Allopurinol Reprograms Uric Acid Self-Assembly by Disrupting Cytotoxic Fibrils and Redirecting Crystal Growth

Abstract

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.

Keywords: allopurinol; crystal polymorphism; cytotoxic fibrils; metabolite aggregation; uric acid self-assembly.

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Early stage of UA self-assembly probed by IMS-MS. (A) Mass spectra of UA in positive (top) and negative (bottom) modes of early UA oligomers (clusters). Each mass spectral peak is annotated with a nominal cluster size (n) to charge (z) ratio. MS peaks with n/z colored in red are those with a mixture of clusters at different charge states. Multiple clusters may share the same n/z ratio. (B) A plot of experimental cross-sections as a function of cluster size. Post-IM dissociation of UA clusters. (C) Illustrations showing neutral losses in clusters with the same charge state and charged losses in clusters with different charge states. These clusters travel through the drift cell at velocities proportional to their size and charge but are detected at the same mass-to-charge (m/z) ratio. (D) Representative IM profiles of UA clusters. 2D plots of drift time vs m/z for selected clusters observed at monoisotopic m/z 169 and m/z 1345.

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PXRD data of UA showing parallel β-sheet structures. (A) X-ray diffraction of UA fibrils (red line) and the fitted pattern (blue line) according to the reported structure. (B) Parallel β-sheet-like layer secondary structure of UA fibrils. (C) Crystal structure of UA by a parallel set of head-to-tail H-bonded chains along the b axes.

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Uric acid assembles into fibrils at low concentrations. Representative TEM images of UA fibrils at (A) 0.5 mg/mL, (B) 1.0 mg/mL, and (C) 1.5 mg/mL. Statistical characterization of fibril widths observed at 0.5 mg/mL. (D) Histogram with kernel density estimation (KDE), which provides a smoothed curve showing the overall distribution shape; multiple peaks suggest possible subpopulations. (E) Empirical cumulative distribution function (ECDF), showing the proportion of fibrils below each measured width and providing a complete view of distribution spread for cross-condition comparisons. (F) Normal Q–Q plot comparing observed quantiles with those expected for a normal distribution; data falling near the diagonal indicate approximate normality, while systematic curvature or tail deviations reveal skewness or heavy tails.

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Uric acid reduces HEK293 cell viability through fibrils and crystals. (A) MTT viability assay in HEK293 cells shows a significant reduction in viability in unfractionated media containing UA fibrils and crystals. Separation of fibrils (supernatant) from crystals (pellet) by centrifugation also reduced viability, though to a lesser extent. Data represent one representative experiment out of three biological repeats; error bars denote SD. Student’s t test, **p < 0.01, *p < 0.05. (B) Representative light microscopy images of media alone, unfractionated UA (1 mg/mL), supernatant, and pellet fractions. Crystals are evident in the unfractionated and pellet fractions. Scale bar: 1 mm. (C) Light microscopy images of media alone, UA (1 mg/mL), and UA (2 mg/mL) after 24, 48, and 72 h (hrs) of incubation at room temperature. Crystals are evident in UA samples, and their abundance markedly increases over time. Scale bar: 1 mm.

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Allopurinol exhibits a unique mode of action. It may remodel the assembly of UA clusters, leading to the observation of new features in the ATDs. Mass spectrum of (A) UA and (B) UA with allopurinol in positive mode showing the presence of allopurinol without significant changes in the intensities of small UA cluster peaks. The inset shows a comparison of IMS-MS data for pure UA and UA with allopurinol. The appearance of new IM features at longer arrival times suggests the formation of new structure types that are otherwise suppressed in pure UA. (C) For comparison, EGCG disrupts the formation of UA clusters.

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(A) Allopurinol exhibits a distinct mode of action, potentially weakening (“redissolving”) UA clusters and leading to the emergence of unique species in the ATDs of UA clusters. These features arise from the post-IM dissociation of large UA clusters, in which the preferred dissociation products depend on initial cluster size but generally favor large clusters because of their initial abundance. This is evident in the drift times of the red-marked features, which remain unchanged with increasing cluster size, suggesting they originate from post-IM dissociation of the same clusters. In contrast, the drift times of early UA cluster features (gray) increase as cluster size grows, following expected trends. Additionally, in the ATD of n/z = 1/1, the protomer of protonated UA (green) appears alongside features corresponding to post-IM dissociated clusters (blue). (B–C) The increase in intensity of features at long arrival times (40–50 ms) in the presence of allopurinol is statistically significant. Examples of the features at 47.76 and 50.45 ms are shown for the ATDs with a nominal n/z ratio of 10/1. *** p < 0.001, n = 5–12.

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Allopurinol and EGCG differentially remodel UA fibrillization and crystallization. Representative transmission electron microscopy (TEM) images (A–D) and optical microscopy images (E–H) show the effects of EGCG and allopurinol on UA aggregation. Statistical analyses of UA fibrils observed in the pure samples and in the presence of allopurinol (I–J). Panels A and E show UA fibrils and crystals formed under control conditions (1 mg/mL UA in PBS); panels B and F show UA with EGCG (1 mg/mL UA + EGCG); panels C and G show UA with DMSO vehicle control (0.005% DMSO); and panels D and H show UA with allopurinol (1 mg/mL UA + allopurinol). TEM images reveal that EGCG-treated samples produce short, clumped fibrils, whereas allopurinol-treated samples yield longer, coarser fibrils. Optical images show that EGCG suppresses crystal formation, leading to fewer but larger, more transparent crystals, whereas allopurinol promotes the formation of flat, elongated crystals. Scale bars: 500 nm (TEM), 50 μm (optical microscopy). (I) Violin and box plots of fibril widths. Distribution of fibril widths measured from three independent data sets of pure UA fibrils and three independent data sets of UA fibrils in the presence of allopurinol. Violin plots depict the full distribution of widths, while overlaid box plots indicate the interquartile range and median. (J) ECDF curves for all six data sets, showing the cumulative fraction of fibrils below a given width. A consistent rightward shift is observed for UA fibrils in the presence of allopurinol relative to pure UA fibrils, indicating thicker fibrils across the entire distribution.