Diflunisal Analogues Stabilize the Native State of Transthyretin. Potent Inhibition of Amyloidogenesis

Adamski‐Werner, Sara L.; Palaninathan, S.K.; Sacchettini, James C.; Kelly, Jeffery W.

doi:10.1021/jm030347n

Cited by 226 publications

(283 citation statements)

References 80 publications

(233 reference statements)

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“…TTR aggregation has been a model system for this approach, because small molecules, including the nonsteroidal anti-inflammatory drug diflunisal and many of its derivatives, are reported to stabilize the native tetrameric structure (31,32,46). In this study, we directly investigated the effect of diflunisal on F-rTTR tetramer structure by analysis of the s isotherms in the presence and absence of drug.…”

Section: Discussionmentioning

confidence: 99%

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Kingsbury

Laue

Klimtchuk

et al. 2008

Journal of Biological Chemistry

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Transthyretin (TTR) is normally a stable plasma protein.However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys 10 ) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescencedetected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.

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Section: Discussionmentioning

confidence: 99%

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Kingsbury

Laue

Klimtchuk

et al. 2008

Journal of Biological Chemistry

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“…Typically, Ͻ1% of TTR in the plasma and cerebrospinal fluid is bound to thyroxine, allowing us to target these sites with other small aromatic molecules to prevent amyloidogenesis (36). By using both focused screening and structure-based design, our laboratory has previously reported several classes of compounds that are capable of inhibiting TTR fibril formation by binding to the thyroxine sites (37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). Good inhibitors bind with high affinity, dissociate slowly, and exhibit high binding selectivity to TTR in the blood.…”

mentioning

confidence: 99%

Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis

Green

Foss

Kelly

2005

Proc. Natl. Acad. Sci. U.S.A.

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110

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The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with three amyloid diseases: senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Small molecules can bind to one or both of the unoccupied TTR thyroid hormone-binding sites, stabilizing the native tetramer more than the dissociative transition state, thereby raising the kinetic barrier for tetramer dissociation. Herein we demonstrate that genistein, the major isoflavone natural product in soy, works in this fashion and is an excellent inhibitor of transthyretin tetramer dissociation and amyloidogenesis, reducing acid-mediated fibril formation to <10% of that exhibited by TTR alone. Genistein also inhibits the amyloidogenesis of the most common familial amyloid polyneuropathy and familial amyloid cardiomyopathy mutations in TTR: V30M and V122I, respectively. Genistein additionally inhibits tetramer dissociation under physiological conditions thought to lead to slow amyloidogenesis in humans. Furthermore, this natural product exhibits highly selective binding to TTR in plasma over all of the other plasma proteins. Isothermal titration calorimetry shows that genistein binds to TTR with negative cooperativity (K d1 ‫؍‬ 40 nM, Kd2 ‫؍‬ 1.4 M). The benefits of using a nutraceutical such as genistein to treat orphan diseases such as the TTR amyloidoses include known oral bioavailability and safety data. It is conceivable that some patients could benefit from simply increasing their intake of soy products or supplements.amyloidogenesis inhibitor ͉ kinetic stabilization F or many years nutritionists and dieticians have noted the health benefits of a soy-based diet, citing the much lower incidence of cancer, including breast cancer, in Asian countries (1-4). The isoflavone genistein (compound 1 in Fig. 1), found in various soy foods at concentrations of 1.9-229 g͞g, is the component of soy implicated in cancer chemoprevention (5). An additional 71-968 g͞g of genistein is present as its O-glucoside conjugate, genistin (2), which is rapidly deglycosylated by intestinal bacteria in vivo. Genistein is being evaluated in preliminary trials for treatment of breast, prostate, and uterine cancers (6, 7), as well as for osteoporosis (8), cardiovascular disease (9), and treatment of menopausal symptoms (10). Toxicity studies reveal that this isoflavone does not appear to cause adverse health effects, even at the relatively high concentrations used (11-13). The isoflavone daidzein (3), lacking the hydroxyl group at the 5 position of genistein, is also found in soy foods, but no chemoprotective effects have been attributed to it.Transthyretin (TTR) is a tetrameric protein composed of identical 127-aa ␤-sheet sandwich subunits (14, 15). TTR functions to transport holo-retinol-binding protein and thyroxine (T4) in the blood and cerebrospinal fluid (16,17). Under denaturing con...

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“…In addition, it is imperative to notice that DES binding mediates several new intersubunit hydrophilic and hydrophobic interactions that will decrease the capacity of the protein to dissociate upon DES binding. In fact, compounds that bind tightly in the TTR binding channel should increase the energetic barrier associated with protein dissociation, and a number of small molecules representative of very distinct structural families are emerging as inhibitors of TTR amyloid formation (13,14,45,46). Furthermore, reported data indicate that the cytotoxic species are oligomers, which occur prior to fibril formation (47).…”

Section: Discussionmentioning

confidence: 99%

“…In vitro studies have shown that binding of the natural ligand T 4 to the TTR central hydrophobic channel stabilizes the tetramer and consequently reduces amyloid fibril formation (12). In the past few years, considerable effort has been directed to the discovery of small molecules that could prevent TTR dissociation by binding in the protein central channel (13,14). Actually, stabilization of the native tetrameric fold, upon high affinity binding of a small compound, is considered to be a potential therapeutic strategy to senile systemic amyloidosis and familial amyloidotic polyneuropathy variants (15,16).…”

mentioning

confidence: 99%

The Crystal Structure of Transthyretin in Complex with Diethylstilbestrol

Morais-de-Sá

Pereira

Saraiva

et al. 2004

Journal of Biological Chemistry

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Transthyretin (TTR) is a homotetrameric plasma protein that, inconditions not yet completely understood, may aggregate, forming the fibrillar material associated with TTR amyloidosis. A number of reported experiments indicate that dissociation of the TTR tetramer occurs prior to fibril formation, and therefore, studies aiming at the discovery of compounds that stabilize the protein quaternary structure, thereby acting as amyloid inhibitors, are being performed. The ability of diethylstilbestrol (DES) to act as a competitive inhibitor for the thyroid hormone binding to TTR indicated a possible stabilizing effect of DES upon binding. Here we report the crystallographic study of DES binding to TTR. The structural data reveal two different binding modes, both located in the thyroxine binding channel. In both cases, DES binds deeply in the channel and establishes interactions with the equivalent molecule present in the adjacent binding site. The most remarkable features of DES interaction with TTR are its hydrophobic interactions within the protein halogen binding pockets, where its ethyl groups are snugly fitted, and the hydrogen bonds established at the center of the tetramer with Ser-117. Experiments concerning amyloid formation in vitro suggest that DES is effectively an amyloid inhibitor in acid-mediated fibrillogenesis and may be used for the design of more powerful drugs. The present study gave us further insight in the molecular mechanism by which DES competes with thyroid hormone binding to TTR and highlights key interactions between DES and TTR that oppose amyloid formation.

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Diflunisal Analogues Stabilize the Native State of Transthyretin. Potent Inhibition of Amyloidogenesis

Cited by 226 publications

References 80 publications

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis

The Crystal Structure of Transthyretin in Complex with Diethylstilbestrol

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