Chemical Space Profiling of SARS-CoV-2 PLpro Using DNA-Encoded Focused Libraries.

DNA-encoded library (DEL) technology is gaining attention for its rapid construction and deconvolution capabilities. Our study explored a novel strategy using rational DELs tailored for the SARS-CoV-2 papain-like protease, which revealed new fragments. Structural changes post-DEL screening mimic traditional medicinal chemistry lead optimization. We unveiled unique aromatic structures offering an alternative optimization path. Notably, we identified superior binding fragments targeting the BL2 groove. Derivative 16 emerged as the most promising by exhibiting IC50 values of 0.25 µM. Derivative 6, which features an aromatic fragment capped with a naphthalene moiety, showed IC50 values of 2.91 µM. Molecular modeling revealed hydrogen bond interactions with Lys157 residue and potential covalent interactions with nearby amino acid residues. This research underscored DEL's potential for fragment-based drug discovery against SARS-CoV-2 protease.

Identification of Dihydrobenzofuran Neolignans as Novel PDE4 Inhibitors and Evaluation of Antiatopic Dermatitis Efficacy in DNCB-Induced Mice Model.

Atopic dermatitis is a chronic relapsing skin disease characterized by recurrent, pruritic, localized eczema, while PDE4 inhibitors have been reported to be effective as antiatopic dermatitis agents. 3',4-O-dimethylcedrusin (DCN) is a natural dihydrobenzofuran neolignan isolated from Magnolia biondii with moderate potency against PDE4 (IC50 = 3.26 ± 0.28 µM) and a binding mode similar to that of apremilast, an approved PDE4 inhibitor for the treatment of psoriasis. The structure-based optimization of DCN led to the identification of 7b-1 that showed high inhibitory potency on PDE4 (IC50 = 0.17 ± 0.02 µM), good anti-TNF-α activity (EC50 = 0.19 ± 0.10 µM), remarkable selectivity profile, and good skin permeability. The topical treatment of 7b-1 resulted in the significant benefits of pharmacological intervention in a DNCB-induced atopic dermatitis-like mice model, demonstrating its potential for the development of novel antiatopic dermatitis agents.

Structure-based development of potent and selective type-II kinase inhibitors of RIPK1.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a key regulator in inflammation and cell death and is involved in mediating a variety of inflammatory or degenerative diseases. A number of allosteric RIPK1 inhibitors (RIPK1i) have been developed, and some of them have already advanced into clinical evaluation. Recently, selective RIPK1i that interact with both the allosteric pocket and the ATP-binding site of RIPK1 have started to emerge. Here, we report the rational development of a new series of type-II RIPK1i based on the rediscovery of a reported but mechanistically atypical RIPK3i. We also describe the structure-guided lead optimization of a potent, selective, and orally bioavailable RIPK1i, 62, which exhibits extraordinary efficacies in mouse models of acute or chronic inflammatory diseases. Collectively, 62 provides a useful tool for evaluating RIPK1 in animal disease models and a promising lead for further drug development.

Pigment Diversity in Leaves of Caladium × hortulanum Birdsey and Transcriptomic and Metabolic Comparisons between Red and White Leaves.

Leaf color is a key ornamental characteristic of cultivated caladium (Caladium × hortulanum Birdsey), a plant with diverse leaf colors. However, the genetic improvement of leaf color in cultivated caladium is hindered by the limited understanding of leaf color diversity and regulation. In this study, the chlorophyll and anthocyanin content of 137 germplasm resources were measured to explore the diversity and mechanism of leaf color formation in cultivated caladium. Association analysis of EST-SSR markers and pigment traits was performed, as well as metabolomics and transcriptomics analysis of a red leaf variety and its white leaf mutant. We found significant differences in chlorophyll and anthocyanin content among different color groups of cultivated caladium, and identified three, eight, three, and seven EST-SSR loci significantly associated with chlorophyll-a, chlorophyll-b, total chlorophyll and total anthocyanins content, respectively. The results further revealed that the white leaf mutation was caused by the down-regulation of various anthocyanins (such as cyanidin-3-O-rutinoside, quercetin-3-O-glucoside, and others). This change in concentration is likely due to the down-regulation of key genes (four PAL, four CHS, six CHI, eight F3H, one F3'H, one FLS, one LAR, four DFR, one ANS and two UFGT) involved in anthocyanin biosynthesis. Concurrently, the up-regulation of certain genes (one FLS and one LAR) that divert the anthocyanin precursors to other pathways was noted. Additionally, a significant change in the expression of numerous transcription factors (12 NAC, 12 bZIP, 23 ERF, 23 bHLH, 19 MYB_related, etc.) was observed. These results revealed the genetic and metabolic basis of leaf color diversity and change in cultivated caladium, and provided valuable information for molecular marker-assisted selection and breeding of leaf color in this ornamental plant.

