Non-covalent Inhibitors Research Articles

SOHO State of the Art Updates and Next Questions: New Targetable Pathways in Chronic Lymphocytic Leukemia.

Regulatory approvals of Bruton tyrosine kinase (BTK) inhibitors and BCL2 inhibitors have transformed the therapeutic paradigm in chronic lymphocytic leukemia (CLL). However, despite significant improvement, treatment discontinuations due to an acquired resistance mutation or intolerance to these agents are common. Those who are refractory and/or intolerant to both these classes of drugs - the "double exposed/refractory" patients - pose a real challenge in clinical practice and are in dire need of novel therapeutic approaches. In this manuscript, we review the ongoing efforts addressing this unmet clinical need including the ongoing development of non-covalent BTK inhibitors, BTK degraders, novel BH3-mimetics, therapeutic antibodies targeting novel antigens and immune cell enabling therapies.

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.

Diphenyl Diselenide and SARS-CoV-2: in silico Exploration of the Mechanisms of Inhibition of Main Protease (Mpro) and Papain-like Protease (PLpro).

The SARS-CoV-2 pandemic has prompted global efforts to develop therapeutics. The main protease of SARS-CoV-2 (Mpro) and the papain-like protease (PLpro) are essential for viral replication and are key targets for therapeutic development. In this work, we investigate the mechanisms of SARS-CoV-2 inhibition by diphenyl diselenide (PhSe)2 which is an archetypal model of diselenides and a renowned potential therapeutic agent. The in vitro inhibitory concentration of (PhSe)2 against SARS-CoV-2 in Vero E6 cells falls in the low micromolar range. Molecular dynamics (MD) simulations and density functional theory (DFT) calculations [level of theory: SMD-B3LYP-D3(BJ)/6-311G(d,p), cc-pVTZ] are used to inspect non-covalent inhibition modes of both proteases via π-stacking and the mechanism of covalent (PhSe)2 + Mpro product formation involving the catalytic residue C145, respectively. The in vitro CC50 (24.61 μM) and EC50 (2.39 μM) data indicate that (PhSe)2 is a good inhibitor of the SARS-CoV-2 virus replication in a cell culture model. The in silico findings indicate potential mechanisms of proteases' inhibition by (PhSe)2; in particular, the results of the covalent inhibition here discussed for Mpro, whose thermodynamics is approximatively isoergonic, prompt further investigation in the design of antiviral organodiselenides.

An in-solution snapshot of SARS-COV-2 main protease maturation process and inhibition

The main protease from SARS-CoV-2 (Mpro) is responsible for cleavage of the viral polyprotein. Mpro self-processing is called maturation, and it is crucial for enzyme dimerization and activity. Here we use C145S Mpro to study the structure and dynamics of N-terminal cleavage in solution. Native mass spectroscopy analysis shows that mixed oligomeric states are composed of cleaved and uncleaved particles, indicating that N-terminal processing is not critical for dimerization. A 3.5 Å cryo-EM structure provides details of Mpro N-terminal cleavage outside the constrains of crystal environment. We show that different classes of inhibitors shift the balance between oligomeric states. While non-covalent inhibitor MAT-POS-e194df51-1 prevents dimerization, the covalent inhibitor nirmatrelvir induces the conversion of monomers into dimers, even with intact N-termini. Our data indicates that the Mpro dimerization is triggered by induced fit due to covalent linkage during substrate processing rather than the N-terminal processing.

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.

Structural Complementarity of Bruton’s Tyrosine Kinase and Its Inhibitors for Implication in B-Cell Malignancies and Autoimmune Diseases

