Network Models of BACE-1 Inhibitors: Exploring Structural and Biochemical Relationships.

This study investigates the clustering patterns of human ß-secretase 1 (BACE-1) inhibitors using complex network methodologies based on various distance functions, including Euclidean, Tanimoto, Hamming, and Levenshtein distances. Molecular descriptor vectors such as molecular mass, Merck Molecular Force Field (MMFF) energy, Crippen partition coefficient (ClogP), Crippen molar refractivity (MR), eccentricity, Kappa indices, Synthetic Accessibility Score, Topological Polar Surface Area (TPSA), and 2D/3D autocorrelation entropies are employed to capture the diverse properties of these inhibitors. The Euclidean distance network demonstrates the most reliable clustering results, with strong agreement metrics and minimal information loss, indicating its robustness in capturing essential structural and physicochemical properties. Tanimoto and Hamming distance networks yield valuable clustering outcomes, albeit with moderate performance, while the Levenshtein distance network shows significant discrepancies. The analysis of eigenvector centrality across different networks identifies key inhibitors acting as hubs, which are likely critical in biochemical pathways. Community detection results highlight distinct clustering patterns, with well-defined communities providing insights into the functional and structural groupings of BACE-1 inhibitors. The study also conducts non-parametric tests, revealing significant differences in molecular descriptors, validating the clustering methodology. Despite its limitations, including reliance on specific descriptors and computational complexity, this study offers a comprehensive framework for understanding molecular interactions and guiding therapeutic interventions. Future research could integrate additional descriptors, advanced machine learning techniques, and dynamic network analysis to enhance clustering accuracy and applicability.

First Insight into the Degradome of Aspergillus ochraceus: Novel Secreted Peptidases and Their Inhibitors.

Aspergillus fungi constitute a pivotal element within ecosystems, serving as both contributors of biologically active compounds and harboring the potential to cause various diseases across living organisms. The organism's proteolytic enzyme complex, termed the degradome, acts as an intermediary in its dynamic interaction with the surrounding environment. Using techniques such as genome and transcriptome sequencing, alongside protein prediction methodologies, we identified putative extracellular peptidases within Aspergillus ochraceus VKM-F4104D. Following manual annotation procedures, a total of 11 aspartic, 2 cysteine, 2 glutamic, 21 serine, 1 threonine, and 21 metallopeptidases were attributed to the extracellular degradome of A. ochraceus VKM-F4104D. Among them are enzymes with promising applications in biotechnology, potential targets and agents for antifungal therapy, and microbial antagonism factors. Thus, additional functionalities of the extracellular degradome, extending beyond mere protein substrate digestion for nutritional purposes, were demonstrated.

Screening of Mpro Protease (SARS-CoV-2) Covalent Inhibitors from an Anthocyanin-Rich Blueberry Extract Using an HRMS-Based Analytical Platform.

BACKGROUND: The viral main protease (Mpro) of SARS-CoV-2 has been recently proposed as a key target to inhibit virus replication in the host. Therefore, molecules that can bind the catalytic site of Mpro could be considered as potential drug candidates in the treatment of SARS-CoV-2 infections. Here we proposed the application of a state-of-the-art analytical platform which combines metabolomics and protein structure analysis to fish-out potential active compounds deriving from a natural matrix, i.e., a blueberry extract. METHODS: The experiments focus on finding MS covalent inhibitors of Mpro that contain in their structure a catechol/pyrogallol moiety capable of binding to the nucleophilic amino acids of the enzyme's catalytic site. RESULTS: Among the potential candidates identified, the delphinidin-3-glucoside showed the most promising results. Its antiviral activity has been confirmed in vitro on Vero E6 cells infected with SARS-CoV-2, showing a dose-dependent inhibitory effect almost comparable to the known Mpro inhibitor baicalin. The interaction of delphinidin-3-glucoside with the Mpro pocket observed was also evaluated by computational studies. CONCLUSIONS: The HRMS analytical platform described proved to be effective in identifying compounds that covalently bind Mpro and are active in the inhibition of SARS-CoV-2 replication, such as delphinidin-3-glucoside.

Host Cell Proteases Involved in Human Respiratory Viral Infections and Their Inhibitors: A Review.

