Bioguided Isolation of Cyclopenin Analogues as Potential SARS-CoV-2 Mpro Inhibitors from Penicillium citrinum TDPEF34.

SARS-CoV-2 virus mutations might increase its virulence, and thus the severity and duration of the ongoing pandemic. Global drug discovery campaigns have successfully developed several vaccines to reduce the number of infections by the virus. However, finding a small molecule pharmaceutical that is effective in inhibiting SARS-CoV-2 remains a challenge. Natural products are the origin of many currently used pharmaceuticals and, for this reason, a library of in-house fungal extracts were screened to assess their potential to inhibit the main viral protease Mpro in vitro. The extract of Penicillium citrinum, TDPEF34, showed potential inhibition and was further analysed to identify potential Mpro inhibitors. Following bio-guided isolation, a series of benzodiazepine alkaloids cyclopenins with good-to-moderate activity against SARS-CoV-2 Mpro were identified. The mode of enzyme inhibition of these compounds was predicted by docking and molecular dynamic simulation. Compounds 1 (isolated as two conformers of S- and R-isomers), 2, and 4 were found to have promising in vitro inhibitory activity towards Mpro, with an IC50 values range of 0.36-0.89 µM comparable to the positive control GC376. The in silico investigation revealed compounds to achieve stable binding with the enzyme active site through multiple H-bonding and hydrophobic interactions. Additionally, the isolated compounds showed very good drug-likeness and ADMET properties. Our findings could be utilized in further in vitro and in vivo investigations to produce anti-SARS-CoV-2 drug candidates. These findings also provide critical structural information that could be used in the future for designing potent Mpro inhibitors.

GABAA receptor agonist cinazepam and its active metabolite 3-hydroxyphenazepam act differently at the presynaptic site.

Cinazepam C19H14BrClN2O5, ("LevanaⓇ ІC") a partial GABAA receptor agonist, and its active metabolite 3-hydroxyphenazepam C15H10BrClN2O2 were comparatively assessed in vitro using nerve terminals isolated from rat cortex (synaptosomes). At the presynaptic site, cinazepam (100 and 200 µM) facilitated synaptosomal transporter-mediated [3H]GABA uptake by enhancing both the initial rate and accumulation, and decreased the ambient level and transporter-mediated release of [3H]GABA. Whereas, 3-hydroxyphenazepam decreased the uptake and did not change the ambient synaptosomal level and transporter-mediated release of [3H]GABA. To exclude GABA transporter influence, NO-711, the transporter blocker, was applied and it was found that exocytotic release of [3H]GABA decreased, whereas tonic release of [3H]GABA was not changed in the presence of both cinazepam or 3-hydroxyphenazepam after treatment of synaptosomes with NO-711. In fluorimetric studies using potential- and pH-sensitive dyes rhodamine 6G and acridine orange, respectively, it was found that cinazepam hyperpolarized the synaptosomal plasma membrane, and increased synaptic vesicle acidification, whereas, 3-hydroxyphenazepam demonstrated opposite effects on these parameters. Therefore, action of cinazepam and its active metabolite 3-hydroxyphenazepam on GABAergic neurotransmission was different. Therapeutic effects of cinazepam can be associated with its ability to hyperpolarize the plasma membrane, to increase synaptic vesicle acidification and capacity of its active metabolite 3-hydroxyphenazepam to inhibit GABA transporter functioning.