Ligand fishing approach to explore Amaryllidaceae alkaloids as potential antiviral candidates targeting SARS-CoV-2 Nsp4.

Ligand fishing, also described as affinity-based assay, represents a convenient and efficient approach to separate potential ligands from complex matrixes or chemical libraries. This approach contributes to the identification of lead compounds that can bind to a specific target. In the context of COVID-19, the search for novel therapeutic agents is crucial. Small molecule-based antiviral drugs, such as Amaryllidaceae alkaloids, have been described as potential candidates because they can inhibit RNA viruses. Among various SARS-CoV-2 proteins, Nsp3, Nsp4, and Nsp6 play a crucial role in the pathogenicity of the virus and are attractive targets for developing COVID-19 treatments. These proteins are responsible for the replication/transcription complex (RTC) within double-membrane vesicles (DMVs), and their inhibition disrupts the virus's infectious cycle. Herein, we have successfully expressed and immobilized the SARS-CoV-2 Nsp4 protein on magnetic beads (Nsp4-MBs) and employed a ligand fishing assay to screen a collection of ten Amaryllidaceae-based alkaloids and applied to Hippeastrum aulicum extract. Remarkably, four out of ten alkaloids, namely 2-α-7-dimethoxyhomolycorine (6), haemanthamine (5), albomaculine (8), and tazettine (9), exhibited selective affinities for Nsp4. Albomaculine (8) and haemanthamine (5) were also identified from extract by the affinity assay. These findings highlight the potential of these alkaloids as model compounds for future drug discovery studies aimed at developing therapeutic interventions against SARS-CoV-2 infections.

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail.

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

Functional characterization of GH7 endo-1,4-ß-glucanase from Aspergillus fumigatus and its potential industrial application.

A gene encoding an endo-1,4-ß-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residues 424-460 and molecular mass of 52 kDa, as estimated by SDS-PAGE. This enzyme was optimally active at pH and temperatures ranging from 4.5 to 5.5 and from 40 to 60 °C, respectively. Mn2+ addition significantly enhanced the Af-EGL7 cellulase activity by 233%, whereas SDS addition fully inhibited this activity. Higher activity was observed toward ß-glucan than toward xyloglucan and CM-Cellulose, suggesting that the enzyme corresponds to a ß-1,3-1,4-glucanase. qRT-PCR in different culture media helped to establish the time-course expression profile. Different polysaccharides induced the gene Af-egl7 in a time-dependent manner; in the particular case of the substrate sugarcane exploded bagasse (SEB), Af-egl7 was induced 2500-fold. Upon addition to a commercial cellulase cocktail, Af-EGL7 significantly improved SEB saccharification, which suggested that the enzyme Af-EGL7 had great potential to hydrolyze complex biomass. From a biotechnological point of view, A. fumigatus Af-EGL7 is a promising candidate to enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing.