4 methylumbelliferyl 4mu

In vitro tests were performed to validate the computational methods. The chitinolytic activity was performed using 4-methylumbelliferyl-β-d-N,N′,N″-triacetylchitotrioside (Sigma-Aldrich Co.) as substrate. A standard curve was constructed using 4-methylumbelliferyl (4MU) (Sigma-Aldrich Co.). Assays were performed in 96-well coated microplates (Greiner CELLSTAR^® Sigma-Aldrich Co.) and consisted of 95 µL McIlvaine buffer pH 6.0, 5 µL of the substrate (0.8 mM), 5 µL Lysing Enzymes from T. harzianum (200 mg/mL in PBS 1 x; Sigma-Aldrich Co.), and 5 µL of the evaluated compound. The reaction was incubated at 37 °C for 15 min. Fluorescence was measured at 355 nm excitation and 460 nm emission using SpectraMax M3. The inhibitory assays were performed with increasing concentrations of the compounds evaluated (compounds 5, 7, and 9) diluted in DMSO. The different concentrations of each compound were used according to the indicated solubility^{28 (link)}. The final concentrations of compound 5 in the assay were: 0.568 ng/µL, 1.13 ng/µL, and 2.27 ng/µL; compound 7 final concentrations were: 5.68 ng/µL, 11.3 ng/µL, and 22.7 ng/µL; and compound 9 final concentrations were: 2.84 ng/µL, 5.68 ng/µL, and 11.3 ng/µL. Quantification of the samples was based on the relative fluorescence units (RFU) using the previously established standard curve^{12 (link),52 (link)}.

Enzymatic activities were measured as previously described^{64 (link)}. Briefly, cells were lysed using 1% Triton X-100, 10% glycerol, 20 mM Tris-HCl pH 7.5 and 150 mM NaCl supplemented with a protease inhibitor cocktail (Roche). Cellular lysates were subjected to three cycles of freeze and thaw and centrifuged for 15 min at 15,000 g at 4 °C. Protein content of cell lysate was quantified using the Micro BCA^TM Protein Assay Kit (Thermo Scientific). Enzymatic reactions were performed in 48-well plates with 20 μl of protein extracts and 100 μl of reaction buffer (100 mM sodium acetate, pH 4.8 containing 10 mM of enzyme substrate) for 1 h at 37 °C. Substrates for β-gluc and for β-hexosaminidase (β-hex) were 4-methylumbelliferyl-β-d-glucuronide and 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (both from Sigma-Aldrich), respectively. Enzymatic reactions were stopped by adding 500 μl of 200 mM sodium carbonate pH 10, and fluorescence intensity was measured at 450 nm using Tecan Infinite® M200 Microplate Reader. Standard curves were performed using serial dilutions of 4-methylumbelliferyl (4-MU; Sigma-Aldrich). Enzyme activity is expressed in nmol 4-MU/μg of protein/h.