Viral tropism is most commonly linked to receptor use, but host cell protease use can be a notable factor in susceptibility to infection. Here we review the use of host cell proteases by human viruses, focusing on those with primarily respiratory tropism, particularly SARS-CoV-2. We first describe the various classes of proteases present in the respiratory tract, as well as elsewhere in the body, and incorporate the targeting of these proteases as therapeutic drugs for use in humans. Host cell proteases are also linked to the systemic spread of viruses and play important roles outside of the respiratory tract; therefore, we address how proteases affect viruses across the spectrum of infections that can occur in humans, intending to understand the extrapulmonary spread of SARS-CoV-2.

Structure-based virtual screening methods for the identification of novel phytochemical inhibitors targeting furin protease for the management of COVID-19.

The coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, is a highly contagious respiratory disease with widespread societal impact. The symptoms range from cough, fever, and pneumonia to complications affecting various organs, including the heart, kidneys, and nervous system. Despite various ongoing efforts, no effective drug has been developed to stop the spread of the virus. Although various types of medications used to treat bacterial and viral diseases have previously been employed to treat COVID-19 patients, their side effects have also been observed. The way SARS-CoV-2 infects the human body is very specific, as its spike protein plays an important role. The S subunit of virus spike protein cleaved by human proteases, such as furin protein, is an initial and important step for its internalization into a human host. Keeping this context, we attempted to inhibit the furin using phytochemicals that could produce minimal side effects. For this, we screened 408 natural phytochemicals from various plants having antiviral properties, against furin protein, and molecular docking and dynamics simulations were performed. Based on the binding score, the top three compounds (robustaflavone, withanolide, and amentoflavone) were selected for further validation. MM/GBSA energy calculations revealed that withanolide has the lowest binding energy of -57.2 kcal/mol followed by robustaflavone and amentoflavone with a binding energy of -45.2 kcal/mol and -39.68 kcal/mol, respectively. Additionally, ADME analysis showed drug-like properties for these three lead compounds. Hence, these natural compounds robustaflavone, withanolide, and amentoflavone, may have therapeutic potential for the management of SARS-CoV-2 by targeting furin.

Therapeutic Potentials of Medicinal Leech in Chinese Medicine.

The use of medicinal leeches in clinical therapy has been employed for a long time, as it was originally recognized for exerting antithrombin effects. These effects were due to the ability of the leech to continuously suck blood while attached to human skin. According to Chinese Pharmacopoei, leeches used in traditional Chinese medicine mainly consist of Whitmania pigra Whitman, Hirudo nipponia Whitman, and Whitmania acranulata, but the latter two species are relatively scarce. The main constituents of leeches are protein and peptide macromolecules. They can be categorized into two categories based on their pharmacological effects. One group consists of active ingredients that directly target the coagulation system, such as hirudin, heparin, and histamine, which are widely known. The other group comprises protease inhibitor components like Decorsin and Hementin. Among these, hirudin secreted by the salivary glands of the leech is the most potent thrombin inhibitor and served as the sole remedy for preventing blood clotting until the discovery of heparin. Additionally, leeches play a significant role in various traditional Chinese medicine formulations. In recent decades, medicinal leeches have been applied in fields including anti-inflammatory treatment, cardiovascular disease management, antitumor treatment, and many other medical conditions. In this review, we present a comprehensive overview of the historical journey and medicinal applications of leeches in various medical conditions, emphasizing their pharmaceutical significance within traditional Chinese medicine. This review offers valuable insights for exploring additional therapeutic opportunities involving the use of leeches in various diseases and elucidating their underlying mechanisms for future research.

Shedding Light on Dark Chemical Matter: The Discovery of a SARS-CoV-2 Mpro Main Protease Inhibitor through Intensive Virtual Screening and In Vitro Evaluation.

The development of specific antiviral therapies targeting SARS-CoV-2 remains fundamental because of the continued high incidence of COVID-19 and limited accessibility to antivirals in some countries. In this context, dark chemical matter (DCM), a set of drug-like compounds with outstanding selectivity profiles that have never shown bioactivity despite being extensively assayed, appears to be an excellent starting point for drug development. Accordingly, in this study, we performed a high-throughput screening to identify inhibitors of the SARS-CoV-2 main protease (Mpro) using DCM compounds as ligands. Multiple receptors and two different docking scoring functions were employed to identify the best molecular docking poses. The selected structures were subjected to extensive conventional and Gaussian accelerated molecular dynamics. From the results, four compounds with the best molecular behavior and binding energy were selected for experimental testing, one of which presented inhibitory activity with a Ki value of 48 ± 5 µM. Through virtual screening, we identified a significant starting point for drug development, shedding new light on DCM compounds.

