Discovery of macrocyclic covalent inhibitors for severe acute respiratory syndrome coronavirus 2 3CL protease.

The coronavirus disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been spread worldwide for more than 3 years. Although the hospitalization rate and mortality have decreased dramatically due to wide vaccination effort and improved treatment options, the disease is still a global health issue due to constant viral mutations, causing negative impact on social and economic activities. In addition, long COVID and complications arising from COVID-19 weeks after infection have become a concern for public health experts. Therefore, better treatments for COVID-19 are still needed. Herein, we describe a class of macrocyclic peptidomimetic compounds that are potent inhibitors of SARS-Cov-2 3CL protease (3CLpro). Significantly, some of the compounds showed a higher stability against human liver microsomes (HLM t1/2 > 180 min) and may be suitable for oral administration without the need for a pharmacokinetic (PK) boosting agent such as ritonavir.

Enhancement of gemcitabine sensitivity in intrahepatic cholangiocarcinoma through Saikosaponin-a mediated modulation of the p-AKT/BCL-6/ABCA1 axis.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) remains a significant challenge in cancer therapy, especially due to its resistance to established treatments like Gemcitabine, necessitating novel therapeutic approaches. METHODS: This study utilized Gemcitabine-resistant cell lines, patient-derived organotypic tumor spheroids (PDOTs), and patient-derived xenografts (PDX) to evaluate the effects of Saikosaponin-a (SSA) on ICC cellular proliferation, migration, apoptosis, and its potential synergistic interaction with Gemcitabine. Techniques such as transcriptome sequencing, Luciferase reporter assays, and molecular docking were employed to unravel the molecular mechanisms. RESULTS: SSA exhibited antitumor effects in both in vitro and PDX models, indicating its considerable potential for ICC treatment. SSA markedly inhibited ICC progression by reducing cellular proliferation, enhancing apoptosis, and decreasing migration and invasion. Crucially, it augmented Gemcitabine's efficacy by targeting the p-AKT/BCL6/ABCA1 signaling pathway. This modulation led to the downregulation of p-AKT and suppression of BCL6 transcriptional activity, ultimately reducing ABCA1 expression and enhancing chemosensitivity to Gemcitabine. Additionally, ABCA1 was validated as a predictive biomarker for drug resistance, with a direct correlation between ABCA1 expression levels and the IC50 values of various small molecule drugs in ICC gene profiles. CONCLUSION: This study highlights the synergistic potential of SSA combined with Gemcitabine in enhancing therapeutic efficacy against ICC and identifies ABCA1 as a key biomarker for drug responsiveness. Furthermore, the introduction of the novel PDOTs microfluidic model provides enhanced insights into ICC research. This combination strategy may provide a novel approach to overcoming treatment challenges in ICC.

Uncovering the mechanism of Kang-ai injection for treating intrahepatic cholangiocarcinoma based on network pharmacology, molecular docking, and in vitro validation.

Objective: Kang-ai injection (KAI) has been a popular adjuvant treatment for solid tumors, but its anti-tumor mechanism in intrahepatic cholangiocarcinoma (ICC) remains poorly understood. This study applied a network pharmacology-based approach to unveil KAI's anti-tumor activity, key targets, and potential pharmacological mechanism in ICC by integrating molecular docking and in vitro validation. Methods: The KAI-compound-target-ICC network was constructed to depict the connections between active KAI compounds and ICC-related targets based on the available data sources. The crucial ingredients, potential targets, and signaling pathways were screened using GO, KEGG enrichment analysis, and the PPI network. Molecular docking was performed to visualize the interactions between hub targets and components. In vitro experiments were carried out to validate the findings. Results: Among the 87 active components of KAI and 80 KAI-ICC-related targets, bioinformatics analysis identified quercetin as a possible candidate. GO and KEGG enrichment analysis indicated that the PI3K-AKT signaling pathway might be essential in ICC pharmacotherapy. The PPI network and its sub-networks screened 10 core target genes, including AKT1 and IL1ß. Molecular docking results showed stable binding between AKT1 and IL1ß with KAI active ingredients. The in vitro experiments confirmed that KAI might suppress the proliferation of ICC cell lines by inhibiting the PI3K/AKT signaling pathway, consistent with the network pharmacology approach and molecular docking predictions. Conclusion: The study sheds light on KAI's biological activity, potential targets, and molecular mechanisms in treating ICC and provides a promising strategy for understanding the scientific basis and therapeutic mechanisms of herbal treatments for ICC. This research has important implications for developing new, targeted therapies for ICC and highlights the importance of network pharmacology-based approaches in investigating complex herbal formulations.