Oral Simnotrelvir for Adult Patients with Mild-to-Moderate Covid-19.

BACKGROUND: Simnotrelvir is an oral 3-chymotrypsin-like protease inhibitor that has been found to have in vitro activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and potential efficacy in a phase 1B trial. METHODS: In this phase 2-3, double-blind, randomized, placebo-controlled trial, we assigned patients who had mild-to-moderate coronavirus disease 2019 (Covid-19) and onset of symptoms within the past 3 days in a 1:1 ratio to receive 750 mg of simnotrelvir plus 100 mg of ritonavir or placebo twice daily for 5 days. The primary efficacy end point was the time to sustained resolution of symptoms, defined as the absence of 11 Covid-19-related symptoms for 2 consecutive days. Safety and changes in viral load were also assessed. RESULTS: A total of 1208 patients were enrolled at 35 sites in China; 603 were assigned to receive simnotrelvir and 605 to receive placebo. Among patients in the modified intention-to-treat population who received the first dose of trial drug or placebo within 72 hours after symptom onset, the time to sustained resolution of Covid-19 symptoms was significantly shorter in the simnotrelvir group than in the placebo group (180.1 hours [95% confidence interval {CI}, 162.1 to 201.6] vs. 216.0 hours [95% CI, 203.4 to 228.1]; median difference, -35.8 hours [95% CI, -60.1 to -12.4]; P = 0.006 by Peto-Prentice test). On day 5, the decrease in viral load from baseline was greater in the simnotrelvir group than in the placebo group (mean difference [±SE], -1.51±0.14 log10 copies per milliliter; 95% CI, -1.79 to -1.24). The incidence of adverse events during treatment was higher in the simnotrelvir group than in the placebo group (29.0% vs. 21.6%). Most adverse events were mild or moderate. CONCLUSIONS: Early administration of simnotrelvir plus ritonavir shortened the time to the resolution of symptoms among adult patients with Covid-19, without evident safety concerns. (Funded by Jiangsu Simcere Pharmaceutical; ClinicalTrials.gov number, NCT05506176.).

Structure-based design of potent FABP4 inhibitors with high selectivity against FABP3.

Fatty-acid binding protein 4 (FABP4) presents an attractive target for therapeutic intervention in metabolic and inflammatory diseases in recent years. However, highly similar three-dimensional structures and fatty acid binding ability of multiple FABP family members pose a significant challenge in design of FABP4-selective inhibitors. Particularly, inhibition of FABP3 raises safety concerns such as cardiac dysfunction and exercise intolerance. Here, we reported the discovery of new FABP4 inhibitors with high selectivity over FABP3 by exploiting the little structural difference in the ligand binding pockets of FABP4 and FABP3. On the basis of our previously reported FABP4 inhibitors with nanomolar potency, different substituents were further introduced to perfectly occupy two sub-pockets of FABP4 that are distinct from those of FABP3. Remarkably, a single methyl group introduction leads to the discovery of compound C3 that impressively exhibits a 601-fold selectivity over FABP3 when maintained nanomolar binding affinity for FABP4. Moreover, C3 also shows good metabolic stability and potent cellular anti-inflammatory activity, making it a promising inhibitor for further development. Therefore, the present study highlights the utility of the structure-based rational design strategy for seeking highly selective and potent inhibitors of FABP4 and the importance of identifying the appropriate subsite as well as substituent for gaining the desired selectivity.

Design, synthesis and biological evaluation of betulinic acid derivatives as potential inhibitors of 3CL-protease of SARS-CoV-2.

During the coronavirus reproduction process, 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) are accountable for the fragmentation of two polyprotein precursors (pp1a/pp1ab) into substructural proteins. These two proteins are vital for the replication and transcription of the viral genome. Therefore, 3CLpro is a key protein and target for the design of coronavirus inhibitors. In previous studies, we found that betulinic acid has an inhibitory effect on 3CLpro, with 51.5 % inhibition of 3CLpro at 20 µM. Then, series of betulinic acid derivatives were designed, synthesized, and evaluated for their inhibition activities. The results showed that BA02 and BA05 showed significant inhibitory activity on 3CLpro with inhibitory rates of 78.1 % and 82.5 % at 20 µM, respectively. Further evaluation of these two compounds shows that their IC50 values are 7.22 ± 0.14 µM and 6.40 ± 0.14 µM, respectively.

