PLpro Inhibitors Research Articles

In silico evaluation of S-adenosyl-L-homocysteine analogs as inhibitors of nsp14-viral cap N7 methyltranferase and PLpro of SARS-CoV-2: synthesis, molecular docking, physicochemical data, ADMET and molecular dynamics simulations studies

A series of S-adenosyl-L-homosysteine (SAH) analogs, with modification in the base and sugar moiety, have been designed, synthesized and screened as nsp14 and PLpro inhibitors of severe acute respiratory syndrome corona virus (SARS-CoV-2). The outcomes of ADMET (Adsorption, Distribution, Metabolism, Excretion, and Toxicity) studies demonstrated that the physicochemical properties of all analogs were permissible for development of these SAH analogs as antiviral agents. All molecules were screened against different SARS-CoV-2 targets using molecular docking. The docking results revealed that the SAH analogs interacted well in the active site of nsp14 protein having H-bond interactions with the amino acid residues Arg289, Val290, Asn388, Arg400, Phe401 and π-alkyl interactions with Arg289, Val290 and Phe426 of Nsp14-MTase site. These analogs also formed stable H-bonds with Leu163, Asp165, Arg167, Ser246, Gln270, Tyr274 and Asp303 residues of PLpro proteins and found to be quite stable complexes therefore behaved as probable nsp14 and PLpro inhibitors. Interestingly, analog 3 showed significant in silico activity against the nsp14 N7 methyltransferase of SARS-CoV-2. The molecular dynamics (MD) and post-MD results of analog 3 unambiguously established the higher stability of the nsp14 (N7 MTase):3 complex and also indicated its behavior as probable nsp14 inhibitor like the reference sinefungin. The docking and MD simulations studies also suggested that sinefungin did act as SARS-CoV-2 PLpro inhibitor as well. This study’s findings not only underscore the efficacy of the designed SAH analogs as potent inhibitors against crucial SARS-CoV-2 proteins but also pinpoint analog 3 as a particularly promising candidate. All the study provides valuable insights, paving the way for potential advancements in antiviral drug development against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Structure-based design of SARS-CoV-2 papain-like protease inhibitors

The COVID-19 pandemic is caused by SARS-CoV-2, an RNA virus with high transmissibility and mutation rate. Given the paucity of orally bioavailable antiviral drugs to combat SARS-CoV-2 infection, there is a critical need for additional antivirals with alternative mechanisms of action. Papain-like protease (PLpro) is one of the two SARS-CoV-2 encoded viral cysteine proteases essential for viral replication. PLpro cleaves at three sites of the viral polyproteins. In addition, PLpro antagonizes the host immune response upon viral infection by cleaving ISG15 and ubiquitin from host proteins. Therefore, PLpro is a validated antiviral drug target. In this study, we report the X-ray crystal structures of papain-like protease (PLpro) with two potent inhibitors, Jun9722 and Jun9843. Subsequently, we designed and synthesized several series of analogs to explore the structure-activity relationship, which led to the discovery of PLpro inhibitors with potent enzymatic inhibitory activity and antiviral activity against SARS-CoV-2. Together, the lead compounds are promising drug candidates for further development.

The development of papain‐like protease from SARS‐CoV‐2, a potential drug target for antiviral screening: A review

The SARS‐CoV‐2 outbreak caused a global pandemic, claiming numerous lives and becoming this century’s most widespread life‐threatening disease. The virus relies on two specific enzymes to facilitate replication, 3‐Chymotrypsin‐like protease (3CLPro) and Papain‐like‐protease (PLpro). These enzymes are crucial in breaking down nonstructural polypeptides into functional proteins. PLpro with LXGG↓X recognition and cleavage sites also play a role in deubiquitylase (DUB) and delSGylase by cleaving after the double glycine residue of ubiquitin (Ub) and ISG15 as a mechanism to suppress the host’s innate immune response. Despite its important role in the viral infection cycle and the potential for drug discovery, no antivirals have been approved as PLpro inhibitors. Therefore, this review focuses on PLpro protein, its recombinant product development and purification, and its application as a protein target in drug discovery for COVID‐19 screening to develop effective COVID‐19 drugs.