3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials.

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pKa of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy.

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme's proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease's active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.

[Improvement of Intestinal and Transmucosal Absorption of Peptide and Protein Drugs].

It is well known that the oral bioavailability of hydrophilic and macromolecular drugs is generally very poor due to their poor membrane permeability characteristics. Among these poorly absorbed drugs, peptide and protein drugs are typical poorly absorbed drugs which have low stability and poor permeability in the gastrointestinal tract. Consequently, the clinical administration of peptide and protein drugs is presently limited to administration by injection. However, such frequent administration subjects the patients to considerable pain, and there is also the possibility of the manifestation of serious side effects. Therefore, various approaches have been examined to overcome the poor absorption characteristics of these drugs. These approaches include (1) to use additives including absorption enhancers and protease inhibitors, (2) to modify the chemical structure of peptide and protein drugs, and (3) to apply dosage forms to these drugs, (4) to develop a novel administration method for these drugs that can serve as an alternative to oral and injection administration. We demonstrated that intestinal and transmucosal absorption of peptide and protein drugs could be improved by using these approaches. These approaches may give us useful basic information to improve the intestinal and transmucosal absorption of peptide and protein drugs.

Identification of novel and potential inhibitors against the dengue virus NS2B/NS3 protease using virtual screening and biomolecular simulations.

Approximately 3.9 billion individuals are vulnerable to dengue infection, a prevalent cause of tropical diseases worldwide. Currently, no drugs are available for preventing or treating Flavivirus diseases, including Dengue, West Nile, and the more recent Zika virus. The highly conserved Flavivirus NS2B-NS3 protease, crucial for viral replication, is a promising therapeutic target. This study employed in-silico methodologies to identify novel and potentially effective anti-dengue small molecules. A pharmacophore model was constructed using an experimentally validated NS2B-NS3 inhibitor, with the Gunner Henry score confirming the model's validity. The Natural Product Activity and Species Source (NPASS) database was screened using the validated pharmacophore model, yielding a total of 60 hits against the NS2B-NS3 protease. Furthermore, the docking finding reveals that our newly identified compounds from the NPASS database have enhanced binding affinities and established significant interactions with allosteric residues of the target protein. MD simulation and post-MD analysis further validated this finding. The free binding energy was computed in terms of MM-GBSA analysis, with the total binding energy for compound 1 (-57.3 ± 2.8 and - 52.9 ± 1.9 replica 1 and 2) indicating a stronger binding affinity for the target protein. Overall, this computational study identified these compounds as potential hit molecules, and these findings can open up a new avenue to explore and develop inhibitors against Dengue virus infection.

Structural basis of rosmarinic acid inhibitory mechanism on SARS-CoV-2 main protease.

The SARS-CoV-2 coronavirus is characterized by high mutation rates and significant infectivity, posing ongoing challenges for therapeutic intervention. To address potential challenges in the future, the continued development of effective drugs targeting SARS-CoV-2 remains an important task for the scientific as well as the pharmaceutical community. The main protease (Mpro) of SARS-CoV-2 is an ideal therapeutic target for COVID-19 drug development, leading to the introduction of various inhibitors, both covalent and non-covalent, each characterized by unique mechanisms of action and possessing inherent strengths and limitations. Natural products, being compounds naturally present in the environment, offer advantages such as low toxicity and diverse activities, presenting a viable source for antiviral drug development. Here, we identified a natural compound, rosmarinic acid, which exhibits significant inhibitory effects on the Mpro of the SARS-CoV-2. Through detailed structural biology analysis, we elucidated the precise crystal structure of the complex formed between rosmarinic acid and SARS-CoV-2 Mpro, revealing the molecular basis of its inhibitory mechanism. These findings not only enhance our understanding of the antiviral action of rosmarinic acid, but also provide valuable structural information and mechanistic insights for the further development of therapeutic strategies against SARS-CoV-2.

X-Ray Film Gel Contact Print Method for Analyzing Differential Expression of Plant Protease Inhibitors Under Abiotic Stress.

