Update of Natural Compounds in Transthyretin Amyloidosis, Years 2020–2025

Abstract

Transthyretin amyloidosis (ATTR) is a disease caused by the deposition of transthyretin-derived fibrils in the body. Despite extensive research conducted over the years, there are currently only four drugs available in clinical use to treat this condition, two of which are repurposed drugs used off-label. However, these treatments present several limitations; therefore, there is an urgent need for new therapeutic options. In this context, dietary supplements containing natural compounds capable of stabilizing the transthyretin (TTR) protein could represent a promising approach to contrast the disease progression, potentially supporting the therapeutic effects of the aforementioned drugs. In light of this, the present review highlights and analyzes the natural compounds that have most recently been reported in the literature as TTR stabilizers. In particular, the studies elucidating the potential of these compounds in the treatment of ATTR, along with the available crystallographic data explaining their binding mode to TTR, are reported. Overall, although the use of natural compounds as supplements shows promise in managing ATTR, further research is still needed to explore its feasibility and confirm its effectiveness. Hopefully, this work will help shed light on these issues and serve as a useful starting point for the development of new strategies to treat this disease.

1. Introduction

In recent years, growing evidence has suggested that natural compounds present in fruit and vegetables can reduce the risk of developing several diseases, ranging from cancer to neurodegenerative disorders [1,2,3,4,5]. In this context, some studies have also investigated their role in transthyretin amyloidosis (ATTR) [6,7,8,9], a disease originating from the dissociation and consequent misfolding of the transthyretin (TTR) protein, which results in the deposition of TTR-derived fibrils in various tissues of the body [10,11,12].

TTR is mainly synthesized by the liver and released in plasma [13], where it acts as a transporter of thyroxine (T₄). However, the main carriers of T₄ are thyroxine-binding protein and albumin; therefore, only 10−20% of T₄ is transported by TTR [14]. TTR can also transport retinol by forming a complex with retinol binding protein (RBP) [15]. Other sites of TTR production are the choroid plexus in the brain [16], the retinal pigment epithelium of the mammalian eye [17], pancreatic cells in the pancreas [18], and trophoblasts in placenta [19].

Experimental evidence suggests that, in the brain, TTR plays a protective role against Alzheimer’s disease (AD) through its interaction with amyloid-β (Aβ) [20,21,22]. In vivo studies in AD patients showed a close connection between TTR and AD physiopathology. Results showed that TTR levels decrease both in plasma and cerebrospinal fluid (CSF) as AD progresses [23].

TTR is characterized by four identical monomers (A, A’, B, B’) organized as a homotetramer (55 kDa) around a 2-fold axis. From a structural point of view, each monomer, composed by 127 amino acids (14 kDa), is formed by eight β-strands labelled from “A” to “H” and one α-helix (located between strands E and F) called the “EF-helix” (Figure 1A). The eight strands are arranged in two twisted β-sheets where strands A and G are parallel while all the others are anti-parallel. The symmetric operations drive the formation of the TTR tetramer, whereby two monomers assemble into a dimer, and two dimers subsequently combine to form the tetramer (Figure 1B). In the middle of the 2-fold symmetry axes, at the dimer–dimer interface, there is a channel called the thyroxin binding pocket (T₄-BP) that spans the entire protein [24,25]. The name T₄-BPs is derived from the binding of the endogenous ligand T₄ to these hydrophobic sites [25]. The two T₄-BPs are characterized by three pairs of symmetric cavities, known as halogen binding pockets (HBP1/1′, 2/2′, 3/3′), which accommodate the iodine atoms of T₄. HBP1 is located in the outer pocket near Lys15, Thr106, and Ala108. HBP2 is placed in the middle pocket, surrounded by Lys15, Ala108, and Leu11. HBP3 resides in the inner pocket, close to Ala108, Leu110, Ser117, and Thr119 (Figure 1C) [25].