SARS-CoV-2 papain-like protease (PLpro) inhibitory and antiviral activity of small molecule derivatives for drug leads

We report here the synthesis and biological evaluation of a series of small molecule SARS-CoV-2 PLpro inhibitors. We compared the activity of selected compounds in both SARS-CoV-1 and SARS-CoV-2 PLpro inhibitory and antiviral assays. We have synthesized and evaluated several new structural variants of previous leads against SARS-CoV-2 PLpro. The replacement of the carboxamide functionality with sulfonamide derivatives resulted in PLpro inhibitors with potent PLpro inhibitory and antiviral activity in VeroE6 cells similar to GRL0617. To obtain molecular insight, we created an optimized model of a potent sulfonamide derivative in the SARS-CoV-2 PLpro active site.

Computer-aided design, syntheses, and ITC binding data of novel flavanone derivatives for use as potential inhibitors of the papain-like protease of COVID-19

The papain-like protease (PLpro) of SARS-CoV-2 (COVID-19) is a high-profile drug target for treating COVID-19 due to its critical role in making essential proteins crucial in viral replication and host immune sensing. The development of small molecule inhibitors of PLpro is an area of ongoing research and interest. To investigate the development of PLpro inhibitors, a series of novel flavanone derivatives were designed usingin silicodocking against the papain-like protease of COVID-19. The most promising targets were synthesized and structurally characterized by NMR and mass spectrometry. Using isothermal calorimetry studies, two synthesized derivatives were found to bind PLpro in the low micromolar to nanomolar range.

Discovery of new non-covalent and covalent inhibitors targeting SARS-CoV-2 papain-like protease and main protease

Global coronavirus disease 2019 (COVID-19) pandemic still threatens human health and public safety, and the development of effective antiviral agent is urgently needed. The SARS-CoV-2 main protease (Mpro) and papain-like protease (PLpro) are vital proteins in viral replication and promising therapeutic targets. Additionally, PLpro also modulates host immune response by cleaving ubiquitin and interferon-stimulated gene product 15 (ISG15) from ISGylated host proteins. In this report, we identified [1,2]selenazolo[5,4-c]pyridin-3(2H)-one and benzo[d]isothiazol-3(2H)-one as attractive scaffolds of PLpro and Mpro inhibitors. The representative compounds 6c and 7e exhibited excellent PLpro inhibition with percent inhibition of 42.9% and 44.9% at 50 nM, respectively. The preliminary enzyme kinetics experiment and fluorescent labelling experiment results determined that 6c was identified as a covalent PLpro inhibitor, while 7e was a non-covalent inhibitor. Molecular docking and dynamics simulations revealed that 6c and 7e bound to Zn-finger domain of PLpro. Compounds 6c and 7e were also identified to potent Mpro inhibitors, and they exhibited potent antiviral activities in SARS-CoV-2 infected Vero E6 cells, with EC50 value of 3.9 μM and 7.4 μM, respectively. In addition, the rat liver homogenate half-life of 6c and 7e exceeded 24 h. These findings suggest that 6c and 7e are promising led compounds for further development of PLpro/Mpro dual-target antiviral drugs.

Screening, Synthesis and Biochemical Characterization of SARS-CoV-2 Protease Inhibitors.

The severe acute respiratory syndrome-causing coronavirus 2 (SARS-CoV-2) papain-like protease (PLpro) and main protease (Mpro) play an important role in viral replication events and are important targets for anti-coronavirus drug discovery. In search of these protease inhibitors, we screened a library of 1300 compounds using a fluorescence thermal shift assay (FTSA) and identified 53 hits that thermally stabilized or destabilized PLpro. The hit compounds structurally belonged to two classes of small molecules: thiazole derivatives and symmetrical disulfide compounds. Compound dissociation constants (Kd) were determined using an enzymatic inhibition method. Seven aromatic disulfide compounds were identified as efficient PLpro inhibitors with Kd values in the micromolar range. Two disulfides displayed six-fold higher potency for PLpro (Kd = 0.5 µM) than for Mpro. The disulfide derivatives bound covalently to both proteases, as confirmed through mass spectrometry. The identified compounds can serve as lead compounds for further chemical optimization toward anti-COVID-19 drugs.

