<|endoftext|>666]\]. Briefly, human fibroblasts were plated in 60-mm dishes and were treated for 24 h with a concentration of 5 μM for each compound and 1 μg/mL and 10 μg/mL of concentration for each extract. After the treatment cells were lysed with a buffer suitable for the isolation of 26S proteasome (0.2% Nonidet P-40, 5 mM ATP, 10% glycerol, 20 mM KCl, 1 mM EDTA, 1 mM dithiothreitol and 20 mM Tris, pH 7.6). Lysates were cleared with centrifugation at 19,000g (4 °C) and 20 μg of proteins were immediately used to determine the main proteasome proteolytic activities \[chymotrypsin-like (CT-L/LLVY) and caspase-like (C-L/LLE)\]. Activities were assayed by recording the hydrolysis of the fluorogenic peptides Suc--Leu--Leu--Val--Tyr--AMC and Z-Leu--Leu--Glu--AMC (Enzo Life Sciences), at 37 °C for 30 min. The fluorescence was measured at a VersaFluorTM Fluorometer System (Bio-Rad laboratories) at excitation and emission wavelengths of 350 and 440 nm, respectively. Each sample was prepared in duplicate.
3.8. Cytotoxicity {#sec3dot8-molecules-25-00666}
-----------------
Cytotoxicity was evaluated on A2058 and HepG2 cell lines by the ������ method, as well as on CCD25sk cell line by the HOECHST assay, following a previously described process \[[@B57-molecules-25-00666],[@B58-molecules-25-00666],[@B59-molecules-25-00666]\].
3.9. Molecular Simulations {#sec3dot9-molecules-25-00666}
--------------------------
Molecular Docking Simulations were run using the crystal structure of pro-cathepsin B (PDBid: 2PBH) and pro-cathepsin L (PDBid: 1CS8). Enzymes we prepared based on the Protein Preparation Wizard as implemented on Maestro 11.5 (Schrödinger, LLC, New York, NY, 2018). Induced fit algorithm was utilized for Molecular Docking as implemented on Maestro 11.5. The active site was defined using the coordinates of residues HisP^28^, LysP^39^ and ArgP^40^ which have been identified as crucial for GAG binding, by mutational analysis \[[@B50-molecules-25-00666]\]. Ligands were designed using Maestro 11.5 and prepared by using the Ligand Preparation workflow as implemented on Maestro 11.5 (Schrödinger, LLC, NY, USA).
3.10. Statistical Analysis {#sec3dot10-molecules-25-00666}
--------------------------
Experiments were performed in duplicates or triplicates as indicated in Figure legends. For statistical analyses MS Excel and the Statistical Package for Social Sciences (IBM SPSS; version 19.0 for Windows, Armonk, NY, USA) were used. Statistical significance was evaluated using one-way analysis of variance (ANOVA). Data points correspond to the mean of the independent experiments and error bars denote standard deviation (SD).
4. Conclusions {#sec4-molecules-25-00666}
==============
Fungal extracts have been used for a long time as a source of antioxidant compounds, as well as for their ability to regulate the immune system, inhibiting tumor cell growth and exhibiting anti-aging effects \[[@B60-molecules-25-00666]\]. Herein, we show that benzoic acid derivatives (**1**--**4**) isolated from *B. adusta* enhance the activity of the two main cell protein degradation systems, namely ALP and UPP (with the exception of compound **2** for UPP) and especially the activity of cathepsins B and L. Our in silico studies support the fact that all the isolated compounds are possible binders of both procathepsin B and L, and thus suggest a likely direct effect on cathepsins activity. Deregulation of both pathways (UPP and ALP) is often associated to aging and age-related diseases. Thus, the development of novel agents able to suppress the age-related decline of UPP and/or ALP activity could be considered as promising candidates for novel formulations with anti-aging properties.
**Sample Availability:** Samples of the compounds **1**--**4** are available from the authors.
######
Click here for additional data file.
The following are available online, Figure S1: HPLC chromatogram of the Hex (A), EtOAc (B), EtOAc-LL (C) and MeOH Xad4 extracts of the strain CF-0902983 at 210 nm., Figure S2: ^1^H-NMR spectrum