Chemical Chaperones Modulate the Formation of Metabolite Assemblies

Abstract

The formation of amyloid-like structures by metabolites is associated with several inborn errors of metabolism (IEMs). These structures display most of the biological, chemical and physical properties of protein amyloids. However, the molecular interactions underlying the assembly remain elusive, and so far, no modulating therapeutic agents are available for clinical use. Chemical chaperones are known to inhibit protein and peptide amyloid formation and stabilize misfolded enzymes. Here, we provide an in-depth characterization of the inhibitory effect of osmolytes and hydrophobic chemical chaperones on metabolite assemblies, thus extending their functional repertoire. We applied a combined in vivo-in vitro-in silico approach and show their ability to inhibit metabolite amyloid-induced toxicity and reduce cellular amyloid content in yeast. We further used various biophysical techniques demonstrating direct inhibition of adenine self-assembly and alteration of fibril morphology by chemical chaperones. Using a scaffold-based approach, we analyzed the physiochemical properties of various dimethyl sulfoxide derivatives and their role in inhibiting metabolite self-assembly. Lastly, we employed whole-atom molecular dynamics simulations to elucidate the role of hydrogen bonds in osmolyte inhibition. Our results imply a dual mode of action of chemical chaperones as IEMs therapeutics, that could be implemented in the rational design of novel lead-like molecules.

Keywords: adenine; amyloid formation; chemical chaperones; hydrophobic compounds; inborn errors of metabolism; metabolite assemblies; osmolytes.

Figure 1

(A) Illustration of the shared properties of metabolite amyloid-like structures and protein or peptide amyloids: nanoscale fibrillar morphology [33,34,35,36], β-sheet secondary structure [37], amyloid-specific fluorescent dye binding [27,33,34], cytotoxicity and apoptosis induction [27,33,34,35,36,38,39], membrane interaction [40,41,42,43,44], inhibition by polyphenols [27,45], cross-seeding with protein amyloids [36,46], immunogenicity [27,33,36,47], intrinsic fluorescence [48,49], sigmoid growth curve [27], piezoelectricity [50], and inhibition by chemical chaperones (this study). Created with BioRender.com. (B) Molecular structure of DMSO, TMAO, DCA, and UDCA.

Figure 2

Inhibition of adenine toxicity by chemical chaperones in a yeast model. Left panel: Growth curves of aah1Δapt1Δ cells under the indicated conditions, at selected chaperones concentrations (see Supplementary Figure S1 for full data). Cells were grown at 30 °C and the absorbance at OD₆₀₀ was measured over time (see Material and Methods). The results are representative of three independent biological repeats. Right panel: Dose-response heat maps representing the difference in optical density between a chaperone-supplemented culture and the control condition (+ADE) when the control cells reached OD₆₀₀ = 0.6. The dose-response heat maps were generated based on the data presented in Supplementary Figure S1. Green, rescue. Red, toxicity. WT and aah1Δapt1Δ yeast were serially diluted and spotted on synthetic defined (SD) media lacking adenine (−ADE), SD media with adenine supplemented at 2 mg/L as a control for DMSO and TMAO, or with 2 mg/L adenine and 0.1% DMSO as a control for DCA and UDCA (+ADE), and SD with 2 mg/L adenine and the indicated concentration of the chemical chaperone. (A) DMSO; (B) TMAO; (C) DCA; (D) UDCA.

Figure 3

Chemical chaperones inhibit adenine aggregation in the salvage yeast model. (A,B) Flow cytometry analysis of wild type (WT) and aah1Δapt1Δ yeast cells under the indicated conditions following ProteoStat staining. Left: Flow cytometry data. Right: Quantification of ProteoStat positive cells under the indicated conditions. * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001 (Student’s t-test). Values are the average ± s.d. of three independent biological repeats. (A) 2 mg/L adenine (+ADE), 2 mg/L adenine and 1 mM TMAO (+TMAO) or 140 mM DMSO (+DMSO). (B) 2 mg/L adenine containing 0.05% DMSO (+ADE), 2 mg/L adenine containing 0.05% DMSO, 10 µM UDCA (+UDCA) or 10 µM DCA (+DCA). (C,D) Representative confocal microscopy images of WT and aah1Δapt1Δ cells under the same conditions as in (A,B). Scale bar is 5 μm.