Ent-labdane-type diterpene glycosides obtained from Rubus chingii Hu and their inhibitory effects on PDE5A activity.

In this study, 16 new ent-labdane-type diterpene glycosides, designated as goshonosides J1-J16 (1-16), along with nine previously known diterpene glycosides (17-25) were extracted from the fruits of Rubus chingii Hu. The structures of goshonosides J1-J16 were elucidated using various analytical techniques, such as nuclear magnetic resonance, electron capture detector ECD, high-resolution electrospray ionization mass spectrometry HREIMS, single-crystal X-ray diffraction, and hydrolysis. Furthermore, the isolates' efficacy in inhibiting the activity of phosphodiesterase type 5 A was evaluated. Goshonosides J1, J2, and G effectively inhibited the activity of the aforementioned enzyme (IC50 values: 6.15 ± 1.76, 3.27 ± 0.65, and 9.61 ± 2.36 µM, respectively). Our findings highlight the remarkable structural diversity of bioactive compounds in R. chingii Hu and offer insights into the use of this shrub.

Repurposing of Rutan showed effective treatment for COVID-19 disease.

Previously, from the tannic sumac plant (Rhus coriaria), we developed the Rutan 25 mg oral drug tablets with antiviral activity against influenza A and B viruses, adenoviruses, paramyxoviruses, herpes virus, and cytomegalovirus. Here, our re-purposing study demonstrated that Rutan at 25, 50, and 100 mg/kg provided a very effective and safe treatment for COVID-19 infection, simultaneously inhibiting two vital enzyme systems of the SARS-CoV-2 virus: 3C-like proteinase (3CLpro) and RNA-dependent RNA polymerase (RdRp). There was no drug accumulation in experimental animals' organs and tissues. A clinical study demonstrated a statistically significant decrease in the C-reactive protein and a reduction of the viremia period. In patients receiving Rutan 25 mg (children) and 100 mg (adults), the frequency of post-COVID-19 manifestations was significantly less than in the control groups not treated with Rutan tablets. Rutan, having antiviral activity, can provide safe treatment and prevention of COVID-19 in adults and children. Clinical Trial Registration: ClinicalTrials.gov, ID NCT05862883.

Drug repurposing and structure-based discovery of new PDE4 and PDE5 inhibitors.

Phosphodiesterase-4 (PDE4) and PDE5 responsible for the hydrolysis of intracellular cAMP and cGMP, respectively, are promising targets for therapeutic intervention in a wide variety of diseases. Here, we report the discovery of novel, drug-like PDE4 inhibitors by performing a high-throughput drug repurposing screening of 2560 approved drugs and drug candidates in clinical trial studies. It allowed us to identify eight potent PDE4 inhibitors with IC50 values ranging from 0.41 to 2.46 µM. Crystal structures of PDE4 in complex with four compounds, namely ethaverine hydrochloride (EH), benzbromarone (BBR), CX-4945, and CVT-313, were further solved to elucidate molecular mechanisms of action of these new inhibitors, providing a solid foundation for optimizing the inhibitors to improve their potency as well as selectivity. Unexpectedly, selectivity profiling of other PDE subfamilies followed by crystal structure determination revealed that CVT-313 was also a potent PDE5 inhibitor with a binding mode similar to that of tadalafil, a marketed PDE5 inhibitor, but distinctively different from the binding mode of CVT-313 with PDE4. Structure-guided modification of CVT-313 led to the discovery of a new inhibitor, compound 2, with significantly improved inhibitory activity as well as selectivity towards PDE5 over PDE4. Together, these results highlight the utility of the drug repurposing in combination with structure-based drug design in identifying novel inhibitors of PDE4 and PDE5, which provides a prime example for efficient discovery of drug-like hits towards a given target protein.

Multi-omics for COVID-19: driving development of therapeutics and vaccines.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has raised global concern for public health and economy. The development of therapeutics and vaccines to combat this virus is continuously progressing. Multi-omics approaches, including genomics, transcriptomics, proteomics, metabolomics, epigenomics and metallomics, have helped understand the structural and molecular features of the virus, thereby assisting in the design of potential therapeutics and accelerating vaccine development for COVID-19. Here, we provide an up-to-date overview of the latest applications of multi-omics technologies in strategies addressing COVID-19, in order to provide suggestions towards the development of highly effective knowledge-based therapeutics and vaccines.