As previously mentioned, recently TTR has gained attention for its pathological role in ATTR [27]. ATTR is a rare, progressive, and fatal disease associated with the presence of either wild-type TTR (wt-TTR) or mutant TTR (mTTR), which gradually lose their three-dimensional structure, leading to the formation of insoluble, toxic protein aggregates. These amorphous, β-sheet-rich aggregates accumulate in organs and tissues, particularly in the heart (leading to a condition known as cardiac amyloidosis) [28,29] and in the peripheral nervous system (causing polyneuropathy) [30,31]. In this regard, the most common point mutations in mTTR are V122I and V30M, which are associated with polyneuropathy and cardiomyopathy, respectively [32,33]. Although it is known that several factors contribute to the acceleration of protein misfolding, including acidic pH, metals dyshomeostasis, and oxidative stress [34], the exact mechanism triggering the onset of amyloidosis remains poorly understood [35,36,37].

Over the years, several therapeutic approaches have been studied for the treatment of ATTR, ranging from liver transplantation to small RNA-interfering drugs, antisense oligonucleotides, and drugs capable of stabilizing the TTR tetramer [38,39,40]. The latter approach is related to the ability of some small molecules to bind the T₄-BPs stabilizing the TTR tetramer structure and contrasting the dissociation of TTR in its corresponding monomers, which is the first step of the amyloidogenic pathway [41]. Currently, four drugs are used in clinical practice to manage this disease, namely, tafamidis [42,43,44], acoramidis [45], diflunisal, and tolcapone [38]—the latter two are repurposed drugs used off-label [46,47]. On the other hand, tafamidis and acoramidis (also called AG10) have been approved by the FDA for the treatment of ATTR [45,48]. Crystallographic studies report that these two drugs bind to the T₄-BPs similarly to the endogenous ligand T₄, both in wt-TTR and related mutants. The carboxylic group is oriented toward Glu54 and Lys15 at the entrance of the pocket, while the aromatic portions are positioned within HBP2 and HBP3, as well as in the endogenous ligand thyroxine (Figure 1C) [49,50].

A comprehensive discussion of these drugs and therapeutic approaches can be found in our recent reviews [38,51]. In this context, the aim of the present work is to provide a concise overview of the natural compounds that have most recently been reported in the literature as stabilizers of the TTR (from 2020 to 2025). For natural TTR stabilizers published prior to this timeframe, we refer readers to previous reviews [51,52]. Noteworthy, in the present paper, we have used paragraph titles analogous to those of previous reviews [51,52] to help readers easily link the compounds discussed here with those presented earlier. The importance of this topic is evident, as natural molecules might prove useful to enhance the therapeutic action of the approved ATTR therapies [51,52,53]. In fact, several studies have highlighted their positive effect in amyloidosis diseases [6,7,8,9]. In this regard, one approach to slow the progression of ATTR might consist of the constant consumption of dietary supplements containing natural compounds capable of inhibiting amyloid fibril formation [54,55]. From this perspective, the following paragraphs summarize the latest natural molecules reported as TTR stabilizing agents. Additionally, we refer readers to the following publications for further insight into the legal frameworks regulating dietary supplements [56,57].

Hopefully, this review will inspire the development of new clinical strategies for treating ATTR.

2. Isoflavone-Derivatives and Flavonoid Metabolites

Natural products encompass a wide array of chemical scaffolds with therapeutic potential against ATTR. Within this diverse chemical space, flavonoids have emerged as particularly promising candidates. As a matter of fact, over the years, they have been extensively investigated as potential TTR inhibitors. Notably, this research also extended to isoflavones, a sub-class of flavonoids [58,59]. In this context, a recent paper examined the activity of a series of isoflavones on ATTR, revealing that millexatin F (1) (Figure 2)—which occurs naturally in Millettia extensa [60,61]—was the most promising compound among those examined. Nevertheless, derrone (2) (Figure 2)—which occurs naturally in Ulex jussiaei (Leguminosae) [62]—also demonstrated notable activity [63]. Interestingly, although both of these compounds are indeed natural products, the authors synthesized them in this study. In an acid-mediated aggregation experiment performed on V30M-TTR, compound 2 exhibited the highest inhibitory activity against amyloid aggregation, followed by compound 1 [63]. Interestingly, in an ex vivo competitive binding assay on human plasma, compound 1 also possessed a higher degree of selectivity toward TTR compared with compound 2, although it was still to a lesser extent than tafamidis. As highlighted by the authors, the selectivity toward TTR is an important feature for a potential drug candidate because there are many other proteins in plasma besides TTR [59]. Given this encouraging results, the authors suggested 1 as a lead compound for structural optimization for the development of new therapeutics for the treatment of ATTR [63].