Long non-coding RNA SNHG22 facilitates hepatocellular carcinoma tumorigenesis and angiogenesis via DNA methylation of microRNA miR-16-5p.

Hepatocellular carcinoma (HCC) is considered as a common malignancy worldwide. Considerable evidence has illustrated that abnormally expressed long noncoding RNAs (lncRNAs) are in a close correlation with the initiation and progression of various tumors, including HCC. LncRNA small nucleolar RNA host gene 22 (SNHG22) has been reported to play important roles in tumor initiation, but its role and mechanism are little known in HCC. In our report, we discovered the high level of SNHG22 in HCC tissues and cells, and the high expression of SNHG22 was correlated with unfavorable clinical outcome in HCC patients. Functional assays implied that SNHG22 deficiency suppressed cell proliferation, migration, invasion, and angiogenesis in vitro. Additionally, it was also confirmed that silenced SNHG22 suppressed tumor growth and angiogenesis in vivo. Mechanistic exploration revealed that SNHG22 recruited DNMT1 to miR-16-5p DNA promoter through EZH2 and inhibited miR-16-5p transcription via DNA methylation. Finally, we verified that the suppression of miR-16-5p countervailed the suppressive effect of SNHG22 deficiency on HCC cell proliferation, migration, invasion, and angiogenesis. Conclusively, this study clarified the SNHG22/EZH2/DNMT1/miR-16-5p axis and revealed that SNHG22 could be an underlying biomarker for HCC.

LncRNA LINC01857 promotes cell growth and diminishes apoptosis via PI3K/mTOR pathway and EMT process by regulating miR-141-3p/MAP4K4 axis in diffuse large B-cell lymphoma.

LINC01857 has been proven to be involved in glioma and breast cancer. However, the biological function of LINC01857 in diffuse large B-cell lymphoma (DLBCL) is poorly investigated. By accessing to the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEX), LINC01857 expression was found upregulated in both DLBCL tissues and cells. Cell proliferation and flow cytometry assays showed that LINC01857 promoted proliferation and cell cycle, but suppressed apoptosis in DLBCL cells. Bioinformatics analysis and luciferase reporter assay confirmed that LINC01857 may serve as a sponge for miR-141-3p and miR-141-3p may target MAP4K4. Mechanically, the regulatory action of miR-141-3p/MAP4K4 on DLBCL cellular behaviors was regulated by LINC01857. In addition, LINC01857 could increase the activity of PI3K/mTOR pathway and facilitate the EMT process in a miR-141-3p-mediated manner in DLBCL. Our data illustrated that the LINC01857/miR-141-3p/MAP4K4 might function as a promising therapeutic avenue for DLBCL treatment.

LINC01232 exerts oncogenic activities in pancreatic adenocarcinoma via regulation of TM9SF2.

Pancreatic adenocarcinoma (PAAD), one of the most prevailing malignant tumors in digestive system, is identified as one of the main culprits of cancer-associated mortality. Despite long intergenic non-protein coding RNA 1232 (LINC01232) is found to be upregulated in TCGA PAAD tissues and associated with poor prognosis, the potential of LINC01232 in PAAD progression still needs more explorations. In this study, LINC01232 was chosen to be the research object in PAAD cellular processes. Functionally, loss-of function assays were carried out and the experimental results indicated that suppression of LINC01232 hindered the deterioration of PAAD by affecting cell proliferation and migration. Furthermore, relationship between LINC01232 and its nearby gene transmembrane 9 superfamily member 2 (TM9SF2) was investigated. The same expression pattern of TM9SF2 in TCGA PAAD samples was observed. It was found that upregulation of LINC01232 could be a crucial factor for the dysregulation of TM9SF2. Mechanistically, LINC01232 recruited EIF4A3 to boost TM9SF2 mRNA stability. Besides, our findings demonstrated that the transcriptional activation of LINC01232 and TM9SF2 was mediated by SP1. Therefore, we concluded that LINC01232 executed carcinogenic properties in PAAD progression via regulation of TM9SF2. In conclusion, this study was the first to unveil the role and molecular mechanism of LINC01232, suggesting LINC01232 as a promising molecular target for pancreatic cancer treatment.

MicroRNA-449b-5p Suppresses Proliferation, Migration, and Invasion of Osteosarcoma by Targeting c-Met.

