Enzyme Assays

The oxygen reactivity of PMOs was measured by a time resolved quantification of H₂O₂ formation in 96-well plates (total volume of 200 μL) using a Perkin Elmer EnSpire Multimode plate reader. All reactions were performed in 100 mM sodium phosphate buffer, pH 6.0 at 22°C. Based on preliminary studies ascorbate and CDH were used in concentrations of 30 μM and 0.3 μM (0.025 mg mL^-1), respectively to prevent a limitation in the PMO reduction step. As electron donor for CDH 500 μM cellobiose was used. When ascorbate was used as reductant, it was added to a final concentration of 30 μM and enzyme assays were started by mixing 20 μL of the respective PMO with 180 μL of the ready-made assay solution containing 30 μM ascorbate, 50 μM Amplex Red and 7.14 U mL^-1 peroxidase in 96-well plate wells. In reference experiments without PMO the background signal (H₂O₂ production by CDH) was measured and subtracted from the assays. When CDH was used as reductant, the PMO assays were started by mixing 20 μL of sample solution and 20 μL CDH solution with 160 μL of the reaction mix containing cellobiose, Amplex Red and peroxidase. Initial fluorescence scans of resorufin showed highest signal intensities and lowest interference with matrix compounds when using an excitation wavelength of 569 nm and an emission wavelength of 585 nm for the selected conditions. The stoichiometry of H₂O₂ conversion to resorufin formation is 1:1. By using a high concentration of Amplex Red (50 μM) the linearity of the detection reaction was ensured and the molar ratio of Amplex Red:H₂O₂ was always greater than 50:1
[22 (link)]. The H₂O₂ concentration in the assays was far below 1 μM, which leads to a linear concentration/activity response of horseradish peroxidase, which has a K_M for H₂O₂ of 1.55 μM. The high final activity of horseradish peroxidase (7.14 U mL^-1) assures a fast conversion of the formed H₂O₂ and prevents the final reaction to be rate limiting. Additionally, it prevents the accumulation of H₂O₂, which could have detrimental effects on enzyme stability in the assay. The stability of resorufin fluorescence under these conditions was tested by addition of varying concentrations of hydrogen peroxide (0.1 – 5 μM) to the assay. A stable signal that remained constant throughout the measured period of 45 minutes was observed and maximum signal intensity was reached already during the mixing period before starting the assay. A linear relation between fluorescence and H₂O₂ concentrations in the range of 0.1 – 2 μM H₂O₂ was observed and the slope (28450 counts μmol^-1) was used for the calculation of an enzyme factor to convert the fluorimeters readout (counts min^-1), into enzyme activity. PMO activity was defined as one μmol H₂O₂ generated per minute under the defined assay conditions.

The data set was prepared based on an extensive literature survey taking IC₅₀ values of in-vitro enzyme inhibition assays against XO and HMGR by various secondary metabolites. Based on IC₅₀ values, sixteen plant- and fungus-based secondary metabolites (Tables 1 and 2) were chosen for the ligand-protein docking study. The docking study was performed against commercial drugs such as atorvastatin, simvastatin, lovastatin, and pravastatin for HMGR. On the other hand, commercial drugs such as allopurinol, febuxostat, topiroxostat, and probenecid were used for molecular docking studies with XO. The structures of the ligand molecules and the control drugs of both enzymes were retrieved from the PubChem database [38 (link)] and verified from SciFinder. The structures were retrieved in SDF format and were converted to PDB and MOL2 format using Discovery Studio Visualizer 4.0 software. The structure and complete chemical properties, torsional energy, van der Waals potential energy, electrostatic energy, weight, log P, total polar surface area (TPSA), donor atoms, and acceptor atoms of the ligands were listed (Supplementary Table 4S) by the help of MOE Module [39 (link)].

GLA activities were determined in Fabry mouse tissues using previously described methods (Desnick et al., 1973 (link)). In brief, tissue samples were homogenized in chilled reporter lysis buffer (Promega) and protease inhibitor (Pierce) was added to the lysates. Protein concentrations were determined using the Bio-Rad Colorimetric Protein Assay Kit. 10 μL of tissue lysate was added to an equal volume of 10 mM 4-methylumbelliferyl-α-D-galactopyranoside (Sigma-Aldrich), dissolved in assay buffer (0.2 M citrate, 0.4 M phosphate buffer, pH 4.4), and 0.1 M N-acetylgalactosamine (Sigma Aldrich), the latter to inhibit α-galactosidase B activity (Mayes et al., 1981 (link)). Following a 30 min incubation at 37°C, reactions were terminated by the addition of 480 μL of 0.1 M ethylenediamine, pH 10.3. The amount of 4-methylumbelliferone (4-MU) produced was determined by measuring fluorescence using a Synergy H1 fluorometer (BioTek). Tissue α-Gal A activities were expressed as nmol of 4-MU produced per h per mg of total protein (nmol/h/mg). Measurement of plasma GLA activities in wildtype mice for PK studies was performed as described above with the following modifications: lysates were incubated with 5 mM 4-methylumbelliferyl α-D-galactopyranoside in assay buffer [20 mM citrate, 30 mM sodium phosphate (pH 4.4), 0.1 M N-acetylgalactosamine, and 4 mg/mL BSA], and the reaction was stopped by addition of stop buffer (0.1 M Glycine, 0.1 N NaOH], as previously described (Shen et al., 2016 (link)).
AGA activity was measured with 1 mM L-aspartic acid β-(7- amido-4-methylcoumarin) in 10% SuperBlock and 90% 50 mM Tris-HC (pH 7.5) for 60 min at 37°C, and then adding 100 µL of stop buffer [0.2 M glycine, 0.175 M NaOH (pH 10.6)], as previously described (Mononen et al., 1993 (link)). GUSB enzyme assay was performed using 10 mM 4-methylumbelliferyl-β-D-glucuronide (Merck) in 0.1 M sodium acetate (pH 4.6) at 37°C for 30 min, and reactions were stopped by 0.1 M sodium carbonate (Grubb et al., 2008 (link)). GAA activity assay was performed with 3 mM 4-methylumbelliferyl-a-D-glucopyranoside (Merck) in assay buffer (30 mM sodium citrate, 40 mM sodium phosphate dibasic, pH 4.0) at 37°C for 3 h (Flanagan et al., 2009 (link)). Reactions were stopped by the addition of an equal volume of 0.4 M glycine, pH 10.8. IDS activity assay was performed with 2.5 mM 4-Methylumbelliferyl sulfate potassium salt (Merck) in 50 mM sodium acetate, at 37°C for 4 h (Dean et al., 2006 (link)). Reactions were stopped with glycine carbonate buffer (pH 10.7). Fluorescence was measured by microplate reader with 360/40 nm excitation and 440/30 nm emission filters.