A novel robust inhibitor of papain-like protease (PLpro) as a COVID-19 drug

Regarding the significance of SARS-CoV-2, scientists have shown considerable interest in developing effective drugs. Inhibitors for PLpro are the primary strategies for locating suitable COVID-19 drugs. Natural compounds comprise the majority of COVID-19 drugs. Due to limitations on the safety of clinical trials in cases of COVID, computational methods are typically utilized for inhibition studies. Whereas papain is highly similar to PLpro and is entirely safe, the current study aimed to examine several plant secondary metabolites to identify the most effective papain inhibitor and validate the results using molecular dynamics and docking. This simulation was conducted identically for PLpro and the optimal inhibitor. The results indicated that the experimental results are comparable to those obtained In-Silico, and the inhibition effects of Chlorogenic acid (CGA) on papain attained in the experiment were validated (IC50=0.54 mM). CGA as an inhibitor was located in the active site of PLpro and papain (total energy −2009410 and −456069 kJ/mol, respectively) at the desired location and distance. The study revealed that CGA and its derivatives are effective PLpro inhibitors against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

Reconstruction of the unbinding pathways of new inhibitors of the SARS-CoV-2 papain-like protease using molecular dynamics simulation

Developing novel antiviral drugs against the SARS-CoV-2 virus and COVID-19 disease is imperative as the vaccines may not offer absolute protection. PLpro plays a crucial role in the viral life cycle, making it an attractive target for drug development. Several PLpro inhibitors have been developed, and their 3D structures in complex with PLpro are available. In this work, we employed Supervised Molecular Dynamics (SuMD), a specific Unbiased Molecular Dynamics (UMD) method, to investigate unbinding pathways of the novel inhibitors of PLpro (PDB IDs: 7LBR, 7RZC, 7SDR and 7E35) and GRL0617 (PDB ID: 7JRN) as a reference. We conducted three simulations for each ligand and achieved unbinding events in the nanosecond timescale in all simulations. We found that unbinding events are commonly affected by altering the conformation of the BL2 loop, which is caused by the natural fluctuations of the loop that are required to trap the substrate and throw out the product. BL2 loop is crucial for keeping the ligand and unbinding and acts as a double-edged sword. Any inhibitor designed to be effective must prevent the loop’s natural fluctuations. We perceived that increasing ligands interactions with the binding pocket interior and the BL2 loop will help prevent natural fluctuation of the BL2 loop, Although the interactions with the binding pocket’s inner side are more critical than the BL2 loop. These findings may be helpful in developing more potent inhibitors against SARS-CoV-2. Communicated by Ramaswamy H. Sarma

NMR Characterization of the Papain-like Protease from SARS-CoV-2 Identifies the Conformational Heterogeneity in Its Inhibitor-Binding Site.

Papain-like protease (PLpro) from severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is a prime target for the development of antivirals for Coronavirus disease 2019 (COVID-19). However, drugs that target the PLpro protein have not yet been approved. In order to gain insights into the development of a PLpro inhibitor, conformational dynamics of PLpro in complex with GRL0617, the most well-characterized PLpro inhibitor, were investigated using nuclear magnetic resonance (NMR) spectroscopy in solution. Although mutational analyses demonstrated that the L162 sidechain interaction is responsible for the affinity for GRL0617, NMR analyses revealed that L162 in the inhibitor-binding pocket underwent conformational exchange and was not fixed in the conformation in which it formed a contact with ortho-methyl group of GRL0617. The identified conformational dynamics would provide a rationale for the binding mechanism of a covalent inhibitor designed based on GRL0617.

Computational study of the binding orientation and affinity of noncovalent inhibitors of the papain-like protease (PLpro) from SARS-CoV-1 considering the protein flexibility by using molecular dynamics and cross-docking.

The papain-like protease (PLpro) from zoonotic coronaviruses (CoVs) has been identified as a target with an essential role in viral respiratory diseases caused by Severe Acute Respiratory Syndrome-associated coronaviruses (SARS-CoVs). The design of PLpro inhibitors has been proposed as an alternative to developing potential drugs against this disease. In this work, 67 naphthalene-derived compounds as noncovalent PLpro inhibitors were studied using molecular modeling methods. Structural characteristics of the bioactive conformations of these inhibitors and their interactions at the SARS-CoV-1 PLpro binding site were reported here in detail, taking into account the flexibility of the protein residues. Firstly, a molecular docking protocol was used to obtain the orientations of the inhibitors. After this, the orientations were compared, and the recurrent interactions between the PLpro residues and ligand chemical groups were described (with LigRMSD and interaction fingerprints methods). In addition, efforts were made to find correlations between docking energy values and experimentally determined binding affinities. For this, the PLpro was sampled by using Gaussian Accelerated Molecular Dynamics (GaMD), generating multiple conformations of the binding site. Diverse protein conformations were selected and a cross-docking experiment was performed, yielding models of the 67 naphthalene-derived compounds adopting different binding modes. Representative complexes for each ligand were selected to obtain the highest correlation between docking energies and activities. A good correlation (R 2 = 0.948) was found when this flexible docking protocol was performed.

