fibrils-earlier onset) by virtue of the amyloid's neurotoxicity and͞or by physically interfering with normal organ function (2-8). A TTR amyloid fibril is Ϸ130Å in diameter and made up of four protofilaments, each having a twisted cross-␤-helix structure (9, 10). TTR amyloid fibril formation is observed during partial acid denaturation from a conformational intermediate formed under conditions simulating a lysosome (pH 5.5 Ϯ 0.5), which has been implicated in fibril formation in vivo (11,12). TTR amyloid fibril formation can be avoided under acidic conditions by working at low TTR concentrations and low temperature (25ЊC) allowing identification of the quaternary, tertiary, and secondary structure of the intermediate(s) that can form amyloid (12). These studies reveal that tetrameric TTR is nonamyloidogenic; however, the dissociation of the tetramer into a monomeric intermediate having an altered, but defined, tertiary structure is capable of amyloid fibril formation and is therefore called the amyloidogenic intermediate (Fig. 1). Several of the 50 FAP-associated TTR single-site mutations still adopt a normal tetrameric s...

Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation: Implications for Wild-Type, V30M, and L55P Amyloid Fibril Formation

“…Amyloid formation is also observed at the lower temperature of 25°C at pH 4.4, albeit in lower yield (20-50%). Sedimentation velocity analysis of TTR at pH 4.4 after an incubation period of 2 days at 25°C is reported in a recent publication where inhibitors of TTR amyloid formation are evaluated (28). Under these conditions, TTR exists as a mixture of sedimenting species with s values ranging from 1.6 to 6.3 S as discerned from the time derivative g(s*) analysis.…”

“…The sedimentation velocity profiles of wild-type TTR at pH 7.0 show the movement of a single boundary across the cell ( Figure 1A). The second moment analysis for the movement of the midpoint of the absorbance boundary (r) vs time (t) was used to obtain the apparent sedimentation coefficient, s, of 4.5 S. This s value corresponds to an s 20,w ) 4.1 S, consistent with the TTR tetramer (28). Further analysis of the data using van Hold-Weischet global fitting also indicates the presence of one single species with s 20,w ) 4.2 S ( Figure 1B,C).…”

Synthesis, Structure, and Activity of Diclofenac Analogues as Transthyretin Amyloid Fibril Formation Inhibitors

“…The activities of diclofenac (1) analogues 2-13 as inhibitors of wild type, V30M, and L55P TTR fibril formation under conditions that simulate those found in the lysosome were determined using a stagnant fibril formation assay. 9,24 In this assay, the compound under evaluation is first incubated for 30 min with TTR at neutral pH (37°C). The pH is then lowered to give the maximal rate of fibril formation for a given variant (4.2 for wild type or 5.0 for V30M and L55P), and the samples are incubated for 72 h at 37°C.…”

“…Experiments have shown that the binding of thyroxine by TTR stabilizes the tetrameric state against acid induced dissociation, thus preventing amyloid formation in vitro. 24 Because of its hormone activity, thyroxine cannot be used to inhibit TTR fibril formation in vivo. However, focused screening and rational design efforts have resulted in the identification of several structurally diverse small molecules that, like thyroxine, bind to and stabilize TTR under fibrillogenic conditions.…”

Quantification of the Thermodynamically Linked Quaternary and Tertiary Structural Stabilities of Transthyretin and Its Disease-Associated Variants: The Relationship between Stability and Amyloidosis

“…Gene therapy mediated by replacing a disease-associated variant TTR/WT TTR expressing liver by a WT TTR/WT TTR expressing liver through transplantation has proven useful as a strategy for treating familial amyloid polyneuropathy (30). We have discovered small molecule TTR tetramer binders that impose kinetic stability on the tetramer, preventing dissociation required for amyloidogenesis (20,(31)(32)(33)(34). Two such small molecules are currently being tested as agents to ameliorate FAP in placebo-controlled clinical trials (see http://www.clinicaltrials.gov/).…”