BACKGROUND The aim of this study was to clarify the biological function of microRNA-449b-5p in the progression of osteosarcoma (OS) and to define the underlying mechanism. MATERIAL AND METHODS Relative levels of microRNA-449b-5p in OS tissues and cell lines was determined by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between microRNA-449b-5p level and pathological characteristics of OS patients was analyzed by chi-square test. Kaplan-Meier analysis was used for survival analysis of OS patients based on their expression level of microRNA-449b-5p. Regulatory effects of microRNA-449b-5p on cellular behaviors of OS cells were evaluated by cell counting kit-8 (CCK-8) and Transwell assay. The binding relationship between microRNA-449b-5p and c-Met was verified through dual-luciferase reporter gene assay, and their interaction in OS progression was further examined through a series of rescue experiments. RESULTS MicroRNA-449b-5p was expressed at low levels in OS. Lower levels of microRNA-449b-5p were seen in OS tissues with worse tumor grade or histological differentiation. OS patients with low levels of microRNA-449b-5p had worse overall survival relative to those with high level of microRNA-449b-5p. Overexpression of microRNA-449b-5p markedly attenuated proliferative, migratory, and invasive abilities of OS cells. C-Met is the downstream target of microRNA-449b-5p, and its level was inhibited in OS cells overexpressing microRNA-449b-5p. Importantly, c-Met partially rescued the inhibitory effects of microRNA-449b-5p on behavior of OS cells. CONCLUSIONS MicroRNA-449b-5p is downregulated in OS, which alleviates the malignant progression of OS by targeting c-Met.

Studies on functional connections between the supraoptic nucleus and the stomach in rats.

The present study was to investigate whether there are functional connections between the hypothalamic supraoptic nucleus (SON) and the stomach, which is the case with the paraventricular nucleus. The rats were divided into four groups. Group I: the neuronal discharge was recorded extracellularly in the NTS, DMV or SON before and after cold physiological saline (4°C) was perfused into the stomach and effused from the duodenum. Group II: the rats were stimulated as for Group I and c-Fos expression in NTS, DMV and SON was examined. Group III: the control to Group II. Group IV: gastric motility was recorded continuously before and after microinjection of L: -Glu into the SON. In Group I, the discharge frequency increased in all the three nuclei, while in Group II, Fos expression in NTS, DMV and SON was, respectively, greater than that of Group III. In Group IV, microinjection of L: -Glu (5 nmol) into SON significantly inhibited gastric motility. These data suggest there are functional connections between SON and stomach.

The role of catecholaminergic neurons in the hypothalamus and medullary visceral zone in response to restraint water-immersion stress in rats.

The activity of catecholaminergic neurons in the hypothalamus and the medullary visceral zone (MVZ) in rats in response to restraint water-immersion stress (RWIS) was measured by use of dual Fos and tyrosine hydroxylase (TH) immunohistochemistry. In RWIS rats Fos immunoreactive (Fos-IR) nuclei dramatically increased in the paraventricular nucleus (PVN), the supraoptic nucleus (SON), the dorsal motor nucleus of the vagus (DMV), the nucleus of the solitary tract (NTS), the area postrema (AP), and the ventrolateral medulla (VLM). A small number of TH-immunoreactive (TH-IR) and Fos/TH double-labeling neurons in the PVN, and their absence from the SON, were observed in both RWIS and nonstressed rats. More TH-IR neurons were observed in the MVZ of RWIS rats than in nonstressed rats. In RWIS and nonstressed rats, the percentage of Fos-IR nuclei in TH-IR neurons was 38.0 and 14.3% in the DMV, 34.4 and 9.7% in the NTS, 18.6 and 4.5% in the AP, and 45.7 and 18.9% in the VLM, respectively. In conclusion, catecholaminergic neurons in the MVZ are involved in the response to RWIS; although the PVN and SON also participate in the response to RWIS, the mechanism is not via catecholaminergic neurons.

[Effects of L-glutamate microinjection into the ventral horn of spinal cord gray matter on gastrocnemius contractility in toad].

A diagram of motor neuron pool of ventral horn of spinal cord gray matter in toad was first delineated. Different concentrations (1, 0.5, 0.1, 0.01 mol/L) of excitatory amino acid L-Glu or physiological saline (0.65% NaCl) were then microinjected into the motor neuron pool in a urethane-anaesthetized toad. The contraction curve of the gastrocnemius was then recorded by the BL-420 Physiological Signal Recording. We took the maximal tension, the duration of rising phase, the velocity of tension variation, and the duration of descending phase as the parameters to study the characteristic of gastrocnemius contractility. It was found that the gastrocnemius contractility of all the 4 groups was tetanus but differed in degree, especially the maximal tension, and velocity of tension variation. In contrast to physiological saline, gastrocnemius contracted by the stimulation of L-Glu, and the contraction parameter showed dose-effect relationships except for the duration of descending phase, which was caused by the combination rate of L-Glu and the receptor.