A Patent Review on SARS Coronavirus Papain-Like Protease (PLpro ) Inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is an unprecedented global health emergency causing more than 6.6 million fatalities by 31 December 2022. So far, only three antiviral drugs have been granted emergency use authorisation or approved by the FDA. The SARS-CoV-2 papain-like protease (PLpro ) is deemed an attractive drug target as it plays an essential role in viral polyprotein processing and pathogenesis although no inhibitors have yet been approved. This patent review discusses coronavirus PLpro inhibitors reported in patents published between 1 January 2003 to 2 March 2023, giving an overview on the inhibitors that have generated commercial interest, especially amongst drug companies.

Discovery of PLpro and Mpro Inhibitors for SARS-CoV-2.

There are very few small-molecule antivirals for SARS-CoV-2 that are either currently approved (or emergency authorized) in the US or globally, including remdesivir, molnupiravir, and paxlovid. The increasing number of SARS-CoV-2 variants that have appeared since the outbreak began over three years ago raises the need for continual development of updated vaccines and orally available antivirals in order to fully protect or treat the population. The viral main protease (Mpro) and the papain-like protease (PLpro) are key for viral replication; therefore, they represent valuable targets for antiviral therapy. We herein describe an in vitro screen performed using the 2560 compounds from the Microsource Spectrum library against Mpro and PLpro in an attempt to identify additional small-molecule hits that could be repurposed for SARS-CoV-2. We subsequently identified 2 hits for Mpro and 8 hits for PLpro. One of these hits was the quaternary ammonium compound cetylpyridinium chloride with dual activity (IC50 = 2.72 ± 0.09 μM for PLpro and IC50 = 7.25 ± 0.15 μM for Mpro). A second inhibitor of PLpro was the selective estrogen receptor modulator raloxifene (IC50 = 3.28 ± 0.29 μM for PLpro and IC50 = 42.8 ± 6.7 μM for Mpro). We additionally tested several kinase inhibitors and identified olmutinib (IC50 = 0.54 ± 0.04 μM), bosutinib (IC50 = 4.23 ± 0.28 μM), crizotinib (IC50 = 3.81 ± 0.04 μM), and dacominitinib (IC50 = IC50 3.33 ± 0.06 μM) as PLpro inhibitors for the first time. In some cases, these molecules have also been tested by others for antiviral activity for this virus, or we have used Calu-3 cells infected with SARS-CoV-2. The results suggest that approved drugs can be identified with promising activity against these proteases, and in several cases we or others have validated their antiviral activity. The additional identification of known kinase inhibitors as molecules targeting PLpro may provide new repurposing opportunities or starting points for chemical optimization.

Structure-Based Design of Potent Peptidomimetic Inhibitors Covalently Targeting SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PLpro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC50 = 0.23 μM) and significant inhibition of SARS-CoV-2 PLpro in the HEK293T cells using a cell-based protease assay (EC50 = 3.61 μM). Moreover, an X-ray crystal structure of SARS-CoV-2 PLpro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PLpro inhibitors and provide an attractive starting point for further optimization.

The SARS-CoV-2 papain-like protease suppresses type I interferon responses by deubiquitinating STING.

