SARS-CoV-2 papain-like protease (PLpro) is a compelling but historically underdeveloped antiviral target. Unlike the viral main protease (Mpro), which rapidly became the focus of intensive drug-discovery efforts and yielded clinical candidates and approved drugs, PLpro posed a more challenging medicinal chemistry problem: a shallow, flexible substrate-recognition surface and a mobile BL2 loop. Nevertheless, PLpro is a high-profile drug target because it is vital for viral replication by processing viral polyproteins and suppresses host innate immunity through deubiquitinating and deISGylating activities. These dual functions make PLpro more than a viral protease; it is a multifunctional immune-evasion enzyme whose inhibition could both block virus replication and restore antiviral host responses. This Account summarizes our group's effort to convert PLpro from a challenging target into a tractable antiviral drug-discovery platform. We began by developing and applying orthogonal assays to identify specific PLpro inhibitors and triage false positives. High-throughput screening and drug-repurposing campaigns yielded early hits, including Jun9722, Jun9754, and tropifexor, but also revealed that biochemical inhibition alone was insufficient to predict cellular antiviral activity. This motivated us to develop a FlipGFP cell-based reporter assay as a BSL-2-compatible bridge between enzymology and live-virus studies. In addition, we later developed a fluorescence polarization assay using a fluorescein-labeled PLpro ligand to enable direct, high-throughput quantification of inhibitor binding. Together with FRET enzymatic assays, thermal shift experiments, cellular FlipGFP assays, and antiviral assays, these tools established a rigorous validation framework for PLpro medicinal chemistry. With this platform in place, we pursued structure-based PLpro inhibitor design. Early cocrystal structures showed that potent noncovalent inhibitors engage the BL2 groove and stabilize inhibitor-bound PLpro conformations. A major conceptual advance came from structural analysis of the Jun11313-bound PLpro complex, which revealed that an inhibitor substituent occupied a hydrophobic surface pocket corresponding to the Val70 position of ubiquitin. We designated this newly recognized region the Val70Ub pocket. Exploiting this pocket transformed PLpro inhibitor design by expanding ligand engagement beyond the canonical BL2 groove and enabling substantial gains in enzymatic inhibition and antiviral activity. This design principle led to orally active noncovalent inhibitors, including Jun12682 and the quinoline lead Jun13296, both of which showed potent enzymatic inhibition, cellular antiviral activity, favorable mouse pharmacokinetics, and protection in SARS-CoV-2 mouse infection models. We further extended the Val70Ub-centered recognition strategy to covalent inhibitor design by appending cysteine-reactive warheads (covalent electrophiles) to optimized noncovalent scaffolds, thereby generating compounds that retained BL2 groove and Val70Ub binding while engaging the catalytic Cys111. Finally, resistance studies identified E167, Y268, and Q269 as drug resistance hotspots, highlighting the need to design inhibitors that engage less mutation-sensitive binding sites. Overall, this Account illustrates how integrated assay development, structural biology, medicinal chemistry, pharmacology, virology, and resistance analysis can transform a challenging viral deubiquitinase into a credible antiviral target. The lessons from PLpro should inform future efforts to design broad-spectrum coronavirus PLpro inhibitors and to target other viral protease-deubiquitinase enzymes with shallow, flexible binding surfaces.

The COVID-19 pandemic spurred the rapid development of nirmatrelvir, a main protease (Mpro) inhibitor now widely prescribed as part of Paxlovid (nirmatrelvir plus ritonavir). However, increasing use has raised concerns about drug resistance. Resistance selection studies have identified multiple Mpro mutations, with E166V emerging as a particularly resistant variant. Sequencing data from COVID-19 patients confirms E166V as a clinically relevant mutation, and importantly, this substitution also confers cross-resistance to several next-generation Mpro inhibitors under development. In response, this study reports the rational design of inhibitors active against nirmatrelvir-resistant E166V/A mutants. The lead candidate, Jun13698, shows potent inhibition of both wild-type Mpro and the E166V/A mutants. Structural studies and molecular dynamics simulations reveal that Jun13698 forms stable complexes with wild-type and mutant proteases, consistent with its potent enzymatic and antiviral activity. Together, these findings position Jun13698 as a promising next-generation Mpro inhibitor capable of overcoming clinically relevant nirmatrelvir resistance.

Enterovirus D68 (EV-D68) is a respiratory virus that often causes mild to moderate respiratory illnesses and, in severe cases, can lead to paralysis and rarely death, mainly in children. There is currently no vaccine or antiviral for EV-D68. Here, we report the rational design of viral 2 C inhibitors for treating EV-D68 infection-induced paralysis in a neonatal mouse model. Viral 2 C protein is a multi-functional protein vital for viral replication. Structure-based drug design identifies Jun6504 showing potent and broad-spectrum antiviral activity against multiple strains of EV-D68, EV-A71, and CVB3, as well as favorable in vitro and in vivo pharmacokinetic properties. In a neonatal mouse model of EV-D68 infection, Jun6504 significantly improves paralysis score and weight gain when administered immediately or 24 hours post-infection. Jun6504 also reduces viral titers in the spinal cord and the infected quadriceps muscle. Collectively, Jun6504 represents a promising candidate for further development as an EV-D68 antiviral.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is the culprit behind the COVID-19 pandemic, which has killed millions of people worldwide. SARS-CoV-2 harbors a crucial papain-like protease domain, named PLpro, in its non-structural protein 3 (nsp3). This enzyme is not only required for processing the viral polyprotein at the nsp1/2, nsp2/3, and nsp3/4 cleavage junctions but also impairs the host immune system by removing ubiquitin and ISG15 modifications from host proteins. Aiming to address the pressing need for additional oral antivirals due to emerging SARS-CoV-2 variants and drug-resistant mutants, we focused on targeting PLpro. Despite its relatively limited exploration compared to other viral proteins, its essential role in viral replication and impact on the host immune response warranted further investigation. In this study, we determined co-crystal structures of PLpro with nine inhibitors using X-ray crystallography and step-by-step modification of the compounds based on their interactions with their binding pocket. First, we discovered a novel binding site (the “Val70Ub site”) for the covalent inhibitor Jun11313 near the established BL2 groove pocket. Comparative analysis with ubiquitin-bound and ISG15-bound PLpro structures (PDB 6XAA and 7RBS) revealed that the thienyl group of Jun11313 occupied the same hydrophobic site as Val70 from ubiquitin and Leu152 from ISG15. Leveraging the Val70Ub site and the BL2 groove, we obtained multiple PLpro inhibitors with inhibitory constants (Ki values) in the two-digit nanomolar range. The co-crystal structures of SARS-CoV-2 PLpro with eight biarylphenyl PLpro inhibitors, Jun11941, Jun12129, Jun12303, Jun12162, Jun12199, Jun12197, Jun12145, and Jun12682, were solved (2.5–3.1 Å resolution). The lead compound, Jun12682, exhibited efficacy against SARS-CoV-2 and its variants (Ki = 38.5 to 63.5 nM), including nirmatrelvir-resistant strains, with EC50 values ranging from 0.44 to 2.02 μM. Twice-daily oral administration of Jun12682 significantly improved survival rates, reduced lung viral loads, and mitigated lesions in a mouse model of SARS-CoV-2 infection. These findings highlight the promise of PLpro inhibitors, particularly Jun12682, as potential oral antiviral candidates against SARS-CoV-2.

Improving in silico compound-protein interaction (CPI) predictability is critical for productive drug discovery. Current deep learning approaches largely rely on end-to-end models trained on limited labeled CPI data, overlooking preexisting, specialized compound-protein input representations. We present Ligand Extra trees-Accelerated Docking (LEAD), a virtual screening framework that accelerates docking by integrating rapid first-pass CPI prediction via ET-Screen. Unlike end-to-end models, ET-Screen uses seven distinct representations, including embeddings from large-scale protein language models and molecular transformers, as well as an original CPI potential fingerprint. ET-Screen's contrastive compression neural network maps the consolidated 2,457-dimensional compound-protein representation into a compact, discriminative form optimized for CPI classification by an ensemble of decision trees. ET-Screen outperforms state-of-the-art end-to-end approaches by up to 23.39% on diverse retrospective benchmarks while being ~100x faster than standard-precision docking. Its speed enables triaging ~10.8 million prospective drug candidates across five targets, reducing them to 10,000 for docking within the LEAD framework and ultimately yielding novel, experimentally-validated hits. ### Competing Interest Statement The authors have declared no competing interest.

The ever-evolving SARS-CoV-2 variants necessitate the development of additional oral antivirals. This study presents the systematic design of quinoline-containing SARS-CoV-2 papain-like protease (PLpro) inhibitors as potential oral antiviral drug candidates. By leveraging the recently discovered Val70Ub binding site in PLpro, we designed a series of quinoline analogs demonstrating potent PLpro inhibition and antiviral activity. Notably, the X-ray crystal structures of 6 lead compounds reveal that the 2-aryl substitution can occupy either the Val70Ub site as expected or the BL2 groove in a flipped orientation. The in vivo lead Jun13296 exhibits favorable pharmacokinetic properties and potent inhibition against SARS-CoV-2 variants and nirmatrelvir-resistant mutants. In a mouse model of SARS-CoV-2 infection, oral treatment with Jun13296 significantly improves survival, reduces body weight loss and lung viral titers, and prevents lung tissue damage. These results underscore the potential of quinoline PLpro inhibitors as promising oral SARS-CoV-2 antiviral candidates, instilling hope for the future of SARS-CoV-2 treatment.

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 (PL

The emergence of SARS-CoV-2 variants and drug-resistant mutants calls for additional oral antivirals. The SARS-CoV-2 papain-like protease (PL pro ) is a promising but challenging drug target. We designed and synthesized 85 noncovalent PL pro inhibitors that bind to a recently discovered ubiquitin binding site and the known BL2 groove pocket near the S4 subsite. Leads inhibited PL pro with the inhibitory constant K i values from 13.2 to 88.2 nanomolar. The co-crystal structures of PL pro with eight leads revealed their interaction modes. The in vivo lead Jun12682 inhibited SARS-CoV-2 and its variants, including nirmatrelvir-resistant strains with EC 50 from 0.44 to 2.02 micromolar. Oral treatment with Jun12682 improved survival and reduced lung viral loads and lesions in a SARS-CoV-2 infection mouse model, suggesting that PL pro inhibitors are promising oral SARS-CoV-2 antiviral candidates.

The COVID-19 pandemic is caused by SARS-CoV-2, a highly transmissible and pathogenic RNA betacoronavirus. Like other RNA viruses, SARS-CoV-2 continues to evolve with or without drug selection pressure, and many variants have emerged since the beginning of the pandemic. The papain-like protease, PLpro, is a cysteine protease that cleaves viral polyproteins as well as ubiquitin and ISG15 modifications from host proteins. Leveraging our recently discovered Val70Ub binding site in PLpro, we designed covalent PLpro inhibitors by connecting cysteine reactive warheads to the biarylphenyl PLpro inhibitors via flexible linkers. Several leads displayed potent enzymatic inhibition (IC50 = 0.1-0.3 μM) and antiviral activity (EC50 = 0.09-0.96 μM). Fumaramide inhibitors Jun13567 (15), Jun13728 (16), and Jun13714 (18) showed favorable in vivo pharmacokinetic properties with intraperitoneal injection. The X-ray crystal structure of PLpro with Jun13567 (15) validated our design strategy, revealing covalent conjugation between the catalytic Cys111 and the fumaramide warhead. The results suggest these covalent PLpro inhibitors are promising SARS-CoV-2 antiviral drug candidates.