Bruton's tyrosine kinase (BTK) is a critical component in B-cell receptor (BCR) signaling and is also expressed in haematogenic and innate immune cells. Inhibition of BTK hyperactivity is implicated in B-cell malignancies and autoimmune diseases. This review derives the structural complementarity of the BTK-kinase domain and its inhibitors from recent three-dimensional structures of inhibitor-bound BTK in the protein data bank (PDB). Additionally, this review analyzes BTK-mediated effector responses of B-cell development and antibody production. Covalent inhibitors contain an α, β-unsaturated carbonyl moiety that forms a covalent bond with Cys481, stabilizing αC-helix in inactive-out conformation which inhibits Tyr551 autophosphorylation. Asn484, located two carbons far from Cys481, influences the stability of the BTK-transition complex. Non-covalent inhibitors engage the BTK-kinase domain through an induced-fit mechanism independent of Cys481 interaction and bind to Tyr551 in the activation kink resulting in H3 cleft, determining BTK selectivity. Covalent and non-covalent binding to the kinase domain of BTK shall induce conformational changes in other domains; therefore, investigating the whole-length BTK conformation is necessary to comprehend BTK's autophosphorylation inhibition. Knowledge about the structural complementarity of BTK and its inhibitors supports the optimization of existing drugs and the discovery of drugs for implication in B-cell malignancies and autoimmune diseases.

Turning high-throughput structural biology into predictive inhibitor design

A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilized advance is the increase in structural biology throughput, which has progressed from an artisanal endeavor to a monthly throughput of hundreds of different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high-throughput crystallography data into predictive models for ligand design. Here, we designed a simple machine learning approach that predicts protein-ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein-ligand complexes and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high-throughput crystallography campaign against the SARS-CoV-2 main protease (MPro), obtaining parallel measurements of over 200 protein-ligand complexes and their binding activities. This allows us to design one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a noncovalent and nonpeptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry.

Imidazole-Based Alkaloids from Marine Sponges (Leucetta and Clathrina) as Potential Inhibitors Targeting SARS-CoV-2 Main Protease: An In Silico Approach

Imidazole-based compounds form a prominent class of heterocyclic compounds, displaying diverse applications, especially with regards to its biological and pharmacological activities. Molecular docking, simulations, and drug-likeness prediction were performed on 45 imidazole-based alkaloids from two species of marine sponges (Leucetta and Clathrina). The study seeks to identify possible inhibitors of the SARS-CoV-2 Main Protease in an effort to battle the prevailing pandemic which has been caused by the widespread infections of the SAR-CoV-2 virus in its varied mutated forms. Computational analysis with MOE 2015.10 program reveals that, among the imidazole-based alkaloids, Naamidines have a high affinity for the target protein (PDB ID:6W63), even interacting with the catalytic dyad, as compared to its non-covalent inhibitor X77. Among all the top-scoring ligands, Naamidine H produced the highest binding score of −8.87078 kcal/mol. MD simulation studies with NAMD confirms the stability of the interactions of Naamidines with the target protein. MM-GBSA calculations were performed on the top binding ligands which further confirms the binding affinity of the top-scoring ligands. Computational and pharmacological investigations in this study proposes Naamidines, as effective inhibitors of Mpro. Naamidine I, Naamidine E, and Pyronaamidine could be potential anti-viral candidates against SAR-CoV-2.

In Silico Evaluation of the Thr58-Associated Conserved Water with KRAS Switch-II Pocket Binders.

The KRAS switch-II pocket (SII-P) has proven to be one of the most successful tools for targeting KRAS with small molecules to date. This has been demonstrated with several KRAS(G12C)-targeting covalent inhibitors, already resulting in two FDA-approved drugs. Several earlier-stage compounds have also been reported to engage KRAS SII-P with other position 12 mutants, including G12D, G12S, and G12R. A highly conserved water molecule exists in the KRAS SII-P, linking Thr58 of switch-II and Gly10 of β1 sheet. This conserved water is also present in the cocrystal structures of most of the disclosed small-molecule inhibitors but is only displaced by a handful of SII-P binders. Here, we evaluated the conserved water molecule energetics by the WaterMap for the SII-P binders with publicly disclosed structures and studied the water behavior in the presence of selected inhibitors by microsecond timescale molecular dynamics (MD) simulations using two water models (total simulation time of 120 μs). Our data revealed the high-energy nature of this hydration site when coexisting with an SII-P binder and that there is a preference for a single isolated hydration site in this location within the most advanced compounds. Furthermore, water displacement was only achieved with a few disclosed compounds and was suboptimal, as for instance a cyanomethyl group as a water displacer appears to introduce repulsion with the native conformation of Thr58. These results suggested that this conserved water should be considered more central when designing new inhibitors, especially in the design of noncovalent inhibitors targeting the SII-P.

Next Generation BTK Inhibitors in CLL: Evolving Challenges and New Opportunities.

Ibrutinib revolutionized the CLL treatment approach and prognosis demonstrating its efficacy and safety even at extended follow-up. During the last few years, several next-generation inhibitors have been developed to overcome the occurrence of toxicity or resistance in patients on continuous treatment. In a head-to-head comparison of two phase III trials, both acalabrutinib and zanubrutinib demonstrated a lower incidence of adverse events in respect to ibrutinib. Nevertheless, resistance mutations remain a concern with continuous therapy and were demonstrated with both first- and next-generation covalent inhibitors. Reversible inhibitors showed efficacy independently of previous treatment and the presence of BTK mutations. Other strategies are currently under development in CLL, especially for high-risk patients, and include BTK inhibitor combinations with BCl2 inhibitors with or without anti-CD20 monoclonal antibodies. Finally, new mechanisms for BTK inhibition are under investigations in patients progressing with both covalent and non-covalent BTK and BCl2 inhibitors. Here we summarize and discuss results from main experiences on irreversible and reversable BTK inhibitors in CLL.

Alleviation of arthritis through prevention of neutrophil extracellular traps by an orally available inhibitor of protein arginine deiminase 4

Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes citrullination of protein and its role in autoimmune diseases has been established through clinical genetics and gene knock out studies in mice. Further, studies with PAD4 – deficient mice have shown that PAD4 deficiency does not lead to increased infection or immune suppression, which makes PAD4 an attractive therapeutic target for auto-immune and inflammatory diseases. PAD4 has critical enzymatic role of promoting chromatin decondensation and neutrophil extracellular traps (NETs) formation that is associated with a number of immune-mediated pathological conditions. Here, we present a non-covalent PAD4 inhibitor JBI-589 with high PAD4 isoform selectivity and delineated its binding mode at 2.88 Å resolution by X-ray crystallography. We confirmed its effectiveness in inhibiting NET formation in vitro. Additionally, by using two mouse arthritis models for human rheumatoid arthritis (RA), the well-known disease associated with PAD4 clinically, we established its efficacy in vivo. These results suggest that JBI-589 would be beneficial for both PAD4 and NET-associated pathological conditions.

Identification of and Mechanistic Insights into SARS-CoV-2 Main Protease Non-Covalent Inhibitors: An In-Silico Study.

The indispensable role of the SARS-CoV-2 main protease (Mpro) in the viral replication cycle and its dissimilarity to human proteases make Mpro a promising drug target. In order to identify the non-covalent Mpro inhibitors, we performed a comprehensive study using a combined computational strategy. We first screened the ZINC purchasable compound database using the pharmacophore model generated from the reference crystal structure of Mpro complexed with the inhibitor ML188. The hit compounds were then filtered by molecular docking and predicted parameters of drug-likeness and pharmacokinetics. The final molecular dynamics (MD) simulations identified three effective candidate inhibitors (ECIs) capable of maintaining binding within the substrate-binding cavity of Mpro. We further performed comparative analyses of the reference and effective complexes in terms of dynamics, thermodynamics, binding free energy (BFE), and interaction energies and modes. The results reveal that, when compared to the inter-molecular electrostatic forces/interactions, the inter-molecular van der Waals (vdW) forces/interactions are far more important in maintaining the association and determining the high affinity. Given the un-favorable effects of the inter-molecular electrostatic interactions-association destabilization by the competitive hydrogen bond (HB) interactions and the reduced binding affinity arising from the un-compensable increase in the electrostatic desolvation penalty-we suggest that enhancing the inter-molecular vdW interactions while avoiding introducing the deeply buried HBs may be a promising strategy in future inhibitor optimization.

Discovery and Characterization of BAY-805, a Potent and Selective Inhibitor of Ubiquitin-Specific Protease USP21.

USP21 belongs to the ubiquitin-specific protease (USP) subfamily of deubiquitinating enzymes (DUBs). Due to its relevance in tumor development and growth, USP21 has been reported as a promising novel therapeutic target for cancer treatment. Herein, we present the discovery of the first highly potent and selective USP21 inhibitor. Following high-throughput screening and subsequent structure-based optimization, we identified BAY-805 to be a non-covalent inhibitor with low nanomolar affinity for USP21 and high selectivity over other DUB targets as well as kinases, proteases, and other common off-targets. Furthermore, surface plasmon resonance (SPR) and cellular thermal shift assays (CETSA) demonstrated high-affinity target engagement of BAY-805, resulting in strong NF-κB activation in a cell-based reporter assay. To the best of our knowledge, BAY-805 is the first potent and selective USP21 inhibitor and represents a valuable high-quality in vitro chemical probe to further explore the complex biology of USP21.

Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor

AbstractBruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell–driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell–driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

Design, synthesis, and biological evaluation of some novel naphthoquinone-glycine/β-alanine anilide derivatives as noncovalent proteasome inhibitors.

A series of novel noncovalent glycine/β-alanine anilide derivatives possessing 2-chloronaphthoquinone structure as a pharmacophoric unit were designed, synthesized, and evaluated for their antiproliferative and antiproteasomal activities against MCF-7 cell line, in vitro. According to biological activity results, all the target compounds showed antiproliferative activity in the range of IC50 = 7.10± 0.10-41.08 ± 0.14 μM and most of them exhibited inhibitory efficacy with varying ratios against the three catalytic subunits (β1, β2, and β5) presenting caspase-like (C-L), trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities of proteasome. The antiproteasomal activity evaluations revealed that compounds preferentially inhibited the β5 subunit compared with β1 and β2 subunits of the proteasome. Among the compounds, compounds 7 and 9 showed the highest antiproliferative activity with an IC50 value of 7.10± 0.10 and 7.43 ± 0.25 μM, respectively. Additionally, compound 7 displayed comparable potency to PI-083 lead compound in terms of β5 antiproteasomal activity with an inhibition percentage of 34.67 at 10μM. This compound showed an IC50 value of 32.30 ± 0.45 μM against β5 subunit. Furthermore, molecular modeling studies of the most active compound 7 revealed key interactions with β5 subunit. The results suggest that this class of compounds may be beneficial for the development of new potent proteasome inhibitors.

Targeting the 'Undruggable' Driver Protein, KRAS, in Epithelial Cancers: Current Perspective.

This review summarizes recent development in synthetic drugs and biologics targeting intracellular driver genes in epithelial cancers, focusing on KRAS, and provides a current perspective and potential leads for the field. Compared to biologics, small molecule inhibitors (SMIs) readily penetrate cells, thus being able to target intracellular proteins. However, SMIs frequently suffer from pleiotropic effects, off-target cytotoxicity and invariably elicit resistance. In contrast, biologics are much larger molecules limited by cellular entry, but if this is surmounted, they may have more specific effects and less therapy-induced resistance. Exciting breakthroughs in the past two years include engineering of non-covalent KRAS G12D-specific inhibitor, probody bispecific antibodies, drug-peptide conjugate as MHC-restricted neoantigen to prompt immune response by T-cells, and success in the adoptive cell therapy front in both breast and pancreatic cancers.

Exploration of SARS-CoV-2 Mpro Noncovalent Natural Inhibitors Using Structure-Based Approaches.