The SARS-CoV-2 papain-like protease (PLpro), which has deubiquitinating activity, suppresses the type I interferon (IFN-I) antiviral response. We investigated the mechanism by which PLpro antagonizes cellular antiviral responses. In HEK392T cells, PLpro removed K63-linked polyubiquitin chains from Lys289 of the stimulator of interferon genes (STING). PLpro-mediated deubiquitination of STING disrupted the STING-IKKε-IRF3 complex that induces the production of IFN-β and IFN-stimulated cytokines and chemokines. In human airway cells infected with SARS-CoV-2, the combined treatment with the STING agonist diABZi and the PLpro inhibitor GRL0617 resulted in the synergistic inhibition of SARS-CoV-2 replication and increased IFN-I responses. The PLpros of seven human coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-HKU1, HCoV-OC43, and HCoV-NL63) and four SARS-CoV-2 variants of concern (α, β, γ, and δ) all bound to STING and suppressed STING-stimulated IFN-I responses in HEK293T cells. These findings reveal how SARS-CoV-2 PLpro inhibits IFN-I signaling through STING deubiquitination and a general mechanism used by seven human coronaviral PLpros to dysregulate STING and to facilitate viral innate immune evasion. We also identified simultaneous pharmacological STING activation and PLpro inhibition as a potentially effective strategy for antiviral therapy against SARS-CoV-2.

Potent and selective covalent inhibition of the papain-like protease from SARS-CoV-2

Direct-acting antivirals are needed to combat coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The papain-like protease (PLpro) domain of Nsp3 from SARS-CoV-2 is essential for viral replication. In addition, PLpro dysregulates the host immune response by cleaving ubiquitin and interferon-stimulated gene 15 protein from host proteins. As a result, PLpro is a promising target for inhibition by small-molecule therapeutics. Here we design a series of covalent inhibitors by introducing a peptidomimetic linker and reactive electrophile onto analogs of the noncovalent PLpro inhibitor GRL0617. The most potent compound inhibits PLpro with kinact/KI = 9,600 M−1 s−1, achieves sub-μM EC50 values against three SARS-CoV-2 variants in mammalian cell lines, and does not inhibit a panel of human deubiquitinases (DUBs) at >30 μM concentrations of inhibitor. An X-ray co-crystal structure of the compound bound to PLpro validates our design strategy and establishes the molecular basis for covalent inhibition and selectivity against structurally similar human DUBs. These findings present an opportunity for further development of covalent PLpro inhibitors.

Large-Scale Virtual Screening for the Discovery of SARS-CoV-2 Papain-like Protease (PLpro) Non-covalent Inhibitors.

The rapid global spread of the SARS-CoV-2 virus facilitated the development of novel direct-acting antiviral agents (DAAs). The papain-like protease (PLpro) has been proposed as one of the major SARS-CoV-2 targets for DAAs due to its dual role in processing viral proteins and facilitating the host's immune suppression. This dual role makes identifying small molecules that can effectively neutralize SARS-CoV-2 PLpro activity a high-priority task. However, PLpro drug discovery faces a significant challenge due to the high mobility and induced-fit effects in the protease's active site. Herein, we virtually screened the ZINC20 database with Deep Docking (DD) to identify prospective noncovalent PLpro binders and combined ultra-large consensus docking with two pharmacophore (ph4)-filtering strategies. The analysis of active compounds revealed their somewhat-limited diversity, likely attributed to the induced-fit nature of PLpro's active site in the crystal structures, and therefore, the use of rigid docking protocols poses inherited limitations. The top hits were assessed against recombinant viral proteins and live viruses, demonstrating desirable inhibitory activities. The best compound VPC-300195 (IC50: 15 μM) ranks among the top noncovalent PLpro inhibitors discovered through in silico methodologies. In the search for novel SARS-CoV-2 PLpro-specific chemotypes, the identified inhibitors could serve as diverse templates for the development of effective noncovalent PLpro inhibitors.

Repurposing 1,2,4-oxadiazoles as SARS-CoV-2 PLpro inhibitors and investigation of their possible viral entry blockade potential