SARS-CoV-2 main protease (Mpro) is a validated antiviral drug target of nirmatrelvir, the active ingredient in Pfizer's oral drug Paxlovid. Drug-drug interactions limit the use of Paxlovid. In addition, drug-resistant Mpro mutants against nirmatrelvir have been identified from cell culture viral passage and naturally occurring variants. As such, there is a need for a second generation of Mpro inhibitors. In this study, we explored several reactive warheads in the design of Mpro inhibitors. We identified Jun11119R (vinyl sulfonamide warhead), Jun10221R (propiolamide warhead), Jun1112R (4-chlorobut-2-ynamide warhead), Jun10541R (nitrile warhead), and Jun10963R (dually activated nitrile warhead) as potent Mpro inhibitors. Jun10541R and Jun10963R also had potent antiviral activity against SARS-CoV-2 in Calu-3 cells with EC50 values of 2.92 and 6.47 μM, respectively. X-ray crystal structures of Mpro with Jun10541R and Jun10221 revealed covalent modification of Cys145. These Mpro inhibitors with diverse reactive warheads collectively represent promising candidates for further development.

Enterovirus D68 (EV-D68) virus is a nonpolio enterovirus that typically causes respiratory illness and, in severe cases, can lead to paralysis and death in children. There is currently no vaccine or antiviral for EV-D68. We previously discovered the viral 2A protease (2Apro) as a viable antiviral drug target and identified telaprevir as a 2Apro inhibitor. 2Apro is a viral cysteine protease that cleaves the viral VP1-2A polyprotein junction. In this study, we report the X-ray crystal structures of EV-D68 2Apro, wild-type, and the C107A mutant and the structure-based lead optimization of telaprevir. Guided by the X-ray crystal structure, we predicted the binding pose of telaprevir in 2Apro using molecular dynamics simulations. We then utilized this model to inform structure-based optimization of the telaprevir's reactive warhead and P1-P4 substitutions. These efforts led to the discovery of 2Apro inhibitors with improved antiviral activity than telaprevir. These compounds represent promising lead compounds for further development as EV-D68 antivirals.

ConspectusSARS-CoV-2 is the etiological pathogen of the COVID-19 pandemic, which led to more than 6.5 million deaths since the beginning of the outbreak in December 2019. The unprecedented disruption of social life and public health caused by COVID-19 calls for fast-track development of diagnostic kits, vaccines, and antiviral drugs. Small molecule antivirals are essential complements of vaccines and can be used for the treatment of SARS-CoV-2 infections. Currently, there are three FDA-approved antiviral drugs, remdesivir, molnupiravir, and paxlovid. Given the moderate clinical efficacy of remdesivir and molnupiravir, the drug–drug interaction of paxlovid, and the emergence of SARS-CoV-2 variants with potential drug-resistant mutations, there is a pressing need for additional antivirals to combat current and future coronavirus outbreaks.In this Account, we describe our efforts in developing covalent and noncovalent main protease (Mpro) inhibitors and the identification of nirmatrelvir-resistant mutants. We initially discovered GC376, calpain inhibitors II and XII, and boceprevir as dual inhibitors of Mpro and host cathepsin L from a screening of a protease inhibitor library. Given the controversy of targeting cathepsin L, we subsequently shifted the focus to designing Mpro-specific inhibitors. Specifically, guided by the X-ray crystal structures of these initial hits, we designed noncovalent Mpro inhibitors such as Jun8-76-3R that are highly selective toward Mpro over host cathepsin L. Using the same scaffold, we also designed covalent Mpro inhibitors with novel cysteine reactive warheads containing di- and trihaloacetamides, which similarly had high target specificity. In parallel to our drug discovery efforts, we developed the cell-based FlipGFP Mpro assay to characterize the cellular target engagement of our rationally designed Mpro inhibitors. The FlipGFP assay was also applied to validate the structurally disparate Mpro inhibitors reported in the literature. Lastly, we introduce recent progress in identifying naturally occurring Mpro mutants that are resistant to nirmatrelvir from genome mining of the nsp5 sequences deposited in the GISAID database. Collectively, the covalent and noncovalent Mpro inhibitors and the nirmatrelvir-resistant hot spot residues from our studies provide insightful guidance for future work aimed at developing orally bioavailable Mpro inhibitors that do not have overlapping resistance profile with nirmatrelvir.

