The supramolecular self-assembly of uric acid (UA), the end product of purine metabolism, underlies crystal deposition in gout and kidney diseases. However, the intermediate states linking soluble UA to crystalline phases remain poorly defined. Here, we report that UA self-assembles into amyloid-like fibrils that coexist with crystalline forms and exhibit cytotoxicity. Native ion mobility spectrometry-mass spectrometry (IMS-MS) reveals discrete UA oligomers up to 60-mers, suggesting a stepwise assembly process. Optical and electron microscopy distinguish between fibrous and crystalline morphologies, with the fibrillar network acting as a potential scaffold for nucleation. We demonstrate that allopurinol, beyond its known function as a xanthine oxidase inhibitor, directly perturbs UA aggregation. Allopurinol alters the thermodynamics of self-assembly, suppressing fibril formation and promoting crystallization into a more stable anhydrous polymorph. In contrast, epigallocatechin gallate (EGCG) suppresses both fibrillation and crystallization. X-ray diffraction confirms the formation of a distinct anhydrous crystal phase in the presence of allopurinol, analogous to that found in patient-derived deposits. These findings expand the chemical understanding of UA phase behavior and polymorphism and establish cytotoxic UA fibrils as drug-modifiable intermediates. Modulating small-molecule-driven metabolite self-assembly provides a mechanistic basis for rational intervention in gout and other disorders characterized by metabolite aggregation.

Phenylketonuria is characterized by the accumulation of phenylalanine, resulting in severe cognitive and neurological disorders if not treated by a remarkably strict diet. There are two approved drugs today, yet both provide only a partial solution. We have previously demonstrated the formation of amyloid-like toxic assemblies by aggregation of phenylalanine, suggesting a new therapeutic target to be further pursued. Moreover, we showed that compounds that halt the formation of these assemblies also prevent their resulting toxicity. Here, we performed high-throughput screening, searching for compounds with inhibitory effects on phenylalanine aggregation. Morin hydrate, one of the most promising hits revealed during the screen, was chosen to be tested in vivo using a phenylketonuria mouse model. Morin hydrate significantly improved cognitive and motor function with a reduction in the number of phenylalanine brain deposits. Moreover, while phenylalanine levels remained high, we observed a recovery in dopaminergic, adrenergic, and neuronal markers. To conclude, the ability of Morin hydrate to halt phenylalanine aggregation without reducing phenylalanine levels implies the toxic role of the phenylalanine assemblies in phenylketonuria and opens new avenues for disease-modifying treatment.

Inborn error of metabolism disorders (IEMs) are a family of diseases resulting from single-gene mutations that lead to the accumulation of metabolites that are usually toxic or interfere with normal cell function. The etiological link between metabolic alteration and the symptoms of IEMs is still elusive. Several metabolites, which accumulate in IEMs, were shown to self-assemble to form ordered structures. These structures display the same biophysical, biochemical, and biological characteristics as proteinaceous amyloid fibrils. Here, we have demonstrated, for the first time, the ability of each of the branched-chain amino acids (BCAAs) that accumulate in maple syrup urine disease (MSUD) to self-assemble into amyloid-like fibrils depicted by characteristic morphology, binding to indicative amyloid-specific dyes and dose-dependent cytotoxicity by a late apoptosis mechanism. We could also detect the presence of the assemblies in living cells. In addition, by employing several in vitro techniques, we demonstrated the ability of known polyphenols to inhibit the formation of the BCAA fibrils. Our study implies that BCAAs possess a pathological role in MSUD, extends the paradigm-shifting concept regarding the toxicity of metabolite amyloid-like structures, and suggests new pathological targets that may lead to highly needed novel therapeutic opportunities for this orphan disease.

Nucleobase crystals demonstrate unique intrinsic fluorescence properties in the visible spectral range. This is in contrast to their monomeric counterparts. Moreover, some nucleobases were found to exhibit red edge excitation shift. This behavior is uncommon in the field of organic supramolecular materials and could have implications in fields such as therapeutics of metabolic disorders and materials science.

Controlling the infectivity of respiratory RNA viruses is critical, especially during the current SARS-CoV-2 pandemic. There is an unmet need for therapeutic agents that can reduce viral replication, preferably independent of the accumulation of viral mutations. Zinc ions have an apparent activity as modulators of intracellular viral RNA replication and thus, appear attractive in reducing viral RNA load and infectivity. However, the intracellular concentration of zinc is usually too low for achieving an optimal inhibitory effect. Various herbal polyphenols serve as excellent zinc ionophores with known antiviral properties. Here, we combined zinc picolinate with a collection of flavonoids, representing commonly used polyphenols. Copper was added to avoid ionic imbalance during treatment and to improve efficacy. Each component separately, as well as their combinations, did not interfere with the viability of cultured A549, H1299, or Vero cells in vitro as determined by MTT assay. The safe combinations were further evaluated to determine antiviral activity. Fluorescence-activated cell sorting and quantitative polymerase chain reaction were used to evaluate antiviral activity of the combinations. They revealed a remarkable (50–95%) decrease, in genome replication levels of a diverse group of respiratory RNA viruses, including the human coronavirus OC43 (HCoV-OC43; a betacoronavirus that causes the common cold), influenza A virus (IAV, strain A/Puerto Rico/8/34 H1N1), and human metapneumovirus (hMPV). Collectively, our results offer an orally bioavailable therapeutic approach that is non-toxic, naturally sourced, applicable to numerous RNA viruses, and potentially insensitive to new mutations and variants.