A first-in-human phase 1 study of simnotrelvir, a 3CL-like protease inhibitor for treatment of COVID-19, in healthy adult subjects.

Safe and efficacious antiviral therapeutics are in urgent need for the treatment of coronavirus disease 2019. Simnotrelvir is a selective 3C-like protease inhibitor that can effectively inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the safety, tolerability, and pharmacokinetics of dose escalations of simnotrelvir alone or with ritonavir (simnotrelvir or simnotrelvir/ritonavir) in healthy subjects, as well as the food effect (ClinicalTrials.gov Identifier: NCT05339646). The overall incidence of adverse events (AEs) was 22.2% (17/72) and 6.3% (1/16) in intervention and placebo groups, respectively. The simnotrelvir apparent clearance was 135-369 L/h with simnotrelvir alone, and decreased significantly to 19.5-29.8 L/h with simnotrelvir/ritonavir. The simnotrelvir exposure increased in an approximately dose-proportional manner between 250 and 750 mg when co-administered with ritonavir. After consecutive twice daily dosing of simnotrelvir/ritonavir, simnotrelvir had a low accumulation index ranging from 1.39 to 1.51. The area under the curve of simnotrelvir increased 44.0 % and 47.3 % respectively, after high fat and normal diet compared with fasted status. In conclusion, simnotrelvir has adequate safety and tolerability. Its pharmacokinetics indicated a trough concentration above the level required for 90 % inhibition of SARS-CoV-2 in vitro at 750 mg/100 mg simnotrelvir/ritonavir twice daily under fasted condition, supporting further development using this dosage as the clinically recommended dose regimen.

Structure-based development and preclinical evaluation of the SARS-CoV-2 3C-like protease inhibitor simnotrelvir.

The persistent pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants accentuates the great demand for developing effective therapeutic agents. Here, we report the development of an orally bioavailable SARS-CoV-2 3C-like protease (3CLpro) inhibitor, namely simnotrelvir, and its preclinical evaluation, which lay the foundation for clinical trials studies as well as the conditional approval of simnotrelvir in combination with ritonavir for the treatment of COVID-19. The structure-based optimization of boceprevir, an approved HCV protease inhibitor, leads to identification of simnotrelvir that covalently inhibits SARS-CoV-2 3CLpro with an enthalpy-driven thermodynamic binding signature. Multiple enzymatic assays reveal that simnotrelvir is a potent pan-CoV 3CLpro inhibitor but has high selectivity. It effectively blocks replications of SARS-CoV-2 variants in cell-based assays and exhibits good pharmacokinetic and safety profiles in male and female rats and monkeys, leading to robust oral efficacy in a male mouse model of SARS-CoV-2 Delta infection in which it not only significantly reduces lung viral loads but also eliminates the virus from brains. The discovery of simnotrelvir thereby highlights the utility of structure-based development of marked protease inhibitors for providing a small molecule therapeutic effectively combatting human coronaviruses.

Discovery of novel cGAS inhibitors based on natural flavonoids.

Cyclic GMP-AMP synthase (cGAS) plays an important role in the inflammatory response. It has been reported that aberrant activation of cGAS is associated with a variety of immune-mediated inflammatory disorders. The development of small molecule inhibitors of cGAS has been considered as a promising therapeutic strategy for the diseases. Flavonoids, a typical class of natural products, are known for their anti-inflammatory activities. Although cGAS is closely associated with inflammation, the potential effects of natural flavonoid compounds on cGAS have been rarely studied. Therefore, we screened an in-house natural flavonoid library by pyrophosphatase (PPiase) coupling assay and identified novel cGAS inhibitors baicalein and baicalin. Subsequently, crystal structures of the two natural flavonoids in complex with human cGAS were determined, which provide mechanistic insight into the anti-inflammatory activities of baicalein and baicalin at the molecular level. After that, a virtual screening based on the crystal structures of baicalein and baicalin in complex with human cGAS was performed. As a result, compound C20 was identified to inhibit both human and mouse cGAS with IC50 values of 2.28 and 1.44 µM, respectively, and its detailed interactions with human cGAS were further revealed by the X-ray crystal structure determination. These results demonstrate the potential of natural products used as hits in drug discovery and provide valuable hints for further development of cGAS inhibitors.