T-type calcium channels activate in response to subthreshold membrane depolarizations and represent an important source of Ca2+ influx near the resting membrane potential. These channels regulate neuronal excitability and have been linked to pain. For this reason, T-type calcium channels are suitable molecular targets for the development of new non-opioid analgesics. Our previous work identified an analogue of benzimidazolonepiperidine, 5bk, that preferentially inhibited CaV3.2 channels and reversed mechanical allodynia. In this study, we synthesized and screened a small library of 47 compounds derived from 5bk. We found several compounds that inhibited the Ca2+ influx in DRG neurons of all sizes. After separating the enantiomers of each active compound, we found two compounds, 3-25-R and 3-14-3-S, that potently inhibited the Ca2+ influx. Whole-cell patch clamp recordings from small- to medium-sized DRG neurons revealed that both compounds decreased total Ca2+. Application of 3-14-3-S (but not 3-25-R) blocked transiently expressed CaV3.1-3.3 channels with a similar IC50 value. 3-14-3-S decreased T-type, but not N-type, Ca2+ currents in DRG neurons. Furthermore, intrathecal delivery of 3-14-3-S relieved tonic, neuropathic, and inflammatory pain in preclinical models. 3-14-3-S did not exhibit any activity against G protein-coupled opioid receptors. Preliminary docking studies also suggest that 3-14-3-S can bind to the central pore domain of T-type channels. Together, our chemical characterization and functional and behavioral data identify a novel T-type calcium channel blocker with in vivo efficacy in experimental models of tonic, neuropathic, and inflammatory pain.

SARS-CoV-2 is the causative agent of the COVID-19 pandemic. The approval of vaccines and small-molecule antivirals is vital in combating the pandemic. The viral polymerase inhibitors remdesivir and molnupiravir and the viral main protease inhibitor nirmatrelvir/ritonavir have been approved by the U.S. FDA. However, the emergence of variants of concern/interest calls for additional antivirals with novel mechanisms of action. The SARS-CoV-2 papain-like protease (PLpro) mediates the cleavage of viral polyprotein and modulates the host's innate immune response upon viral infection, rendering it a promising antiviral drug target. This Perspective highlights major achievements in structure-based design and high-throughput screening of SARS-CoV-2 PLpro inhibitors since the beginning of the pandemic. Encouraging progress includes the design of non-covalent PLpro inhibitors with favorable pharmacokinetic properties and the first-in-class covalent PLpro inhibitors. In addition, we offer our opinion on the knowledge gaps that need to be filled to advance PLpro inhibitors to the clinic.

The marine hydrographic measurement system is used to complete the information measurement system of seabed topography, sea water sound velocity profile, flow velocity, flow direction profile, pressure and so on. In this paper, a marine hydrographic measurement system is described.

TGF-β type I receptor (also known as activin-like kinase 5 or ALK5) plays a critical role in the progression of fibrotic diseases and tumor invasiveness and metastasis, as well. The development of small inhibitors targeting ALK5 has been validated as a potential therapeutic strategy for fibrotic diseases and cancer. Here, we developed various 4-((1-cyclopropyl-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-yl) oxy) pyridine-2-yl) amino derivatives as ALK5 inhibitors. The optimization led to identification of potent and selective ALK5 inhibitors 12r. The compound 12r exhibited strong inhibitory activity both in vitro and in vivo, and pharmacokinetics study showed an oral bioavailability of 57.6%. Thus, compound 12r may provide as new therapeutic option as ALK5 TGF-βR1 inhibitor.