Structure-based design of pan-coronavirus inhibitors targeting host cathepsin L and calpain-1.

Respiratory disease caused by coronavirus infection remains a global health crisis. Although several SARS-CoV-2-specific vaccines and direct-acting antivirals are available, their efficacy on emerging coronaviruses in the future, including SARS-CoV-2 variants, might be compromised. Host-targeting antivirals provide preventive and therapeutic strategies to overcome resistance and manage future outbreak of emerging coronaviruses. Cathepsin L (CTSL) and calpain-1 (CAPN1) are host cysteine proteases which play crucial roles in coronaviral entrance into cells and infection-related immune response. Here, two peptidomimetic α-ketoamide compounds, 14a and 14b, were identified as potent dual target inhibitors against CTSL and CAPN1. The X-ray crystal structures of human CTSL and CAPN1 in complex with 14a and 14b revealed the covalent binding of α-ketoamide groups of 14a and 14b to C25 of CTSL and C115 of CAPN1. Both showed potent and broad-spectrum anticoronaviral activities in vitro, and it is worth noting that they exhibited low nanomolar potency against SARS-CoV-2 and its variants of concern (VOCs) with EC50 values ranging from 0.80 to 161.7 nM in various cells. Preliminary mechanistic exploration indicated that they exhibited anticoronaviral activity through blocking viral entrance. Moreover, 14a and 14b exhibited good oral pharmacokinetic properties in mice, rats and dogs, and favorable safety in mice. In addition, both 14a and 14b treatments demonstrated potent antiviral potency against SARS-CoV-2 XBB 1.16 variant infection in a K18-hACE2 transgenic mouse model. And 14b also showed effective antiviral activity against HCoV-OC43 infection in a mouse model with a final survival rate of 60%. Further evaluation showed that 14a and 14b exhibited excellent anti-inflammatory effects in Raw 264.7 mouse macrophages and in mice with acute pneumonia. Taken together, these results suggested that 14a and 14b are promising drug candidates, providing novel insight into developing pan-coronavirus inhibitors with antiviral and anti-inflammatory properties.

Direct Construction of C-Alkyl Glycosides from Non-Activated Olefins via Nickel-Catalyzed C(sp3)─C(sp3) Coupling Reaction.

Among C-glycosides, C-alkyl glycosides are significant building blocks for natural products and glycopeptides. However, research on efficient construction methods for C-alkyl glycosides remains relatively limited. Compared with Michael acceptors, non-activated olefins are more challenging substrates and have rarely been employed in the construction of C-glycosides. Here, a highly efficient and convenient approach for the synthesis of C-alkyl glycosides through a nickel-catalyzed C(sp3)-C(sp3) coupling reaction is presented. A distinctive feature of this method is its utilization of non-activated olefins as the anomeric radical acceptors for hydroalkylation, allowing for the direct formation of C-glycoside bonds in a single step. Furthermore, this method demonstrates excellent compatibility with a broad scope of highly reactive functional groups. Mechanistic investigations suggest that the reaction proceeds via a free radical pathway, leading predominantly to the formation of products with α-configuration. Overall, this innovative methodology offers a versatile and practical approach for the synthesis of C-alkyl glycosides, offering new avenues for the production of intricate glycosides with potential applications in drug discovery and chemical biology.

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.

Comparative transcriptome analysis reveals the effects of different feeding times on the hepatopancreas of Chinese mitten crabs.

Feeding rhythms affect a range of physiological functions in crustaceans. To investigate their effect on the physiological functions of Eriocheir sinensis, herein, we analyzed the influence of different feeding times on the hepatopancreas transcriptome via high-throughput sequencing. We harvested the hepatopancreas of crabs at 12:00 on day 11 of the experiment. We weighted the crabs before and after the experiment and found that those in the 06:00 group had the highest weight gain rate. In addition, 512 differentially expressed genes (DEGs) were grouped into nine distinct clusters. Functional enrichment analysis of DEGs showed that E. sinensis metabolic and immune processes were affected by the feeding time. Furthermore, we mapped the DEGs involved in retinol metabolism and the lysosome pathway. To our knowledge, this is the first comparative transcriptomic analysis of the hepatopancreas of E. sinensis based on different feeding times, which provides multi-level information to reveal the mechanism underlying the regulation of feeding rhythms in E. sinensis.

Cobalt(II)-Catalyzed C(sp3 )-C(sp3 ) Coupling for the Direct Stereoselective Synthesis of 2-Deoxy-C-Glycosides from Glycals.

We report a ligand-controlled CoII -catalyzed C(sp3 )-C(sp3 ) coupling hydroalkylation for direct and ß-selective synthesis of 2-deoxy-C-glycosides from glycals. This reaction proceeds by a radical pathway for alkyl halide activation and is ß-selective through ligand control. This approach may inspire the development of further stereoselective coupling reactions with potential application in the field of carbohydrates.

Deep sequencing of microRNAs reveals circadian-dependent microRNA expression in the eyestalks of the Chinese mitten crab Eriocheir sinensis.

MicroRNAs (miRNAs) are small endogenous non-coding RNAs. In crustaceans, miRNAs might be involved in the regulation of circadian rhythms. Many physiological functions of crustaceans including immunity and hormone secretion exhibit circadian rhythms, but it remains unclear whether specific miRNAs contribute to the alteration of crustacean physiological processes under circadian rhythms. This study investigated the mechanisms of miRNA regulation of circadian rhythms in the Chinese mitten crab (Eriocheir sinensis), one of China's most important aquaculture species. We obtained eyestalks from crab specimens at four time points (6:00; 12:00; 18:00; 24:00) during a 24-h period. We identified 725 mature miRNAs, with 23 known miRNAs differentially expressed depending on the time of day. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the putative target genes for differentially expressed miRNAs were significantly enriched in the immune response and endocrine-related pathways. Numerous putative target genes are involved in the circadian-related pathways and enriched on circadian-control genes. These results suggest that the expression of miRNAs regulates some specific physiological functions in E. sinensis under circadian cycles. We also profiled various putative target genes enriched under the circadian-related pathway. This study performed miRNA expression in the eyestalks of E. sinensis during a 24-h daily cycle, providing insights into the molecular mechanism underlying crustacean circadian rhythms and suggesting miRNAs' role in studying crustacean physiology should not be overlooked.

Discovery, synthesis and mechanism study of 2,3,5-substituted [1,2,4]-thiadiazoles as covalent inhibitors targeting 3C-Like protease of SARS-CoV-2.

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC50 values ranging from 0.118 to 0.582 µM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC50 value of 7.249 µM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a, one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study.

Hepatopancreas immune response during different photoperiods in the Chinese mitten crab, Eriocheir sinensis.

Photoperiod plays an important role in the growth, development, and metabolism of crustaceans. The growth and reproduction of crabs are closely related to the photoperiod. The hepatopancreas is an important source of innate immune molecules; however, hepatopancreatic patterns of gene expression depending on the photoperiod-which may underlie changes in immune mechanisms-remain unknown. To study the molecular basis of immune regulation in the Chinese mitten crab (Eriocheir sinensis) under different light conditions, a new generation of high-throughput Illumina sequencing technology was used, and functional genes associated with immune function in the hepatopancreas of this crab were explored via assembly of high-quality sequences, gene annotation, and classification. A total of 383,899,798 clean reads from the hepatopancreas of the normal group (12 h/12 h L:D), 387,936,676 clean reads from the continuous light group (24 h/0 h L:D), and 384,872,734 clean reads from the continuous darkness group (0 h/24 h L:D) were obtained. Compared with the normal group, 141, 152, 60, 87, 90, and 101 differentially expressed genes were identified in the groups exposed to continuous light for 2 days, continuous darkness for 2 days, continuous light for 4 days, continuous darkness for 4 days, continuous light for 6 days, and continuous darkness for 6 days, respectively. The results of this study revealed that under continuous light and dark conditions, the crabs were most affected by light on day 2, but the interference gradually decreased with time. We suggest that long-term light or dark treatment makes crabs adaptable to fluctuations in the photoperiod. The expression of genes associated with immune response patterns was found to change during different photoperiods. Prophenoloxidase (proPO) and serine proteinase (kazal-type serine proteinase inhibitor 1 and serine proteinase inhibitor-3) in the proPO-activating system were significantly upregulated in the 2-day continuous light group. Glutathione peroxidase 3 was significantly downregulated under continuous light exposure, while cyclooxygenase was upregulated in the continuous light and dark environments. These results provide insights into the molecular mechanism underlying the effects of the photoperiod on immune regulation and the physiological activity of E. sinensis.

Intestinal bacterial community composition of juvenile Chinese mitten crab Eriocheir sinensis under different feeding times in lab conditions.

Feeding time is an important factor affecting the physiological activity and feeding rhythm of crustaceans. However, little is known about the factors and mechanisms contributing to variations in feeding time in aquatic species or their impacts. Moreover, the gut microbiome largely affects host physiology and is associated with diet. To investigate the effects of different feeding times on the composition of intestinal bacterial communities, high-throughput 16S rRNA sequencing was used to monitor the gut bacteria of the Chinese mitten crab Eriocheir sinensis over a 10-day period under different feeding times: 06:00 h, 12:00 h, 18:00 h, and 24:00 h. Weight gain of the day-fed groups was significantly higher than that of the night-fed groups. Two probiotics, Akkermansia muciniphila and Faecalibacterium prausnitzii, were detected in the intestines of crabs in the 12:00 group. In addition, the diversity and richness of the flora in the 12:00 group were slightly higher than those in the other treatment groups. These results collectively indicate that different feeding times change the intestinal flora composition of Chinese mitten crabs, and further identified specific feeding times associated with a more significant weight gain effect. Our findings provide important insights into improving farming strategies for Chinese mitten crabs.

Comparative transcriptome analysis reveals the impact of the daily rhythm on the hemolymph of the Chinese mitten crab (Eriocheir sinensis).

The daily rhythm affects a series of physiological functions in crustaceans. To study its effect on the physiological function in Eriocheir sinensis, a crustacean species of high economic value, we analyzed the hemolymph transcriptome during the daily rhythm by high-throughput sequencing. We sampled the hemolymph from crabs at four time points in a single day (06:00, 12:00, 18:00, and 24:00 h) and identified 3,01,661 and 1,03,998 transcripts and unigenes, respectively; some of the unigenes were annotated as core clock genes. Moreover, 15,564 differentially expressed genes (DEGs) were divided into nine different clusters. Functional enrichment analysis of DEGs indicated that the molting, metabolism, and immunity processes in E. sinensis were impacted by its daily rhythm. In addition, we mapped the DEGs involved in the daily entrainment pathway. To the best of our knowledge, this is the first comparative transcriptome analysis of crustacean hemolymph during the day-night cycle, and provides multi-level information for unraveling the finer regulatory effects of the daily cycle in crustaceans.

Comparative transcriptome analysis of Chinese grass shrimp (Palaemonetes sinensis) hepatopancreas under ectoparasitic isopod (Tachaea chinensis) infection.

Tachaea chinensis, a parasitic isopod, negatively affects the production of several commercially important shrimp species. To better understand the interaction between shrimp immunity and isopod infection, we performed a transcriptome analysis of the hepatopancreas of Palaemonetes sinensis challenged with T. chinensis. After assembly and annotation, 75,980 high-quality unigenes were obtained using RNA-seq data. Differential gene expression analysis revealed 896 significantly differently expressed genes (DEGs) after infection, with 452 and 444 upregulated and downregulated genes, respectively. Specifically, expression levels of genes involved in detoxification, such as the interferon regulatory factor, venom carboxylesterase-6, serine proteinase inhibitor, and cytochrome P450, were upregulated. Furthermore, expression levels of genes corresponding to retinol dehydrogenase, triosephosphate isomerase, variant ionotropic glutamate receptor, and phosphoenolpyruvate carboxykinase were significantly upregulated after isopod parasitization, indicating that the shrimp's visual system was influenced by isopod parasitization. Moreover, quantitative real-time PCR of 10 DEGs helped validate the RNA-seq findings. These results provide a valuable basis for future studies on the elucidation of immune responses of P. sinensis to T. chinensis infection.

Activated carbon/Brønsted acid-promoted aerobic benzylic oxidation under "on-water" condition: Green and efficient synthesis of 3-benzoylquinoxalinones as potent tubulin inhibitors.

Green chemistry is becoming the favored approach to preparing drug molecules in pharmaceutical industry. Herein, we developed a clean and efficient method to synthesize 3-benzoylquinoxalines via activated carbon promoted aerobic benzylic oxidation under "on-water" condition. Moreover, biological studies with this class of compounds reveal an antiproliferative profile. Further structure modifications are performed and the investigations exhibited that the most active 12a could inhibit the microtubule polymerization by binding to tubulin and thus induce multipolar mitosis, G2/M phase arrest, and apoptosis of cancer cells. In addition, molecular docking studies allow the rationalization of the pharmacodynamic properties observed. Our systematic studies provide not only guidance for applications of O2/AC/H2O system, but also a new scaffold targeting tubulin for antitumor agent discovery.