Search Results (3)

The ability of Mycobacterium tuberculosis to derive lipids from the host, store them intracellularly, and then break them down into energy requires a battery of serine hydrolases. Serine hydrolases are a large, diverse enzyme family with functional roles in dormant, active, and reactivating mycobacterial cultures. To rapidly measure substrate-dependent shifts in mycobacterial serine hydrolase activity, we combined a robust mycobacterial growth system of nitrogen limitation and variable carbon availability with nimble in-gel fluorogenic enzyme measurements. Using this methodology, we rapidly analyzed a range of ester substrates, identified multiple hydrolases concurrently, observed functional enzyme shifts, and measured global substrate preferences. Within every growth condition, mycobacterial hydrolases displayed the full, dynamic range of upregulated, downregulated, and constitutively active hydrolases independent of the ester substrate. Increasing the alkyl chain length of the ester substrate also allowed visualization of distinct hydrolase activity likely corresponding with lipases most responsible for lipid breakdown. The most robust expression of hydrolase activity was observed under the highest stress growth conditions, reflecting the induction of multiple metabolic pathways scavenging for energy to survive under this high stress. The unique hydrolases present under these high-stress conditions could represent novel drug targets for combination treatment with current front-line therapeutics. Combining diverse fluorogenic esters with in-gel activity measurements provides a rapid, customizable, and sensitive detection method for mycobacterial serine hydrolase activity. Full article

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CL^pro. The kinetic parameters were determined by microtiter plate-reading fluorimeter using a fluorogenic substrate. The cytotoxic activity was tested on murine Sertoli TM4 cells. In silico analysis was performed to ascertain the nature of the binding with the 3CL^pro. The compounds are slow-binding inactivators of 3CL^pro with a K_i of 3.95 μM and 3.77 μM for protocetraric and salazinic acid, respectively, and inhibitory efficiency k_inact/K_i at about 3 × 10⁻⁵ s⁻¹µM⁻¹. The mechanism of inhibition shows that both compounds act as competitive inhibitors with the formation of a stable covalent adduct. The viability assay on epithelial cells revealed that none of them shows cytotoxicity up to 80 μM, which is well below the K_i values. By molecular modelling, we predicted that the catalytic Cys145 makes a nucleophilic attack on the carbonyl carbon of the cyclic ester common to both inhibitors, forming a stably acyl-enzyme complex. The computational and kinetic analyses confirm the formation of a stable acyl-enzyme complex with 3CL^pro. The results obtained enrich the knowledge of the already numerous biological activities exhibited by lichen secondary metabolites, paving the way for developing promising scaffolds for the design of cysteine enzyme inhibitors. Full article

Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration. Full article