The regulation of proteolytic enzymes by protease inhibitors is crucial for maintaining the balance between protein synthesis and degradation, preventing uncontrolled proteolysis and fine-tuning cellular processes essential for optimal function and survival of the plants. It is known that the plant protease inhibitors activities are induced in defense of biotic as well as abiotic stresses. Thus, beyond their fundamental physiological functions, their involvement in stress responses, such as drought, cold, and salinity, is of equally significant. The X-ray film contact print method is an effective method for assessing various protease inhibitors exposed to stress conditions. In this approach, initially plant protease inhibitors will be separated using electrophoresis, and then the gel is treated with trypsin, which inhibits protease inhibitors. This gel when placed on X-ray film, the trypsin will digest the gelatin layer present on the film and the gelatinolytic activity stalled at the premises of protease inhibitors. This will provide the impression of the differentially expressed protease inhibitors in stress-treated plants.

Computational Insights into SARS-CoV-2 Main Protease Mutations and Nirmatrelvir Efficacy: The Effects of P132H and P132H-A173V.

Nirmatrelvir, a pivotal component of the oral antiviral Paxlovid for COVID-19, targets the SARS-CoV-2 main protease (Mpro) as a covalent inhibitor. Here, we employed combined computational methods to explore how the prevalent Omicron variant mutation P132H, alone and in combination with A173V (P132H-A173V), affects nirmatrelvir's efficacy. Our findings suggest that P132H enhances the noncovalent binding affinity of Mpro for nirmatrelvir, whereas P132H-A173V diminishes it. Although both mutants catalyze the rate-limiting step more efficiently than the wild-type (WT) Mpro, P132H slows the overall rate of covalent bond formation, whereas P132H-A173V accelerates it. Comprehensive analysis of noncovalent and covalent contributions to the overall binding free energy of the covalent complex suggests that P132H likely enhances Mpro sensitivity to nirmatrelvir, while P132H-A173V may confer resistance. Per-residue decompositions of the binding and activation free energies pinpoint key residues that significantly affect the binding affinity and reaction rates, revealing how the mutations modulate these effects. The mutation-induced conformational perturbations alter drug-protein local contact intensities and the electrostatic preorganization of the protein, affecting noncovalent binding affinity and the stability of key reaction states, respectively. Our findings inform the mechanisms of nirmatrelvir resistance and sensitivity, facilitating improved drug design and the detection of resistant strains.

Structural basis for the inhibition of the HCoV-NL63 main protease Mpro by X77.

Human coronaviruses are a group of pathogens that primarily cause respiratory and intestinal diseases. Infection can easily cause respiratory symptoms, as well as a variety of serious complications. There are several types of human coronaviruses, such as SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and SARS-CoV-2. The prevalence of COVID-19 has led to a growing focus on drug research against human coronaviruses. The main protease (Mpro) from human coronaviruses is a relatively conserved that controls viral replication. X77 was discovered to have extremely high inhibitory activity against SARS-CoV-2 Mpro through the use of computer-simulated docking. In this paper, we have resolved the crystal structure of the HCoV-NL63 Mpro complexed with X77 and analyzed their interaction in detail. This data provides essential information for solving their binding modes and their structural determinants. Then, we compared the binding modes of X77 with SARS-CoV-2 Mpro and HCoV-NL63 Mpro in detail. This study illustrates the structural basis of HCoV-NL63 Mpro binding to the inhibitor X77. The structural insights derived from this study will inform the development of new drugs with broad-spectrum resistance to human coronaviruses.

Design, Synthesis, and Biological Evaluation of 1,2,4-Oxadiazole Derivatives Containing an Aryl Carboxylic Acid Moiety as Potent Sarbecovirus Papain-like Protease Inhibitors.

Papain-like protease (PLpro) is a promising therapeutic target for its pivotal role in the life cycle of SARS-CoV-2. A series of 1,2,4-oxadiazole derivatives was designed and synthesized via a ring formation strategy based on SARS-CoV-2 PLpro-GRL0617 complex structure. Systematic structure-activity relationship studies revealed that introducing oxadiazole and aryl carboxylic acid moieties to GRL0617 enhanced the enzymatic inhibition activity, affinity, and deubiquitination capacity toward PLpro. 1,2,4-Oxadiazole compounds 13f and 26r, which had PLpro inhibition activity (IC50 = 1.8 and 1.0 µM) and antiviral activity against SARS-CoV-2 (EC50 = 5.4 and 4.3 µM), exhibited good metabolic stability (t1/2 > 93.2 min) and higher plasma exposure (AUC0-t = 17,380.08 and 24,289.76 ng·h/mL) in mice. Especially, compound 26r with moderate oral bioavailability of 39.1% and potent antiviral activity is worthy of further studies in vivo. Our findings provide a new insight for the discovery of antiviral agents targeting PLpro.