Cross-Compatibility in Interspecific Hybridization of Different Curcuma Accessions.

Curcuma is extensively cultivated as a medicinal and ornamental plant in tropical and subtropical regions. Due to the bright bract color, distinctive inflorescence and long blooming period, it has become a new favorite in terms of the urban landscape, potted flowers and cut flowers. However, little research on breeding new cultivars using traditional plant breeding methods is available on the genus Curcuma. In the present study, pollen viability and stigma receptivity evaluation were performed, and the genetic relationship of 38 Curcuma accessions was evaluated, then 5 C. alismatifolia Gagnep. (Ca), 2 C. hybrid (Ch), 2 C. sparganiifolia Gagnep. cultivars and 4 Curcuma native species were selected as parents for subsequent interspecific cross-breeding. A total of 132 reciprocal crosses were carried out for interspecific hybridization, including 70 obverse and 62 inverse crosses. Obvious discrepancies among fruit-setting rates were manifested in different combinations and in reciprocal crosses. Results showed that the highest fruit-setting rate (87.5%) was observed in the Ca combinations. There were 87 combinations with a fruit-setting rate of 0%, which meant nearly 65.9% was incompatible. We concluded that C. alismatifolia 'Siam Shadow' (Ch34) was suitable as a male parent and C. petiolata Roxb. (Cpet) was suitable as a female parent to improve the fruit-setting rates. The maximum number of seeds per fruit (45.4) was obtained when C. alismatifolia 'Chiang Mai Pink' (Ca01) was used as a female parent followed by C. attenuata Wall. ex Baker (Catt) (42.8) and C. alismatifolia 'Splash' (Ca63) (39.6) as male parents. The highest germination rate was observed for the Ca group followed by Catt and C. sparganiifolia 'Maetang Sunrise' (Csms). The germination rates of Ca accessions ranged from 58.2% (C. alismatifolia 'Siam Scarlet' (Ca06) as a male parent) to 89.3% (C. alismatifolia 'Sitone' (Ca10) as a male parent) with an average value of 74.0%. Based on the results of hybrid identification, all the individuals from the four combinations exhibited paternal-specific bands, indicating that the true hybrid rates of crossings were 100%. Our results would facilitate the interspecific hybridization and introduction of genetic variation from wild species into the cultivars in Curcuma in the future, which could be helpful in realizing the sustainable application in urban green areas.

Discovery and Mechanism Study of SARS-CoV-2 3C-like Protease Inhibitors with a New Reactive Group.

3CLpro is an attractive target for the treatment of COVID-19. Using the scaffold hopping strategy, we identified a potent inhibitor of 3CLpro (3a) that contains a thiocyanate moiety as a novel warhead that can form a covalent bond with Cys145 of the protein. Tandem mass spectrometry (MS/MS) and X-ray crystallography confirmed the mechanism of covalent formation between 3a and the protein in its catalytic pocket. Moreover, several analogues of compound 3a were designed and synthesized. Among them, compound 3h shows the best inhibition of 3CLpro with an IC50 of 0.322 µM and a kinact/Ki value of 1669.34 M-1 s-1, and it exhibits good target selectivity for 3CLpro against host proteases. Compound 3c inhibits SARS-CoV-2 in Vero E6 cells (EC50 = 2.499 µM) with low cytotoxicity (CC50 > 200 µM). These studies provide ideas and insights to explore and develop new 3CLpro inhibitors in the future.

Efficacy and safety of SIM0417 (SSD8432) plus ritonavir for COVID-19 treatment: a randomised, double-blind, placebo-controlled, phase 1b trial.