Another member of this class of molecules that has been subject of research is glabridin (3) (Figure 2), a natural isoflavone that occurs naturally in G. glabra L. (Favaceae) [55]. In this context, building on the fact that it had already been proven to be capable of stabilizing the TTR tetramer in previous in vitro experiments [64], Matsushita et al. performed additional studies on it and also investigated the activity of glavonoid, a natural supplement whose main constituent is glabridin [55,65,66,67]. However, it should be noted that the authors also hypothesized a possible contribution of the other polyphenols present in glavonoid to its stabilizing activity of the TTR tetramer observed in their study. In the study by Matsushita et al., glabridin, glavonoid, and Kaneka Glavonoid (the latter being a soft gel capsule used in human studies) were purchased from appropriate suppliers [55].

According to the authors, the results point to a possible employment of glavonoid in ATTR as a dietary supplement. Indeed, not only does it not cause significant side effects, but in vitro tests on human plasma from healthy patients also showed that, contrary to compound 3, glavonoid is capable of enhancing the tetramer levels while reducing those of the monomer, thus suggesting that it is capable of stabilizing the TTR tetramer. In addition, glavonoid decreased the monomer/tetramer ratio of TTR in healthy patients both at 300 mg/day and 600 mg/day oral dosage (although the effect appeared earlier in the 600 mg/day regimen). Notably, the plasma concentration of compound 3 after a 12-week oral administration of glavonoid at 300 mg/day to healthy patients (n = 7) was found to be approximately 2.5 ng/mL. In light of this, it is possible to hypothesize the use of glavonoid as a dietary supplement for the prevention of ATTR [55].

In parallel, others studies explored flavonoids metabolites, particularly glycosylated derivatives of quercetin (quercetin 3-O-β-D-glucuronopyranoside (4), quercetin 3-O-β-D-galactopyranoside (5), and quercetin 3-O-β-D-glucopyranoside (6), Figure 3) as potential TTR inhibitors [68,69], building on previous evidence that quercetin itself can bind to the T₄-BPs, thereby reducing TTR misfolding and aggregation [70]. The 3-O-glycosylated derivatives, as well as quercetin, are naturally present in several commonly consumed foods, including apple, orange, onion, black tea, molokheya, broccoli, kale, and blueberry [68]. In an acid turbidimetric assay on wt-TTR, compounds 4–6 (isolated from Cornus mas, Cornus sanguinea, and Ruprechtia polystacya [71,72]) showed a moderate capability of inhibiting the amyloid fibril formation (the percentage of fibril formation was 45% in the presence of compounds 4 and 6 and 40% in the presence of compound 5), thus suggesting that they have a low affinity for the T₄ binding sites, which was confirmed using the ANS displacement binding assay [73,74]. These results were further validated using thioflavin T binding assays [75] on wt-TTR, in which the percentage of fibril formation in the presence of compounds 4–6 was assessed to be between 23% and 38% (with respect to TTR alone, in which the percentage of fibril formation was 100%). Moreover, all compounds were also shown to have a strong antioxidant activity, which, according to the authors, could help preserve the TTR protein in its tetrameric state by avoiding the oxidation of Cys10 (an amino- acid with an important role in the native structure of TTR [76,77]). The in vitro tests were performed in triplicate (n = 3) [69].

Structural insights into the binding mode of these quercetin derivatives with TTR were obtained from the crystal structure of the TTR–compound 5 (3-O-β-D-galactopyranoside) complex. Crystallographic analysis revealed that compound 5 binds to only one of the T₄-BPs, positioning its dihydroxyphenyl group in the outer cavity, while the dihydroxyphenyl ring fits into the inner pocket near Ser117 and Thr119. This binding mode is opposite to that observed for quercetin, likely due to the steric hindrance caused by the galactopyranoside moiety of compound 5 (Figure 4).

All in all, these results suggest that patients suffering from ATTR might possibly benefit to some extent from dietary intake of these compounds. However, further research is still needed in this field to determine their real usefulness.

3. Natural Derivatives of Resveratrol

Previous studies have shown that resveratrol (7) (Figure 5)—which naturally occurs in peanuts, raspberries, grape skins, blueberries, and mulberries—is an interesting compound in the field of ATTR [54,78,79,80]. However, its use is limited by some drawbacks, such as its rapid elimination following oral administration [54,78,79]. In light of this, natural derivatives of resveratrol have been investigated with the aim of finding compounds with improved properties. In this context, Yokoyama et al. evaluated two natural derivatives of resveratrol (pinostilbene (8) and pterostilbene (9), (Figure 5)) for their ability to inhibit amyloid fibril formation, using an acid-induced aggregation assay on V30M-TTR [54,81]. In particular, compound 8 is a metabolite of compound 9, which in turn is derived from compound 7. Noteworthy, compound 9 naturally occurs in berries and peanuts and is also available as a dietary supplement [54,81,82]. However, it should be noted that in the study by Yokoyama et al. compounds (7, 8, and 9) were purchased from appropriate companies. The results of Yokoyama and colleagues showed that the inhibitory potencies of compounds 8 and 9 are similar to that of tafamidis, as evidenced by their inhibition constants (3.4 µM, 3.5 µM, and 3.1 µM for 8, 9, and tafamidis, respectively). Subsequently, the apparent dissociation constant of these compounds (1.2 µM and 1.9 µM for 8 and 9, respectively) was measured using an intrinsic tryptophan fluorescence quenching assay, which revealed that these derivatives inhibit the amyloid fibril formation by binding to the TTR. In addition, isothermal titration calorimetry (ITC) experiments were performed to assess the binding affinity of these compounds to V30M-TTR (the values of the dissociation constant are 1.5 ± 0.38 µM, 2.4 ± 0.27 µM, and 4.6 ± 0.29 µM for 7, 8, and 9, respectively) [54].

The binding modes of compound 7 and of its derivatives 8 and 9 were further investigated using X-ray crystallography [54]. The authors performed their study on the TTR mutant V30M, obtaining high-resolution crystal complexes structurally very similar to that already reported in literature for wt-TTR [83,84]. The crystals of V30M-TTR in complex with compounds 7, 8, and 9 belonged to the space group P2₁2₁2 with a dimer in the asymmetric unit. The symmetric operation generates the tetramer with the two T₄-BPs that form a channel that crosses the whole protein [54].

Compound 7, as revealed by the electron density maps, exhibited dual binding modes with the V30M mutant, as it did with wt-TTR [54,84]. In the first, known as the “canonical binding mode”, the resorcinic ring is located in the outer binding pocket with its hydroxyl groups oriented toward Lys15/15′, while the phenolic ring resides in the inner binding pocket with its hydroxy moiety pointing toward Ser117/117′ (Figure 6A, ligand in yellow). In the second mode, compound 7 is flipped. The resorcinol ring is inserted into the inner binding pocket with its hydroxy moieties oriented toward Ser117/117′, while the phenolic ring is positioned in the outer cavity (Figure 6A, ligand in green). This dual binding behavior was also observed for compound 8 (ligand in lime in Figure 6B and ligand in light pink in Figure 6C) [54].

Regarding compound 9, only the “canonical binding mode” was observed. The aromatic ring bearing the methoxy groups is located in the outer cavity, while the phenolic ring is positioned deep within the T₄-BSs (Figure 6C, ligand in yellow) [54].

According to the authors, these encouraging results highlight the potential of resveratrol derivatives for the development of novel therapeutic approaches in which resveratrol analogies could be used as a supplement during the ATTR treatment.

4. Other Natural Stabilizers from Various Sources

Omega-3 poly-unsaturated fatty acids (PUFAs) are recognized to have multiple positive effects on human health, such as the reduction of blood pressure and of hypertriglyceridemia [85,86]. Within this class of molecules are eicosapentaenoic acid (EPA) (10) (Figure 7) and docosahexaenoic acid (DHA) (11) (Figure 7), which naturally occur, for example, in oil from fish [87].