Exploring Cannabinoids as Potential Inhibitors of SARS-CoV-2 Papain-like Protease: Insights from Computational Analysis and Molecular Dynamics Simulations.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global COVID-19 pandemic, challenging healthcare systems worldwide. Effective therapeutic strategies against this novel coronavirus remain limited, underscoring the urgent need for innovative approaches. The present research investigates the potential of cannabis compounds as therapeutic agents against SARS-CoV-2 through their interaction with the virus's papain-like protease (PLpro) protein, a crucial element in viral replication and immune evasion. Computational methods, including molecular docking and molecular dynamics (MD) simulations, were employed to screen cannabis compounds against PLpro and analyze their binding mechanisms and interaction patterns. The results showed cannabinoids with binding affinities ranging from -6.1 kcal/mol to -4.6 kcal/mol, forming interactions with PLpro. Notably, Cannabigerolic and Cannabidiolic acids exhibited strong binding contacts with critical residues in PLpro's active region, indicating their potential as viral replication inhibitors. MD simulations revealed the dynamic behavior of cannabinoid-PLpro complexes, highlighting stable binding conformations and conformational changes over time. These findings shed light on the mechanisms underlying cannabis interaction with SARS-CoV-2 PLpro, aiding in the rational design of antiviral therapies. Future research will focus on experimental validation, optimizing binding affinity and selectivity, and preclinical assessments to develop effective treatments against COVID-19.

Extensive targeting of chemical space at the prime side of ketoamide inhibitors of rhomboid proteases by branched substituents empowers their selectivity and potency.

Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipidic cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesized here containing a branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2.

Rational Approach toward COVID-19's Main Protease Inhibitors: A Hierarchical Biochemoinformatics Analysis.

This study investigated the potential of selected compounds as inhibitors of SARS-CoV-2 Mpro through pharmacokinetic and toxicological analyses, molecular docking, and molecular dynamics simulations. In silico molecular docking simulations revealed promising ligands with favorable binding affinities for Mpro, ranging from -6.2 to -9.5 kcal/mol. Moreover, molecular dynamics simulations demonstrated the stability of protein-ligand complexes over 200 ns, maintaining protein secondary structures. MM-PBSA analysis revealed favorable interactions between ligands and Mpro, with negative binding energy values. Hydrogen bond formation capacity during molecular dynamics was confirmed, indicating consistent interactions with Mpro catalytic residues. Based on these findings, selected ligands show promise for future studies in developing COVID-19 treatments.

Characterization and noncovalent inhibition of the K63-deubiquitinase activity of SARS-cov-2 PLpro.

SARS-CoV-2 papain-like protease (PLpro) could facilitate viral replication and host immune evasion by respectively hydrolyzing viral polyprotein and host ubiquitin conjugates, thereby rendering itself as an important antiviral target. Yet few noncovalent PLpro inhibitors of SARS-CoV-2 have been reported with improved directed towards pathogenic deubiquitinating activities inhibition. Herein, we report that coronavirus PLpro proteases have distinctive substrate bias and are conserved to deubiquitylate K63-linked polyubiquitination, thereby attenuating host type I interferon response. We identify a noncovalent compound specifically optimized towards halting the K63-deubiquitinase activity of SARS-CoV-2 PLpro, but not other coronavirus (CoV) counterparts or host deubiquitinase. Contrasting with GRL-0617, a SARS-CoV-1 PLpro inhibitor, SIMM-036 is 50-fold and 7-fold (half maximal inhibitory concentration (IC50)) more potent to inhibit viral replication during SARS-CoV-2 infection and restore the host interferon-ß (IFN-ß) response in human angiotensin-converting enzyme 2 (hACE2)-HeLa cells, respectively. Structure-activity relationship (SAR) analysis further reveals the importance of BL2 groove of PLpro, which could determine the selectivity of K63-deubiquitinase activity of the enzyme.