With the emergence of antibody-evasive omicron subvariants (BA.2.12.1, BA.4, and BA.5), which can compromise the efficacy of vaccination, it is of utmost importance to widen the finite therapeutic options for COVID-19. Although more than 600 co-crystal complexes of Mpro with inhibitors have been revealed, utilizing them to search for novel Mpro inhibitors remains limited. Although there were two major groups of Mpro inhibitors, covalent and noncovalent inhibitors, noncovalent inhibitors were our main focus due to the safety concerns with their covalent counterparts. Hence, this study aimed to explore Mpro noncovalent inhibition ability of phytochemicals extracted from Vietnamese herbals by combining multiple structure-based approaches. By closely inspecting 223 complexes of Mpro with noncovalent inhibitors, a 3D-pharmacophore model representing typical chemical features of Mpro noncovalent inhibitors was generated with good validation scores (sensitivity = 92.11%, specificity = 90.42%, accuracy = 90.65%, and goodness-of-hit score = 0.61). Afterward, the pharmacophore model was applied to explore the potential Mpro inhibitors from our in-house Vietnamese phytochemical database, revealing 18 substances, 5 of which were in vitro assayed. The remaining 13 substances were then examined by induced-fit molecular docking, revealing 12 suitable compounds. A machine-learning activity prediction model was developed to rank the hit, suggesting nigracin and calycosin-7-O-β-glucopyranoside as promising Mpro natural noncovalent inhibitors.

LSD1 inhibitors for cancer treatment: Focus on multi-target agents and compounds in clinical trials.

Histone lysine-specific demethylase 1 (LSD1/KDM1A) was first identified in 2004 as an epigenetic enzyme able to demethylate specific lysine residues of histone H3, namely H3K4me1/2 and H3K9me1/2, using FAD as the cofactor. It is ubiquitously overexpressed in many types of cancers (breast, gastric, prostate, hepatocellular, and esophageal cancer, acute myeloid leukemia, and others) leading to block of differentiation and increase of proliferation, migration and invasiveness at cellular level. LSD1 inhibitors can be grouped in covalent and non-covalent agents. Each group includes some hybrid compounds, able to inhibit LSD1 in addition to other target(s) at the same time (dual or multitargeting compounds). To date, 9 LSD1 inhibitors have entered clinical trials, for hematological and/or solid cancers. Seven of them (tranylcypromine, iadademstat (ORY-1001), bomedemstat (IMG-7289), GSK-2879552, INCB059872, JBI-802, and Phenelzine) covalently bind the FAD cofactor, and two are non-covalent LSD1 inhibitors [pulrodemstat (CC-90011) and seclidemstat (SP-2577)]. Another TCP-based LSD1/MAO-B dual inhibitor, vafidemstat (ORY-2001), is in clinical trial for Alzheimer's diseases and personality disorders. The present review summarizes the structure and functions of LSD1, its pathological implications in cancer and non-cancer diseases, and the identification of LSD1 covalent and non-covalent inhibitors with different chemical scaffolds, including those involved in clinical trials, highlighting their potential as potent and selective anticancer agents.

Synthesis and Evaluation of 2-Amine-4-oxyphosaniline Pyrimidine Derivatives as EGFR L858R/T790M/C797S Mutant Inhibitors.

Epidermal growth factor receptor (EGFR) C797S mutation leads to Osimertinib drug resistance by disturbing the covalent biding of Michael acceptor group to the Cys797 residue in the ATP biding cleft. In this manuscript, a class of 2-amine-4-oxyphosaniline pyrimidine derivatives were designed, synthesized and evaluated as new noncovalent reversible EGFR inhibitors against L858R/T790M/C797S (CTL) triple mutant. The kinases inhibitiory activity evaluation showed that four compounds exhibited significant inhibitory activities against CTL (IC50 < 30 nM). In particularly, the most promising compound 7a showed excellent enzymatic inhibitory activity against CTL with IC50 value of 9.9 nM, which was more potent than control compound Osimertinib. Moreover, cell proliferation assays indicated that 7a effectively inhibited H1975-EGFR L858R/T790M/C797S with IC50 value of 0.33 µM. Furthermore, compound 7a displayed good metabolic stabilities in human, rat and mouse liver microsomes, and the putative biding mode of compound 7a with ATP was revealed by molecular docking study. These findings strongly indicated that compound 7a was a promising L858R/T790M/C797S mutant EGFR inhibitor.

Development of Highly Potent Noncovalent Inhibitors of SARS-CoV-2 3CLpro.

The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, noncovalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment.