Combinatorial Chemistry & High Throughput Screening - Volume 26, Issue 3, 2023

With the continuous development of structural biology, the requirement for accurate threedimensional structures during functional modulation of biological macromolecules is increasing. Therefore, determining the dynamic structures of bio-macromolecular at high resolution has been a highpriority task. With the development of cryo-electron microscopy (cryo-EM) techniques, the flexible structures of biomacromolecules at the atomic resolution level grow rapidly. Nevertheless, it is difficult for cryo-EM to produce high-resolution dynamic structures without a great deal of manpower and time. Fortunately, deep learning, belonging to the domain of artificial intelligence, speeds up and simplifies this workflow for handling the high-throughput cryo-EM data. Here, we generalized and summarized some software packages and referred algorithms of deep learning with remarkable effects on cryo-EM data processing, including Warp, user-free preprocessing routines, TranSPHIRE, PARSED, Topaz, crYOLO, and self-supervised workflow, and pointed out the strategies to improve the resolution and efficiency of three-dimensional reconstruction. We hope it will shed some light on the bio-macromolecular dynamic structure modeling with the deep learning algorithms.

The ongoing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic has been proven to be more severe than the previous coronavirus outbreaks due to the virus’ high transmissibility. With the emergence of new variants, this global phenomenon took a more dramatic turn, with many countries recently experiencing higher surges of confirmed cases and deaths. On top of this, the inadequacy of effective treatment options for COVID-19 aggravated the problem. As a way to address the unavailability of target-specific viral therapeutics, computational strategies have been employed to hasten and systematize the search. The objective of this review is to provide initial data highlighting the utility of polyphenols as potential prophylaxis or treatment for COVID-19. In particular, presented here are virtually screened polyphenolic compounds which showed potential as either antagonists to viral entry and host cell recognition through binding with various receptor-binding regions of SARS-CoV-2 spike protein or as inhibitors of viral replication and post-translational modifications through binding with essential SARS-CoV-2 non-structural proteins.

Introduction: TLRs are fundamental elements in the orchestration of the innate immune system. These receptors seem to be responsible for the inflammation and fibrosis in chronic dacryocystitis. The aim of the present study was to investigate the role of the toll-Like receptors (TLR2 and TLR4) signaling pathway and its downstream effector chemokine genes in the pathogenesis of chronic dacryocystitis. Methods: This study was conducted on 20 patients diagnosed with chronic dacryocystitis and underwent external dacryocystorhinostomy. Estimation of gene expression of TLR2, TLR4, CCL2, CCL4, CXCL3, CXCR4, and c-FOS genes in the lacrimal sac tissues was performed together with the assessment of the inflammatory markers TNFα, IL-1β, IFN-γ, and IL-22. Histopathological examination of the lacrimal sac walls using hematoxylin and eosin (H) stain, in addition to immunohistochemical staining of the CD68 and CD163 macrophage markers, was also performed. Results: Our results showed that TLR2, TLR4, and c-FOS gene expressions were significantly increased in the chronic dacryocystitis group with a subsequent increase in their downstream effector chemokine genes CCL2, CCL4, and CXCL3. This up-regulation of genes was accompanied by macrophage shift of polarization toward the M1 pro-inflammatory phenotype (increased CD68 and decreased CD163 expression), leading to increased levels of the pro-inflammatory cytokines (TNF- α, IL-1β and IFN-γ) and decreased anti-inflammatory marker IL-22 with chronic dacryocystitis. Conclusion: It is essential to fine-tune TLR activation through emerging therapeutic approaches. Targeting TLR signaling at the level of receptors or downstream adaptor molecules represents a new challenge for treating chronic dacryocystitis.

Background: Intestinal ischemia-reperfusion (I/R) injury occurs in several clinical situations and after intestinal transplantation. This study aimed to examine the role of rhubarb peony decoction (RPD) in intestinal I/R injury. Methods: Different concentrations of RPD were set to treat IEC-6 and Caco-2 cells. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry assays. High-throughput transcriptome sequencing was performed on IEC-6 cells treated with hypoxia-reoxygenation (HR) or HR and RPD. Results: RPD treatment significantly promoted the proliferation of IEC-6 and Caco-2 cells and inhibited apoptosis. Sequencing results identified 109 significantly up-regulated genes and 36 significantly down-regulated genes in the RPD group. In addition, the results of western blot suggested that HR induced the expression of c-Fos, and the treatment of RPD prevented the HR-induced c- Fos expression. Importantly, knockdown of c-Fos rescued the HR-inhibited cell proliferation and HR-induced apoptosis. Conclusions: In conclusion, RPD was beneficial in protecting the survival of intestinal epithelial cells under HR stress. Furthermore, the increase in c-Fos expression after HR stress was closely related to the proliferation and apoptosis of intestinal epithelial cells.

Aims: This study aimed to investigate the correlation between gene expression and immune cell infiltration and the overall survival rate in tumor tissues, which may contribute to the therapy and prognosis of small cell lung cancer (SCLC) patients. Background: SCLC is the most aggressive type of lung neoplasm. There is no proper marker for the treatment and prediction of prognosis in SCLC. Objectives: Three gene expression profiles of SCLC patients were obtained from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified between normal lung samples and SCLC lung samples. Methods: Functional enrichment analysis of all DEGs was performed to explore the linkage among DEGs, the tumor immune microenvironment, and SCLC tumorigenesis. The common genes among the 3 groups in the Venn diagram and hub genes in protein-protein interaction (PPI) networks were considered potential key genes in SCLC patients. The TIMER (tumor immune estimation resource) database calculation and Kaplan–Meier survival curves were used to investigate the association between potential key genes and immune infiltrate prognosis of SCLC patients. Results: A total of 750 (top 250 from each study) differentially expressed genes (DEGs) were identified. CLDN18 and BRIP1 were significantly related to immune infiltration in the tumor microenvironment. SHCBP1 and KIF23 were related mostly to prognosis in SCLC patients. Conclusion: The present study may provide some potential biomarkers for the therapy and prognosis of SCLC.

Aim and Objective: Nanotechnology attempts to solve the problem of antibiotic resistance. Zinc oxide nanoparticles and curcumin have been shown to be antimicrobial agents and promising anticancer agents, both on their own as well as in combination, and this incorporation will likely improve these properties via a possible additive effect. Materials and Methods: In this study, the synthesis of zinc oxide nanoparticles was done by the distilled extract of Stachys byzantina via the co-precipitation method, which is an economical and eco-friendly green synthesis method. Then, curcumin was loaded to zinc oxide nanoparticles. Antibacterial efficacy of the synthesized nanoparticles was evaluated against five intracellular bacteria; moreover, cytotoxicity was evaluated on breast cancer cells. Results: To confirm the synthesis and characterization of the nanoparticles, some techniques, such as XRD, FTIR, FESEM, and EDX were used. In addition, the antimicrobial activity of biosynthetic zinc oxide/curcumin nanocomposites was evaluated against selected bacterial strains. The uniform spherical nature of the zinc oxide nanoparticles was observed in the FESEM images, with the particle sizes ranging from 20 to 40 nm. The EDX spectrum showed the presence of C, O, and Zn and curcumin uptake on zinc oxide nanoparticles. Conclusion: The zinc oxide/curcumin nanocomposites demonstrated an effective antibacterial effect in the disk diffusion method against five bacterial species. Furthermore, the zinc oxide/ curcumin nanocomposites showed a significant inhibitory effect on the growth of breast cancer cells in the MTT test. Thus, it seems that the synthesized zinc oxide/curcumin nanocomposites have promising high potential antimicrobial and cytotoxic effects.

Background: Gastric cancer (GC) is the most common malignancy of the human digestive system and represents the second leading cause of cancer-related deaths. As early GC is generally mild or asymptomatic and advanced GC is commonly diagnosed, early detection has a significant impact on clinical outcomes. This study aimed to identify epigenetic factors (EFs) as potential GC biomarkers. Methods: We identified 3572 differential expressed genes (DEGs) from 436 GC tissues and 41 non-tumor adjacent samples through The Cancer Genome Atlas (TCGA) datasets. Among them, a total of 57 overlapped genes were identified as differentially expressed EFs (DE-EFs), including 25 up-regulated DE-EFs and 32 down-regulated DE-EFs. Results: Then, Gene Ontology (GO) enrichment analysis revealed that the DE-EFs were mainly associated with histone modification, chromatin remodeling, histone binding, modificationdependent protein binding, etc. Meanwhile, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results suggested that RNA degradation, thermogenesis, shigellosis, insulin resistance, AMPK, and FoxO signaling pathways play roles in the progression of GC. Subsequently, Cox regression and Kaplan-Meier analysis showed that higher expression levels of the three hub EFs, including BRCC3, USP12, and WAC, were associated with better patients’ OS. We also found that GC patients in the TCGA dataset with the earlier stage of TNM stage, invasion, depth of tumor, lymph node metastasis, distant metastasis, and younger age had significantly better GC patients’ OS. Discussion: Furthermore, as the pathway enrichment analysis showed that BRCC3 participated in NOD-like receptors (NLRs)-mediated signaling and the homologous recombination (HR) pathways, strong and statistically significant positive relationships were found between BRCC3 with genes in NLRs signaling and HR pathways, including BRCA1, BRCA2, Rad51, BRE, TOPBP1, HSP90AA1, CASP1, NEK7, and SUGT1, respectively. Conclusion: We found three hub EFs, namely BRCC3, USP12, and WAC, which were downregulated in GC tissues compared to normal tissues, associated with the overall survival of GC patients and could be used as potential biomarkers to predict prognosis in GC patients. The regulation of hub genes in GC may promote the exploration of the epigenetic mechanisms associated with tumorigenesis and provide potential targets for GC diagnosis and treatment.

Background: Although head and neck squamous cell carcinoma (HNSCC) is a common malignancy, the molecular biology landscape underlying its occurrence and development remains poorly understood. The family with sequence similarity (FAM) 3 family of proteins includes four family members, namely FAM3A, FAM3B, FAM3C and FAM3D. In particular, FAM3C has been previously reported to be closely associated with various human malignancies. Methods: Combining analyses using The Cancer Genome Atlas, Gene Expression Profiling Interactive Analysis, Tumor Immune Estimation Resource and MethSurv databases, coupled with the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes bioinformatics tools, the possible biological function and key pathways regulated by the FAM3 family in HNSCC were probed. Results: High FAM3A expression was found to increase HNSCC mitochondrial biosynthesis and energy metabolism, inhibit immune cell infiltration in the HNSCC tumor microenvironment, and be associated with poor prognosis. By contrast, lower expression levels of FAM3B in HNSCC were associated with a poorer prognosis in patients with HNSCC. This was most likely due to the finding that FAM3B can inhibit the development of HNSCC by increasing immune cell infiltration, inhibiting epithelial-mesenchymal transition (EMT) and the cytochrome P450 pathway. FAM3C was overexpressed in oral squamous cell carcinoma (OSCC) and associated with increased OSCC cell stemness, immune escape and EMT. In the present study, FAM3C expression was associated with poor prognosis for patients with HNSCC by suppressing tumor immune cell infiltration. FAM3C expression was also positively correlated with the expression of epithelial and mesenchymal markers such as E-cadherin, N-cadherin, Vimentin and ZO-1, which may promote the partial EMT status in HNSCC and greatly increase its malignancy. FAM3D is a maintenance factor of the epithelial phenotype in HNSCC that can inhibit the progression of EMT, promote tumor immune cell infiltration and inhibit HNSCC progression. In addition, methylation levels of the FAM3 gene family were correlated with the overall survival rate of HNSCC. Conclusion: The FAM3 family may be applied as a biomarker and potential therapeutic target for HNSCC.

Objective: Huanglian Jiedu Decoction (HLJDD) was shown to exert a therapeutic effect on pneumonia for a long time in China. However, its pharmacological mechanism remains to be elucidated. Methods: The active compounds and target proteins of HLJDD were screened from TCMSP, and the pneumonia targets were obtained from GeneCards. GO, and KEGG enrichment was applied in this study. Cytoscape established networks with R-Bioconductor. The affinity between components and targets was detected by molecular docking. Finally, active ingredients and targets were selected to be verified in an inflammatory model established in LPS-induced A549 cells. CCK8 proliferation assay and western blot were performed to test the relative indicators. Results: 102 bioactive components and 205 targets from 4 herbs in HLJDD were collected. 68 potential therapeutic targets and 55 corresponding compounds were screened to establish the networks. 4 active compounds (quercetin, wogonin, kaempferol and baicalein) and 5 hub genes (IL6, AKT1, CXCL8, CCL2 and IL1B) were then selected to make molecular docking. The results indicated that quercetin and wogonin had a better affinity with CXCL8, CCL2 or IL1B. In vitro experiments revealed that quercetin and wogonin could decrease the proliferation inhibiting and apoptosis of A549 cells injured by LPS. CXCL8, CCL2 or IL1B were downregulated after quercetin or wogonin treatment, compared with LPS-induced A549 cells (P < 0.01). Conclusion: The current study suggested that the mechanism of HLJDD treating pneumonia might inhibit apoptosis by targeting inflammatory factors, mainly quercetin and wogonin.

