In a recent study posted to the bioRxiv* preprint server, researchers identified a papain-like protease inhibitor for treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections.

Study: Identification of a Papain-Like Protease Inhibitor with Potential for Repurposing in Combination with an Mpro Protease Inhibitor for Treatment of SARS-CoV-2. Image Credit: MIA Studio/Shutterstock

SARS-CoV-2 important protease (Mpro) inhibitors were the first approach in the event of treatment of coronavirus disease 2019 (COVID-19) which led to the authorization of nirmatrelvir (Paxlovid) for emergency use. Mpro has been a promising goal for novel drugs on account of its dissimilarity to human proteases and its importance during infections. Nevertheless, most research has only focused on the event of single Mpro inhibitors.

Concerning the study

In the current study, researchers identified a papain-like protease (PLpro) which could possibly be potentially used together with a Mpro inhibitor in a protease inhibitor cocktail for SARS-CoV-2 treatment.

The team assessed small molecules from a custom clinical library comprising 58 compounds that were collected from different sources based on their inhibitory activity against the PLpro enzyme. The effectiveness of the inhibitors against PLpro was analyzed by estimating the dose-response inhibition of PLpro together with its deISGylase and deubiquitinase activities. Moreover, the team assessed whether PLpro inhibitors could rescue interferon-beta (IFN-β) induction. This was achieved by co-transfecting HEK-293T cells with the PLpro corresponding to the SARS-CoV-2 wild-type strain and an IFN-β luciferase reporter. The co-transfection was further enhanced with polyinosinic:polycytidylic acid (poly I:C) to perform just like the double strand ribonucleic acid (RNA).

The team also assessed whether DDL-701 could possibly be a component of a possible protease inhibitor cocktail and perform effectively against COVID-19 infection of the central nervous system (CNS) by conducting pharmacokinetic study in mice. The mice were treated with DDL-701 or DDL-750 alone or together, followed by assessment of concentrations within the plasma and the brain.

Results

The study results showed that screening of small molecules resulted in 4 hits that showed PLpro inhibitory activity of greater than 80%, namely eltrombopag (DDL-701), a cysteinyl leukotriene receptor antagonist called zafirlukast (DDL-715), thrombopoietin receptor agonist-1 (TPO-1 agonist, DDL-713), and GRL-0617. Moreover, an antipsychotic drug called fluspirilene and a leukotriene receptor antagonist called montelukast increased PLpro activity.

Validation of DDL-701 inhibitory activity against PLpro showed that the IC50 value for the dose-response inhibition of PLpro was 13 μM, the deubiquitinase activity was 21 μM and the deISGylase activity was 8 μM. Furthermore, the dose-response curves generated for DDL-713, DDL-715, and GRL-0617 revealed weaker inhibitory potency as in comparison with that of DDL-701. The team also noted that the PLpro inhibition by DDL-701 was not affected when employed inside a protease assay together with DDL-750. Moreover, the Mpro inhibitory activity of DDL-750 was sustained even within the presence of DDL-701 while DDL-701 itself displayed no Mpro inhibitory activity.   

The team also found that the PLpro expression reduced induction of IFN-β by greater than 50% while treatment with DDL-701 substantially rescued IFN-β induction. Nevertheless, DDL-715 and DDL-750 didn’t rescue IFN-β induction. PLpro activity has also been found to antagonize nuclear factor kappa B (NF-κB) signaling but there was no evidence of NF-κB rescue by any of the tested compounds.

Assessment of DDL-701 or DDL-750-treated mice showed that the concentrations of DDL-701 and DDL-750 were almost  μM and roughly 0.18 μM after two hours of treatment, respectively. Nevertheless, DDL-701 displayed five times higher penetration within the brain as in comparison with DDL-750. Moreover, DDL-701 and DDL-750 concentrations within the plasma were roughly 14 μM and 0.5 μM, respectively. When used together, the degrees of the 2 drugs were substantially lower within the plasma and the brain.

Docking of DDL-701 with PLpro indicated that neither GRL-0617 nor DDl-701 interacted with the cysteine-111 residue energetic site but were certain to tyrosine-268 which is present outside the tunnel that comprises the cysteine-111 residue. Moreover, DDL-715 and losartan, a PLpro inhibitor, was found to bind the enzyme at tyrosine-268. 

Conclusion

The study findings showed that DDL-701 is an efficient PLpro inhibitor which displayed sustained inhibitory activity even within the presence of nirmatrelvir, an Mpro inhibitor. DDL-701 can also be reported to stimulate plasma levels that might elicit inhibition of PLpro in addition to improve antiviral efficacy. Due to this fact, the researchers imagine that DDL-701 is a possible candidate to be employed together with a Mpro inhibitor for COVID-19 treatment.

*Essential notice

bioRxiv publishes preliminary scientific reports that should not peer-reviewed and, due to this fact, mustn’t be thought to be conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:

• Jesus Campagna, Barbara Jagodzinska, Pablo Alvarez, Constance Yeun, Patricia Spilman, Kathryn M Enquist, Whitaker Cohn, Pavla Fajtov, Anthony J O’Donoghue, Vaithilingaraja Arumugaswami, Melody M.H. Li, Robert Damoiseaux, Varghese John. (2022). Identification of a Papain-Like Protease Inhibitor with Potential for Repurposing in Combination with an Mpro Protease Inhibitor for Treatment of SARS-CoV-2. bioRxiv. doi: https://doi.org/10.1101/2022.07.18.500363 https://www.biorxiv.org/content/10.1101/2022.07.18.500363v1