The formation of ordered protein and peptide assemblies is a phenomenon related to a wide range of human diseases. However, the mechanism of assembly at the molecular level remains largely unknown. Minimal models enable the exploration of the underlying interactions that are at the core of such self-assembly processes. In particular, the ability of phenylalanine, a single aromatic amino acid, to form an amyloid-like structure has challenged the previous dogma viewing a peptide backbone as a prerequisite for assembly. The driving forces controlling the nucleation and assembly in the absence of a peptide backbone remain to be identified. Here, aiming to unravel these forces, we explored the kinetics and thermodynamics of three phenylalanine-containing molecules during their assembly process: the amino acid phenylalanine, which accumulates in phenylketonuria patients, the diphenylalanine core-motif of the amyloid β peptide related to Alzheimer's disease, and the extended triphenylalanine peptide which forms a range of distinct nanostructures in vitro. We found that the aggregation propensity, regarding the critical monomer concentration, strongly increases with size, with triphenylalanine being the most aggregation-prone species under our experimental conditions. In the context of classical nucleation theory, this increase in aggregation propensity can be attributed to the larger free energy decrease upon aggregation of larger peptides and is not due to the presence/absence of a peptide bond per se. Taken together, this work provides insights into the aggregation processes of chemically simple systems and suggests that both backbone-containing peptides and backbone-lacking amino acids assemble through a similar mechanism, thus supporting the classification of amino acids in the continuum of amyloid-forming building blocks.

The skin is a key site for drug administration because of its large surface area and noninvasive accessibility. However, the dermal architecture serves as an excellent barrier, protecting from external mechanical, chemical, microbial, and physical perturbations. Most drugs display poor permeability through this barrier, thus making dermal and subdermal delivery challenging. Cell-penetrating peptides (CPPs), a diverse group of relatively short cationic and amphipathic membrane-interacting peptides, are fast becoming an important class of drug carriers and could potentially be developed for the dermal delivery of active molecules. However, the mechanism of CPP transdermal delivery is not fully understood, and there is a genuine need for a minimal model to understand this important phenomenon. Here, we demonstrate the potent membrane interactions of a minimal four-amino-acid-long CPP as well as the significance of guanidinium patterning and cationic nature of this palindromic peptide on its bioactivity. Furthermore, we demonstrate the biocompatibility of this peptide as well as its rapid cellular uptake and endosomal distribution. Finally, by utilizing a porcine full-thickness skin model, we demonstrate the substantial independent dermal and sonophoresis-based transdermal penetration of this minimal model. These results provide a minimal model for CPPs which can be easily manipulated for further biophysical and biochemical evaluations as well as a potent functional CPP with excellent skin permeability, which can be utilized for a wide variety of cosmetic and medical applications.

Phenylalanine was the minimalistic and first of numerous nonproteinaceous building blocks to be demonstrated to form amyloid-like fibrils. This unexpected organization of such a simple building block into canonical architecture, which was previously observed only with proteins and peptides, has numerous implications for medicine and supramolecular chemistry. However, the morphology of phenylalanine fibrils and their mechanical properties was never characterized in solutions. Here, using electron and atomic force microscopy, we analyze the morphological and mechanical properties of phenylalanine fibrils in both air and fluids. The fibrils demonstrate an exceptionally high Young’s modulus (up to 30 GPa) and are found to be composed of intertwined protofilaments in a helical or twisted ribbon morphology. In addition, X-ray scattering experiments provide convincing evidence of an amyloidal cross-β-like secondary structure within the nanoassemblies. Furthermore, increasing the phenylalanine concentration results in the formation of highly homogenous, noncrystalline, self-healing hydrogels that display storage and loss moduli significantly higher than similar noncovalently cross-linked biomolecular nanofibrillar scaffolds. These remarkably stiff nanofibrillar hydrogels can be harnessed for various technological and biomedical applications, such as self-healing, printable, structural, load-bearing 3D scaffolds. The properties of this simple but quite remarkable hydrogel open a possibility to utilize it in the biomaterial industry.

The formation of metabolite fibrillar assemblies represents a paradigm shift in the study of human metabolic disorders. Yet, direct clinical relevance has been attributed only to metabolite crystals. A notable example for metabolite crystallization is calcium oxalate crystals observed in various diseases, including primary hyperoxaluria. We unexpectedly observed retinal damage among young hyperoxaluria patients in the absence of crystals. Exploring the possible formation of alternative supramolecular organizations and their biological role, here we show that oxalate can form ordered fibrils with no associated calcium. These fibrils inflict intense retinal cytotoxicity in cultured cells. A rat model injected with oxalate fibrils recaptures patterns of retinal dysfunction observed in patients. Antibodies purified from hyperoxaluria patient sera recognize oxalate fibrils regardless of the presence of calcium. These findings highlight a new molecular basis for oxalate-associated disease, and to our knowledge provide the first direct clinical indication for the pathogenic role of metabolite fibrillar assemblies.

The formation of ordered nanostructures by metabolites is gaining increased interest due to the simplicity of the building blocks and their natural occurrence. Specifically, aromatic amino acids possess the ability to form ordered supramolecular interactions due to their limited solubility in aqueous solution. Unexpectedly, l-tyrosine (l-Tyr) is almost 2 orders of magnitude less soluble in water compared to l-phenylalanine (l-Phe). However, the underlying mechanism is not fully understood as l-Tyr is more polar. Here, we explore the utilization of insoluble tyrosine assemblies for technological applications and their molecular basis by manipulating the basic building blocks of tightly packed dimers. We show that the addition of an amyloid inhibition agent increases l-Tyr solubility due to the disruption of the dimer formation. The molecular organization grants the l-Tyr crystal higher thermal stability and mechanical properties between three amino acids. Additionally, l-Tyr crystals are shown to generate high and stable piezoelectric power outputs under mechanical pressure in a sandwich device. By incorporating the rigid l-Tyr crystals into a soft polymer, a mechano-responsive bending composite was fabricated. Furthermore, the l-Tyr crystalline needles exhibit an active photowaveguiding property, making them promising candidates for the generation of photonic biomaterial-based devices. The present work exemplifies a feasible strategy to explore physical properties of supramolecular self-assemblies comprises minimalistic naturally occurring building blocks and their applications in energy harvesting, photonic devices, stretchable electronics, and soft robotics.

The extension of the amyloid hypothesis to include non-protein metabolite assemblies invokes a paradigm for the pathology of inborn error of metabolism disorders. However, a direct demonstration of the assembly of metabolite amyloid-like structures has so far been provided only in vitro. Here, we established an in vivo model of adenine self-assembly in yeast, in which toxicity is associated with intracellular accumulation of the metabolite. Using a strain blocked in the enzymatic pathway downstream to adenine, we observed a non-linear dose-dependent growth inhibition. Both the staining with an indicative amyloid dye and anti-adenine assemblies antibodies demonstrated the accumulation of adenine amyloid-like structures, which were eliminated by lowering the supplied adenine levels. Treatment with a polyphenol inhibitor reduced the occurrence of amyloid-like structures while not affecting the dramatic increase in intracellular adenine concentration, resulting in inhibition of cytotoxicity, further supporting the notion that toxicity is triggered by adenine assemblies.

Here we report the influence of amino acid sequence order on the self-assembly and biological functions of the core recognition motif of Amyloid β.

The formation of ordered amyloid fibrils by proteins and polypeptides is associated with human disorders. A recent extension of the amyloidogenic building block family includes several small metabolites, which form assemblies with structural and functional similarities to well-established amyloids. Here we investigate whether generic amyloid polyphenolic inhibitors can also restrict the formation of metabolite fibrils. We reveal that epigallocatechin gallate and tannic acid inhibit amyloid-like fibrillation of adenine, phenylalanine, and tyrosine. Moreover, the compounds reduce the cytotoxicity triggered by these assemblies. In contrast, acetylsalicylic acid, used as a control does not have an inhibitory effect. The compounds’ differential effects at various time points is consistent with molecular dynamics simulations, providing information about the inhibition mechanisms and inhibitors’ key interactions with the monomeric and subsequent crystalline fibril states. Taken together, we provide additional evidence for the fundamental similarities between protein- and metabolite-based amyloids, the inhibition process and dynamics of association.

The amino acid tyrosine forms cytotoxic amyloid-like fibrils by molecular self-assembly. However, the production of antibodies towards tyrosine assemblies, reflecting their presentation to the immune system, was not demonstrated yet. Here, we describe the production of antibodies that specifically recognize tyrosine in its fibrillated form. The antibodies were demonstrated to specifically bind self-assembled tyrosine, in contrast to its non-aggregated form or disintegrated fibrils. The antibodies could be used for immunostaining of tyrosine fibrils in cultured cells. Furthermore, confocal microscopy allowed a demonstration of the intracellular presence of the metabolite amyloids in a neuroblastoma cell model. Finally, pre-incubation of tyrosine fibrils with the antibodies resulted in significant reduction in their cytotoxicity. Taken together, we provide an experimental proof for the immunogenicity of tyrosine amyloid fibrillary assemblies. These specific antibodies against tyrosine structures could be further used as a research tool to study the dynamics, toxicity and cellular localization of the assemblies.

Amyloid-like structure formation by various metabolites represents a significant extension of the amyloidogenic building block family. Similar to protein amyloids, metabolite amyloids induce apoptotic toxicity, a process that was linked to membrane association. Here, we demonstrate that metabolite amyloids interact with model membranes and study the mechanism by molecular dynamics.