An efficient treatment against a COVID-19 disease, caused by the novel coronavirus SARS-CoV-2 (CoV2), remains a challenge. The papain-like protease (PLpro) from the human coronavirus is a protease that plays a critical role in virus replication. Moreover, CoV2 uses this enzyme to modulate the host’s immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. We used Approximate Bayesian Computation tools, molecular modelling and enzyme activity studies to identify highly active inhibitors of the PLpro. We discovered organoselenium compounds, ebselen and its structural analogues, as a novel approach for inhibiting the activity of PLproCoV2. Furthermore, we identified, for the first time, inhibitors of PLproCoV2 showing potency in the nanomolar range. Moreover, we found a difference between PLpro from SARS and CoV2 that can be correlated with the diverse dynamics of their replication, and, putatively to disease progression.

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Since December 2019 a novel a coronavirus identified as SARS-CoV-2 or COV2 has been spreading around the world. On the 16th of May around 4.5 million people got infected and over 300,000 died due to the infection of COV2. The effective treatment remains a challenge. Targeted therapeutics are still under investigation. The papain-like protease (PL Pro ) from the human SARS-CoV-2 coronavirus is a cysteine protease that plays a critical role in virus replication. Its activity is required to process the viral polyprotein into functional, mature subunits. Moreover, COV2 uses this enzyme to modulate the host's immune system to its own benefit. Therefore, it represents a highly promising target for the development of antiviral drugs. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity in mammalian cells and cytotoxicity in lower organisms, is a highly active inhibitor of PL Pro CoV2. We proved that ebselen is a covalent, fast-binding inhibitor of PL Pro CoV2 exhibiting a low micromolar potency. Furthermore, we identified a difference between PL Pro from SARS-CoV-1 (the corona virus which caused the 2002-2004 outbreak, SARS) and SARS-CoV-2 that allows to explain the difference in dynamics of the replication, and, thus, the disease progression. Namely, we present that they show differences in the binding affinity of substrates that we observed through kinetics and molecular docking studies. Using a novel Approximate Bayesian Computation method we were able to find kinetic constants for both enzymes. Molecular modeling study on the structure of the active site and binding mode of the ebselen with SARS and COV2 showed also significant differences that could explain our observation that ebselen is less active and slower bounding with SARS than COV2. In conclusion, we show that ebselen inhibits the activity of the essential viral enzyme papain-like protease (PLpro) from SARS-COV-2 in low micromolar range.

A series of hydrazide-hydrazones 1–3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24–674 µM with the predicted and desirable competitive type of inhibition. The structure–activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8–233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives’ interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.

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A library of phosphonic acid analogs of phenylalanine substituted with fluorine, chlorine and trifluoromethyl moieties on the aromatic ring was synthesized and evaluated for inhibitory activity against human (hAPN) and porcine (pAPN) aminopeptidases. Fluorogenic screening indicated that these analogs are micromolar or submicromolar inhibitors, both enzymes being more active against hAPN. In order to better understand the mode of the action of the most active compounds, molecular modeling was used. It confirmed that aminophosphonic portion of the enzyme is bound nearly identically in the case of all the studied compounds, whereas the difference in activity results from the placement of aromatic side chain of an inhibitor. Interestingly, both enantiomers of the individual compounds are usually bound quite similarly.