Although vaccines are obviously mitigating the COVID-19 pandemic diffusion, efficient complementary antiviral agents are urgently needed to combat SARS-CoV-2. The viral papain-like protease (PLpro) is a promising therapeutic target being one of only two essential proteases crucial for viral replication. Nevertheless, it dysregulates the host immune sensing response. Here we report repositioning of the privileged 1,2,4-oxadiazole scaffold as promising SARS-CoV-2 PLpro inhibitor with potential viral entry inhibition profile. The design strategy relied on mimicking the general structural features of the lead benzamide PLpro inhibitor GRL0617 with isosteric replacement of its pharmacophoric amide backbone by 1,2,4-oxadiazole core. Inspired by the multitarget antiviral agents, the substitution pattern was rationalized to tune the scaffold's potency against other additional viral targets, especially the spike receptor binding domain (RBD) that is responsible for the viral invasion. The Adopted facial synthetic protocol allowed easy access to various rationally substituted derivatives. Among the evaluated series, the 2-[5-(pyridin-4-yl)-1,2,4-oxadiazol-3-yl]aniline (5) displayed the most balanced dual inhibitory potential against SARS-CoV-2 PLpro (IC50=7.197 μM) and spike protein RBD (IC50 = 8.673 μM), with acceptable ligand efficiency metrics, practical LogP (3.8) and safety profile on Wi-38 (CC50 = 51.78 μM) and LT-A549 (CC50 = 45.77 μM) lung cells. Docking simulations declared the possible structural determinants of activities and enriched the SAR data for further optimization studies.

Effects of diarylbutane lignans from Schisandra chinensis fruit on SARS-CoV-2 3CLpro and PLpro and their in vitro anti-inflammatory properties

BackgroundSchisandra chinensis fruit is a well-known traditional Chinese medicine (TCM), whose extract has a potent inhibitory effect on the severe acute respiratory syndrome-coronavirus-2 (SARS‑CoV‑2) 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). PurposeThis work aims to find the active components from the fruit of S. chinensis against SARS‑CoV‑2 3CLpro and PLpro. Materials and methodsThe chemical constituents of the fruit of S. chinensis were retrieved based on the electronic databases, such as Web of Science, PubMed, Medline Plus, and CNKI. Molecular docking was used to screen the active components against SARS‑CoV‑2 3CLpro and PLpro. Potential hit compounds were further evaluated by enzymatic activity assay. Furthermore, the anti-inflammatory activities of the active compounds were further explored using the phorbol-12-myristate-13-acetate (PMA)-induced THP1 cells model. ResultsIn this work, we retrieved 75 components of S. chinensis fruit, including 62 dibenzocyclooctadiene-type lignans, 3 diarylbutane-type lignans, 2 tetrahydrofuran-type lignans, and 8 nortriterpenoids. Combining molecular docking study and in vitro experiments, we found that pregomisin (63), meso‑dihydroguaiaretic acid (64), and nordihydroguaiaretic acid (65) could potently inhibit 3CLpro with IC50 values of 3.07 ± 0.38, 4.12 ± 0.38, and 6.06 ± 0.62 μM, respectively, and inhibit PLpro with IC50 values of 5.23 ± 0.33, 4.24 ± 0.46, and 16.28 ± 0.54 μM, respectively. Interestingly, compounds 63, 64, and 65 also have potent activities of regulating the inflammatory response in vitro. ConclusionOur results suggest that compounds 63, 64, and 65 may be promising SARS-CoV-2 3CLpro and PLpro inhibitors and anti-inflammatory.

In vitro metabolic characterization of the SARS-CoV-2 papain-like protease inhibitors GRL0617 and HY-17542.

The SARS-CoV-2 pandemic requires a new therapeutic target for viral infection, and papain-like protease (Plpro) has been suggested as a druggable target. This in-vitro study was conducted to examine the drug metabolism of the GRL0617 and HY-17542, Plpro inhibitors. Metabolism of these inhibitors was studied to predict the pharmacokinetics in human liver microsomes. The hepatic cytochrome P450 (CYP) isoforms responsible for their metabolism were identified using recombinant enzymes. The drug-drug interaction potential mediated by cytochrome P450 inhibition was estimated. In human liver microsomes, the Plpro inhibitors had phase I and phase I + II metabolism with half-lives of 26.35 and 29.53min, respectively. Hydroxylation (M1) and desaturation (-H2, M3) of the para-amino toluene side chain were the predominant reactions mediated with CYP3A4 and CYP3A5. CYP2D6 is responsible for the hydroxylation of the naphthalene side ring. GRL0617 inhibits major drug-metabolizing enzymes, including CYP2C9 and CYP3A4. HY-17542 is structural analog of GRL0617 and it is metabolized to GRL0617 through non-cytochrome P450 reactions in human liver microsomes without NADPH. Like GRL0617 and HY-17542 undergoes additional hepatic metabolism. The in-vitro hepatic metabolism of the Plpro inhibitors featured short half-lives; preclinical metabolism studies are needed to determine therapeutic doses for these inhibitors.