These molecules were already known for their interesting properties (such as their antioxidant activity); however, more recently, they have also been investigated for their capability of preventing the misfolding of the TTR protein [87,88,89]. To perform the study, compounds 10 and 11 were purchased from an appropriate supplier; all the following tests were performed in triplicate (n = 3). The turbidimetric assay on wt-TTR showed that 10 and 11 can effectively inhibit the amyloid fibril formation (the percentage of fibril formation was 40% and 36%, respectively). In addition, further studies on wt-TTR using thioflavin T fluorescence spectroscopy revealed that 11 has a similar efficacy to diflunisal in reducing the amyloid β-sheet content (42% vs. 54% for 11 and diflunisal, respectively), while 10 is much less active (22%). According to the authors, these results might depend on the fact that small protofibrils can be clearly detected only using the thioflavin T assay and not the turbidimetric one. In order to clarify the mechanism of action of these molecules, an ANS displacement biding assay on wt-TTR was also carried out, and the results revealed that both 10 and 11 can bind to the TTR-binding sites with an IC₅₀ of 13.0 ± 0.08 µM and 12.1 ± 0.07 µM, respectively. This interaction likely leads to a stabilization of the TTR tetramer and justifies the inhibition of the amyloid fibril formation. In light of this, the authors have suggested the use of these molecules in association with appropriate drugs for the treatment of ATTR [87].

Another source of natural compounds that has been investigated for new natural TTR inhibitors is Santalum album L., a plant that has been traditionally exploited to produce perfumes [90,91]. In particular, this plant contains two natural sesquiterpenes, α-santalol (12) (Figure 8) and β-santalol (13) (Figure 8), that have recently attracted attention for their activity in ATTR [90].

In their study, Mohankumar at al. purified compounds 12 and 13 from sandalwood (Santalum album L.) oil [90]. Both compounds were proven to be capable of inhibiting the aggregation of both wt-TTR (EC₅₀ values were 36.7 ± 1.2 µM and 31.1 ± 2.4 µM for 12 and 13, respectively) and V30M-TTR (EC₅₀ values were 44.3 ± 3.5 µM and 34.5 ± 2.8 µM for 12 and 13, respectively) under acidic conditions, albeit, in both cases, less effectively than tolcapone (EC₅₀ values were 4.6 ± 0.5 µM and 4.8 ± 0.7 µM for wt-TTR and V30M-TTR, respectively). Notably, these results were obtained from four independent biological replicates, each performed in triplicate. In addition, the capability of these compounds to stabilize the TTR protein was verified using a urea-induced denaturation assay on wt-TTR and V30M-TTR. Moreover, in in vivo experiments on C. elegans worms expressing wt-TTR and V30M-TTR (both at full-length and fused with a fluorescent protein), both 12 and 13 were proven to be capable of inhibiting the formation of TTR aggregates as effectively as tolcapone. Noteworthy, the compounds were more effective as the worms grew older, and a synergistic effect was observed when they were administrated together each at its half optimal dose (16 μM and 8 μM for 12 and 13, respectively) [90].

5. Conclusions

ATTR is a complex disease caused by the dissociation and consequent misfolding of the TTR protein, which results in the deposition of TTR-derived fibrils in different areas of the body. One of the currently available therapeutic strategies for managing this disease consists of the administration of TTR-stabilizing drugs, the purpose of which is to avoid fibril formation. However, despite their proven clinical benefits, these therapeutics are not resolutive, making further research in this field essential. In this scenario, dietary supplements containing natural compounds capable of stabilizing the TTR protein could contribute to the therapeutic action of the drugs used in therapy, thus representing a new helpful tool in the clinical management of this disease. In light of this, here, we reported the natural compounds with such activity that have most recently been reported (years 2020–2025) in the literature as transthyretin stabilizers. Their therapeutic potential in the treatment of ATTR has been described, and the available crystallographic data elucidating their binding mode to TTR have been illustrated. Overall, the use of natural compounds in the form of dietary supplements appears to be a promising strategy for improving the clinical management of the ATTR disorder. However, additional research is needed to determine its feasibility and its actual therapeutic effectiveness. Hopefully, this review will help shed some light on these issues and assist future researchers in designing new and improved protocols for treating this disease.