Protocetraric and Salazinic Acids as Potential Inhibitors of SARS-CoV-2 3CL Protease: Biochemical, Cytotoxic, and Computational Characterization of Depsidones as Slow-Binding Inactivators.

The study investigated the inhibitory activity of protocetraric and salazinic acids against SARS-CoV-2 3CLpro. 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 3CLpro. The compounds are slow-binding inactivators of 3CLpro with a Ki of 3.95 µM and 3.77 µM for protocetraric and salazinic acid, respectively, and inhibitory efficiency kinact/Ki 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 Ki 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 3CLpro. 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.

Structurally Related Liposomes Containing N-Oxide Surfactants: Physicochemical Properties and Evaluation of Antimicrobial Activity in Combination with Therapeutically Available Antibiotics.

Although liposomes are largely investigated as drug delivery systems, they can also exert a pharmacological activity if devoid of an active principle as a function of their composition. Specifically, charged liposomes can electrostatically interact with bacterial cells and, in some cases, induce bacterial cell death. Moreover, they also show a high affinity toward bacterial biofilms. We investigated the physicochemical and antimicrobial properties of liposomes formulated with a natural phospholipid and four synthetic l-prolinol-derived surfactants at 9/1 and 8/2 molar ratios. The synthetic components differ in the nature of the polar headgroup (quaternary ammonium salt or N-oxide) and/or the length of the alkyl chain (14 or 16 methylenes). These differences allowed us to investigate the effect of the molecular structure of liposome components on the properties of the aggregates and their ability to interact with bacterial cells. The antimicrobial properties of the different formulations were assessed against Gram-negative and Gram-positive bacteria and fungi. Drug-drug interactions with four classes of available clinical antibiotics were evaluated against Staphylococcus spp. The target of each class of antibiotics plays a pivotal role in exerting a synergistic effect. Our results highlight that the liposomal formulations with an N-oxide moiety are required for the antibacterial activity against Gram-positive bacteria. In particular, we observed a synergism between oxacillin and liposomes containing 20 molar percentage of N-oxide surfactants onStaphylococcus haemolyticus, Staphylococcus epidermidis, andStaphylococcus aureus. In the case of liposomes containing 20 molar percentage of the N-oxide surfactant with 14 carbon atoms in the alkyl chain for S. epidermidis, the minimum inhibitory concentration was 0.125 µg/mL, well below the breakpoint value of the antibiotic.

New and simplified method for drug combination studies by checkerboard assay.

To evaluate the effect of two combined antimicrobial drugs, one method currently in use is the checkerboard assay in a 96-well microplate, which gives a good in vivo estimation of the drug-drug combination effect. Appropriate and consolidated methods are described in numerous scientific publications which are, however, in turn, laborious and time-spending, specifically for the setting of the 96-well microplate preparation. Each drug of every combination must be prepared and dispensed individually in several steps, often limiting its use in terms of consumed materials and working time. In our method, the strengths of the previous consolidated techniques are kept, although the toughness and the execution time are drastically reduced. No special laboratory apparatuses are needed. All the procedures of our method can be referred to the CLSI or EUCAST guideline. The method provides few main steps, which can be summarised in:•Preparation of the microorganism inoculum and three concentrations of antimicrobial drugs.•Easy dispensing of all reagents into the microplates with a multichannel pipette.•Evaluation of the microorganism optical density (OD) by a microplate reader, and calculation of growth percentage for each of the 77 combinations.

Cyclic and Acyclic Amine Oxide Alkyl Derivatives as Potential Adjuvants in Antimicrobial Chemotherapy against Methicillin-Resistant Staphylococcus aureus with an MDR Profile.

The dramatic intensification of antimicrobial resistance occurrence in pathogenic bacteria concerns the global community. The revitalisation of inactive antibiotics is, at present, the only way to go through this health system crisis and the use of antimicrobial adjuvants is turning out the most promising approach. Due to their low toxicity, eco-friendly characteristics and antimicrobial activity, amphoteric surfactants are good candidates. This study investigated the adjuvant potentialities of commercial acyclic and newly cyclic N-oxide surfactants combined with therapeutically available antibiotics against MDR methicillin-resistant Staphylococcus aureus (MRSA). The safety profile of the new cyclic compounds, compared to commercial surfactants, was preliminarily assessed, evaluating the cytotoxicity on human peripheral mononuclear blood cells and the haemolysis in human red blood cells. The compounds show an efficacious antimicrobial activity strongly related to the length of the carbon atom chain. In drug-drug interaction assays, all surfactants act synergistically, restoring sensitivity to oxacillin in MRSA, with dodecyl acyclic and cyclic derivatives being the most effective. After evaluating the cytotoxicity and considering the antimicrobial action, the most promising compound is the L-prolinol amine-oxide C12NOX. These findings suggest that the combination of antibiotics with amphoteric surfactants is a valuable therapeutic option for topical infections sustained by multidrug-resistant S. aureus.