Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

One Pot Synthesis of Micromolar BACE-1 Inhibitors Based on the Dihydropyrimidinone Scaffold and Their Thia and Imino Analogues.

A library of dihydropyrimidinones was synthesized via a "one-pot" three component Biginelli reaction using different aldehydes in combination with ß-dicarbonyl compounds and urea. Selected 2-thiooxo and 2-imino analogs were also obtained with the Biginelli reaction from thiourea and guanidine hydrochloride, respectively. The products were screened in vitro for their ß-secretase inhibitory activity. The majority of the compounds resulted to be active, with IC50 in the range 100 nM-50 µM.

In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells.

BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.