It has been almost four years since the beginning of the COVID-19 pandemic. The COVID-19 pandemic is caused by SARS-CoV-2, a β-coronavirus that leads to respiratory infections with disease severities ranging from respiratory symptoms to death [1]. SARS-CoV-2 is highly contagious and can be transmitted through the airway or by contact with a virus-contaminated surface. Unlike SARS-CoV, a large percentage of the SARS-CoV-2 infected patients are asymptomatic and continue to shed the virus, which makes it challenging to contact tracing and contain the spread of the virus [1]. One solution to help curb the pandemic is coupling rapid testing with oral bioavailable antivirals [2,3]. Ideally, people in the high-risk group with a positive COVID-19 test will have immediate access to COVID-19 drugs at the onset of symptoms or being asymptomatic, so they can take the pills at home to prevent the disease from progressing to severe infections, thereby alleviating the burden of hospitals and healthcare systems.

Currently, there are three FDA-approved COVID-19 antivirals, including two viral RNA-dependent RNA polymerase (RdRp) inhibitors, remdesivir and molnupiravir, and the viral main protease (Mpro) (also called 3CL protease, 3CLpro) inhibitor nirmatrelvir [4,5]. Remdesivir is administrated by intravenous injection and is reserved for hospitalized patients with severe infections. Although remdesivir is effective in lowering the risk of hospitalization and death when given prophylactically [6], the therapeutic benefit of remdesivir in delayed treatment is controversial, as shown by the results of multiple clinical trials [[7], [8], [9]]. Molnupiravir is a mutagen that inhibits SARS-CoV-2 replication by ramping the virus's mutation rate [10]. One potential concern for molnupiravir is that it might similarly cause mutations in the host, which is supported by the results from the mammalian cell culture experiment [11,12]. Paxlovid combines the Mpro inhibitor nirmatrelvir and the CYP-3A4 inhibitor ritonavir. Nirmatrelvir is a rationally designed reversible covalent Mpro inhibitor with a nitrile warhead [13]. One limitation of Paxlovid is the potential drug-drug interactions [12], and the development of a newer generation of Mpro inhibitors without the co-administration of ritonavir is actively pursued [14]. In addition, nirmatrelvir drug-resistant mutants have been identified in cell culture under drug selection pressure as well as in Paxlovid-treated patients [[15], [16], [17], [18], [19], [20]].

Although the SARS-CoV-2 RdRp has the proof-reading function, SARS-CoV-2 continues to mutate at an alarming rate, and several variants of concern (VOC) and variants of interest (VOI) have emerged since the pandemic. Leveraging the lessons learned from combating HIV and HCV infections [21], additional antiviral drugs with novel mechanisms of action are needed to manage COVID-19 infection and drug resistance. The viral papain-like protease (PLpro) is a validated antiviral drug target [22,23]. PLpro mediates the cleavage of viral polyproteins pp1a and pp1ab during viral replication. In addition, PLpro antagonizes the host immune response by cleaving ubiquitin and ISG15 from host proteins [24]. SARS-CoV-2 PLpro shares 82.9% sequence identify with SARS-CoV PLpro. As such, most SARS-CoV-2 PLpro inhibitors are developed based on previously reported SARS-CoV-2 PLpro inhibitors [22,23,25]. One prominent PLpro inhibitor is the naphthalene-containing compound GRL0617 with the amide linker (Fig. 1) [26]. We recently reported the discovery of potent PLpro inhibitors Jun9722 and Jun9843 containing the amine linker, and both showed improved enzymatic inhibition and antiviral activity than GRL0617 (Fig. 1) [27]. Additional notable PLpro inhibitors include the 2-phenylthiophene series, such as XR8-23, and the covalent PLpro inhibitors, such as compound 7 (Fig. 1) [28,29]. For additional examples, please refer to recent reviews and original articles [22,23,25,[30], [31], [32], [33]]. In this study, we report the X-ray crystal structures of PLpro with two inhibitors containing the amine linker and the structure-activity relationship studies of PLpro inhibitors. Several compounds have been identified with potent enzymatic inhibition (IC50 < 1 μM), antiviral activity (EC50 ≤ 6 μM), and favorable stability in mouse microsomes (T1/2 > 50 min). The mechanism of action was characterized by enzymatic assay and thermal shift assay.