Figure 4

Inhibition of adenine fibril formation by different chemical chaperones in vitro. (A–D) Kinetic analysis of adenine aggregation as measured in a ThT binding assay. Adenine was dissolved at 6 mg/mL in PBS and heated to 90 °C to obtain a monomeric solution, followed by the addition of the chemical chaperones at the indicated concentrations (see Materials and Methods). ThT was added to a final concentration of 40 µM, and fluorescence was measured over time with excitation and emission wavelengths of 450 nm and 480 nm, respectively. The results are representative of three independent repeats. (E,F) Representative TEM micrographs of 1 mg/mL adenine in the presence or absence of the indicated chemical chaperone at 1.4 mM DMSO, 0.1 mM TMAO, 50 µM DCA, and 50 µM UDCA. Scale bar is 1 µm. The images are representatives of three double-blinded repeats.

Figure 5

The effect of different DMSO derivatives on adenine self-assembly inhibition. (A) Physicochemical properties of different DMSO derivatives calculated by StarDrop (Optibrium). Oxygen atoms are shown in red; sulfur atoms are shown in yellow. (B) Kinetic analysis of adenine aggregation as measured in a ThT binding assay. Adenine was dissolved at 6 mg/mL in PBS and heated to 90 °C to obtain a monomeric solution, followed by the addition of DMSO or its derivatives at the indicated concentrations (see Supplementary Figure S3). The controls for each compound were prepared as described in the Materials and Methods. ThT was added to a final concentration of 40 µM, and fluorescence was measured over time with excitation and emission wavelengths of 450 nm and 480 nm, respectively. The relative concentration was calculated at an endpoint of 1000 min, and the concentration of the inhibitor was divided by the percentage of inhibition. The relative concentration represents the minimal concentration required to inhibit 1% of adenine fibrilization (relative con. (mM/1% inhibition)) (see material and method section and supplementary Figure S3). ns, not significant; * p < 0.02; ** p < 0.01 (Student’s t-test). Values are the average ± s.d. of three independent biological repeats.

Figure 6

USAFDO-38 inhibits adenine toxicity and self-assembly. (A) Growth curves of aah1Δapt1Δ cells under the indicated conditions. The absorbance at OD₆₀₀ was measured over time. The results are representative of three independent biological repeats. (B) Dose-response heat map representing the difference in optical density between chaperone-supplemented and the control condition (+ADE) when the control cells reached OD₆₀₀ = 0.6. The dose-response heat map was generated based on data shown in the kinetics graphs presented in Supplementary Figure S4A. Green, rescue. Red, toxicity. (C) Kinetic analysis of adenine aggregation as measured in a ThT binding assay. Adenine was dissolved at 6 mg/mL in PBS and heated to 90 °C to obtain a monomeric solution, followed by the addition of USAFDO-38 at the indicated concentrations. The control (ADE) was prepared as described in the Materials and Methods. ThT was added to a final concentration of 40 µM, and fluorescence was measured over time with excitation and emission wavelengths of 450 nm and 480 nm, respectively. The results are representative of three independent repeats. (D) Representative TEM micrographs of 1 mg/mL adenine in the presence or absence of 10 µM USAFDO-38. Scale bar is 2 µm. The images are representatives of three repeats. (E) Distribution of the angles between the normal vectors to the planes of monomers in contact, in the absence and the presence of USAFDO-38, as observed in the MD simulations (Supplementary Figure S4). (F) Representative structures obtained from MD simulations in the presence or absence of USAFDO-38. The MD simulation in the presence of USAFDO-38 is magnified and the area is marked with dashed lines. Oxygen atoms are shown in red; sulfur atoms are shown in yellow. Hydrogen bonds are represented as yellow dashed lines.