The COVID-19, caused by the SARS-CoV-2 virus, was first reported in December 2019 (Huang et al., 2020) and rapidly spread globally. By October 2024, the World Health Organization (WHO) had reported over 770 million confirmed COVID-19 cases and more than 6.9 million deaths globally. A recent WHO report indicated an increasing trend in SARS-CoV-2 cases, with 295,000 cases and 5200 deaths reported in the 28 days leading up to September 22, 2024. SARS-CoV-2 is a highly contagious, single-stranded, positive-sense RNA virus that affects humans (Machhi et al., 2020). While COVID-19 remains ongoing, the virus continues to evolve. New, more virulent or vaccine-resistant variants could trigger future waves or pandemics if global immunity wanes.

In a recent address, WHO Director-General Dr. Tedros Adhanom Ghebreyesus emphasized that "the next pandemic will not wait," cautioning that it could be triggered by various pathogens, including coronaviruses, which have high lethality and pandemic potential if they mutate to spread efficiently among humans (WHO, 2024). The virus is deemed high-risk due to its potential for rapid transmission, high mortality rates, or both, making them the focus of global research and preparedness efforts.

The SARS-CoV-2 structural proteins S, E, and M form the viral envelope, while N encapsulates the RNA genome (Rastogi et al., 2020). ORF1a and ORF1b encode 16 non-structural proteins (nsp1–nsp16), which include RNA-dependent RNA polymerase (RdRp) crucial for viral replication, the main protease (Mpro), and papain-like protease (PLpro) for polyprotein cleavage (Romano et al., 2020). Mpro, also known as nsp5, and PLpro play distinct roles in SARS-CoV-2 polyprotein processing. PLpro is within the multidomain nsp3 protein and cleaves three conserved sites to release nsp1, nsp2, and nsp3, while Mpro cleaves 11 conserved sites to generate nsp4–nsp16 (Fan et al., 2004).

PLpro remains a crucial target for antiviral drug development because of its vital role in viral replication and its ability to interfere with the host’s immune response. It achieves this by cleaving the interferon-stimulated gene product-15 (ISG15) modifier protein, which plays a key role in detecting cytosolic viral RNA and triggering the innate immune response (Klemm et al., 2020, Shin et al., 2020) Several inhibitors targeting the papain-like protease (PLpro) of SARS-CoV-2 have been evaluated and reported to exhibit low IC50 or EC50 values, indicating their potential effectiveness in inhibiting viral replication (Calleja et al., 2022, Brewitz et al., 2022, Ma and Jun Wang, 2022).

GRL0617 is a small-molecule inhibitor initially identified as a non-covalent inhibitor of SARS-CoV-1 PLpro, with an IC50 of approximately 0.6 µM (Ratia et al., 2008). It has been shown to block PLpro's deubiquitinating and viral replication activities in vitro. The IC50 of GRL-0617 against SARS-CoV-2 PLpro has been reported to be approximately 2.1 µM (Fu et al., 2021).

Tan et al. (2024) reported highly potent nanomolar inhibitors, which remain part of the GRL0617-derived compound class, with IC50 values ranging from 66.2 to 164.6 nM. The in vivo lead Jun12682 inhibited SARS-CoV-2 and nirmatrelvir-resistant strains (EC50: 0.44–2.02 µM) and improved survival and reducing lung viral loads in a mouse infection model, supporting PLpro inhibitors as promising antiviral candidates.

Ebselen is a selenoorganic compound with low molecular weight, recognized for its powerful antioxidant, anti-inflammatory, anti-atherosclerotic, and cell-protective effects (Siegfried et al., 2022). This compound demonstrated inhibition against Mpro and PLpro of SARS-CoV-2 with the IC50 values of 0.67 and 2.0 μM, respectively (Weglarz-Tomczak et al., 2021), slightly more potent than disulfiram. In a cell-based assay, ebselen demonstrates notable antiviral activity, with an EC50 of 4.67 μM (Jin et al., 2020).

Currently, the PLpro inhibitors mentioned above have been studied extensively in efficacy and toxicity, but need to complete pre-clinical studies. As of now, no drugs specifically targeting SARS-CoV-2 PLpro have been approved by the U.S. Food and Drug Administration (FDA). Most FDA-approved treatments for COVID-19, such as remdesivir (targeting RNA-dependent RNA polymerase), Paxlovid (which includes nirmatrelvir targeting Mpro), and monoclonal antibodies, focus on other viral targets. Nonetheless, PLpro remains a promising target for antiviral drug development.

In this study, we discovered four new PLpro inhibitors through docking studies, which were confirmed by in vitro experiments. This is similar to many studies aginst SARS-CoV-2 (Singh et al., 2022, Singh et al., 2021a, Singh et al., 2021b, Singh and Purohit, 2024, Sharma et al., 2021, Bhardwaj et al., 2021). They exhibited low IC50 values and small dissociation constants (KD), indicating high affinity for PLpro binding. We also evaluated hydrogen bonding and hydrophobic interactions between each inhibitor and PLpro, revealing that each compound interacts with key amino acid residues. These four inhibitors may have the potential to serve as COVID-19 drug candidates, pending further preclinical and clinical evaluations (Michelle et al., 2024, M. Bader et al., 2025).