Background: SIM0417 (SSD8432) is an orally administered coronavirus main proteinase (3CLpro) inhibitor with potential anti-SARS-CoV-2 activity. This study aimed to evaluate the efficacy and safety of SIM0417 plus ritonavir (a pharmacokinetic enhancer) in adults with COVID-19. Methods: This was a randomised, double-blind, placebo-controlled, phase 1b study in China. Adults with asymptomatic infection, mild or moderate COVID-19 were randomly assigned (3:3:2) to receive either 750 mg SIM0417 plus 100 mg ritonavir, 300 mg SIM0417 plus 100 mg ritonavir or placebo every 12 h for 10 doses. The main efficacy endpoints included SARS-CoV-2 viral load, proportion of participants with positive SARS-CoV-2 nucleic acid test and time to alleviation of COVID-19 symptoms. This trial is registered with ClinicalTrials.gov, NCT05369676. Findings: Between May 12 and August 29, 2022, 32 participants were enrolled and randomised to high dose group (n = 12), low dose group (n = 12) or placebo (n = 8). The viral load change from baseline in high dose group was statistically lower compared with placebo, with a maximum mean difference of -2.16 ± 0.761 log10 copies/mL (p = 0.0124) on Day 4. The proportion of positive SARS-CoV-2 in both active groups were lower than the placebo. The median time to sustained alleviation of COVID-19 symptoms was 2.0 days in high dose group versus 6.0 days in the placebo group (HR = 3.08, 95% CI 0.968-9.818). SIM0417 plus ritonavir were well tolerated with all adverse events in grade 1. Interpretation: SIM0417 plus ritonavir was generally well tolerated. The efficacy of SIM0417 showed a monotonic dose-response relationship, and the 750 mg SIM0417 plus 100 mg ritonavir was selected as the recommended clinical dose. Funding: The study was funded by Jiangsu Simcere Pharmaceutical Co., Ltd.

Structural insights into dimerization and activation of the mGlu2-mGlu3 and mGlu2-mGlu4 heterodimers.

Heterodimerization of the metabotropic glutamate receptors (mGlus) has shown importance in the functional modulation of the receptors and offers potential drug targets for treating central nervous system diseases. However, due to a lack of molecular details of the mGlu heterodimers, understanding of the mechanisms underlying mGlu heterodimerization and activation is limited. Here we report twelve cryo-electron microscopy (cryo-EM) structures of the mGlu2-mGlu3 and mGlu2-mGlu4 heterodimers in different conformational states, including inactive, intermediate inactive, intermediate active and fully active conformations. These structures provide a full picture of conformational rearrangement of mGlu2-mGlu3 upon activation. The Venus flytrap domains undergo a sequential conformational change, while the transmembrane domains exhibit a substantial rearrangement from an inactive, symmetric dimer with diverse dimerization patterns to an active, asymmetric dimer in a conserved dimerization mode. Combined with functional data, these structures reveal that stability of the inactive conformations of the subunits and the subunit-G protein interaction pattern are determinants of asymmetric signal transduction of the heterodimers. Furthermore, a novel binding site for two mGlu4 positive allosteric modulators was observed in the asymmetric dimer interfaces of the mGlu2-mGlu4 heterodimer and mGlu4 homodimer, and may serve as a drug recognition site. These findings greatly extend our knowledge about signal transduction of the mGlus.

Discovery of Polyphenolic Natural Products as SARS-CoV-2 Mpro Inhibitors for COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has forced the development of direct-acting antiviral drugs due to the coronavirus disease 2019 (COVID-19) pandemic. The main protease of SARS-CoV-2 is a crucial enzyme that breaks down polyproteins synthesized from the viral RNA, making it a validated target for the development of SARS-CoV-2 therapeutics. New chemical phenotypes are frequently discovered in natural goods. In the current study, we used a fluorogenic assay to test a variety of natural products for their ability to inhibit SARS-CoV-2 Mpro. Several compounds were discovered to inhibit Mpro at low micromolar concentrations. It was possible to crystallize robinetin together with SARS-CoV-2 Mpro, and the X-ray structure revealed covalent interaction with the protease's catalytic Cys145 site. Selected potent molecules also exhibited antiviral properties without cytotoxicity. Some of these powerful inhibitors might be utilized as lead compounds for future COVID-19 research.

Design, synthesis and biological evaluation of covalent peptidomimetic 3CL protease inhibitors containing nitrile moiety.

In this paper, a series of peptidomimetic SARS-CoV-2 3CL protease inhibitors with new P2 and P4 positions were synthesized and evaluated. Among these compounds, 1a and 2b exhibited obvious 3CLpro inhibitory activities with IC50 of 18.06 nM and 22.42 nM, respectively. 1a and 2b also showed excellent antiviral activities against SARS-CoV-2 in vitro with EC50 of 313.0 nM and 170.2 nM, respectively, the antiviral activities of 1a and 2b were 2- and 4-fold better than that of nirmatrelvir, respectively. In vitro studies revealed that these two compounds had no significant cytotoxicity. Further metabolic stability tests and pharmacokinetic studies showed that the metabolic stability of 1a and 2b in liver microsomes was significantly improved, and 2b had similar pharmacokinetic parameters to that of nirmatrelvir in mice.