Background: The competing endogenous RNA (ceRNA) network plays an important role in the occurrence and development of a variety of diseases. This study aimed to construct a ceRNA network related to exosomes in diabetic retinopathy (DR). Methods: We explored the Gene Expression Omnibus (GEO) database and then analyzed the RNAs of samples to obtain differentially expressed lncRNAs (DELs), miRNAs (DEMs) and mRNAs (DEGs) alongside the progress of DR. Next, Gene Set Enrichment Analysis (GSEA) analysis of DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of up-DEGs were performed. In addition, a ceRNA network related to exosomes in DR was constructed on the base of DELs, DEMs and DEGs. Finally, the function of the ceRNA network was explored by GO and KEGG enrichment analysis. Results: Through our analysis, 267 DELs (93 up and 174 down), 114 DEMs (64 up and 50 down) and 2368 DEGs (1252 up and 1116 down) were screened. The GSEA analysis results show that these genes were mainly related to cytokine-cytokine receptor interaction, hippo signaling pathway and JAK-STAT signaling pathway. The GO and KEGG results show that these up-DEGs were mainly enriched in viral gene expression, components of ribosomes, mineral absorption, Wntprotein binding, and TGF-β signaling pathway. Besides, a ceRNA network, including 15 lncRNAs (e.g., C1orf145, FGF14-IT1, and PRNT), 3 miRNAs (miR-10a-5p, miR-1297 and miR-507) and 11 mRNAs (NCOR2, CHAC1 and LIX1L, etc.) was constructed. Those 5 lncRNAs were up-regulated, 1 miRNA was down-regulated and 5 mRNAs were up-regulated in DR, while 10 lncRNAs were downregulated, 2 miRNAs were up-regulated and 6 mRNAs were down-regulated in DR. Conclusion: The novel ceRNA network that we constructed will provide new insights into the underlying molecular mechanisms of exosomes in DR.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects the lower respiratory tract by binding to angiotensin-converting enzyme 2 (ACE2) via its S-protein. Recent emerging SARS-CoV-2 variants from the United Kingdom (B.1.1.7) and South Africa (501Y.V2) are spreading worldwide at an alarming rate. The new variants have manifested amino acid substitution K417N, E484K, and N501Y on the RBD domain that binds to ACE2. These mutations may influence the binding of the S-protein to ACE2 and affect viral entry into the host cell. Methods: In this study, we modelled the amino acid substitutions on the S-protein and utilised the HADDOCK server to assess the S-protein RBD domain binding with ACE2. Additionally, we calculated the binding affinity of ACE2 to S-protein WT, B.1.1.7 and 501Y.V2 variants using Molecular Mechanics-Generalized Born Surface Area (MM/GBSA). Results: We demonstrated that the S-protein of both variants possesses a higher binding affinity to ACE2 than WT, with the South African 501Y.V2 being more infective than the B.1.1.7 that originated in the United Kingdom. Conclusion: The South African 501Y.V2 variant presents three amino acid substitutions that changed the H-bonding network, resulting in a higher affinity to ACE2, indicating that the 501Y.V2 strain is more infective than the B.1.1.7 strain.

Background: Endometrial cancer is ranked fourth in women's cancers worldwide. SUMOylation is a process of post-translational modification and some evidence indicate that SUMOylation may influence the occurrence and development of cancer. Until now, the prognostic value of SUMOylation-related genes in endometrial cancer remains unclear. Therefore, we aimed at exploring the prognostic value of SUMOylation-related genes in endometrial cancer in this study. Methods: The transcriptome of endometrial cancer from TCGA database was downloaded and then differentially expressed SUMOylation-related genes were extracted. The risk model was constructed with the use of the least absolute shrinkage and selection operator Cox regression. Samples were divided into low-risk and high-risk group based on the risk score. Survival analysis and Cox analysis were performed between groups. A validation cohort from Fudan University Shanghai Cancer Center were obtained to verify the model. Gene ontology and Kyoto Encylopedia of Genes and Genomes analyses were conducted based on differentially expressed genes between groups. Results: Samples in low-risk group possess better outcome than in high-risk group. (P<0.001) The results of univariate (P<0.001) and multivariate (P=0.018) analysis showed that the risk score was independently correlated to worse outcome for patients with endometrial cancer. In Fudan University Shanghai Cancer Center validation cohort, the low-risk group possessed better survival outcome than the high-risk group (P=0.0393). Functional analysis demonstrated that most of the immune cell infiltration levels and immune pathways activity in low-risk group were higher than in high-risk group. Conclusions: In short, the SUMOylation-related signature had good predictability in endometrial cancer and SUMOylation-related genes play important roles in tumour immunity. Also, our study might have some merits in elucidating potential mechanism of SUMOylation in endometrial cancer.

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease, which does not have a specific drug presently. Huanglian jiedu decoction (HJD) is one of the effective traditional Chinese medicine prescriptions. The real material and mechanisms of HJD for AD are not clear. Objective: Network pharmacology and in vivo experiments were used to explore the real material and mechanisms of HJD for AD. Methods: A systems’ pharmacology approach that provides a comprehensive analysis of bioactive compounds, targets, and pathway interactions was employed to elucidate the molecular pathogenesis of HJD for AD. First, the compound databases were constructed for HJD, and compound targets were predicted. Then, the hub targets of HJD were selected by degree centrality analysis and validated using the molecular docking method. Finally, Compound-Target and Target-Pathway networks were constructed to explore the latent mechanism of HJD for AD. Then, animal models of AD were established, the pathology of the skin lesions was observed, and RT-PCR and ELISA methods were used to verify the key targets in the serum of AD mice. Results: The results showed that 60 bioactive compounds (palmatine, wogonin, cavidine, etc.) of HJD interacting with 169 related hub targets (PTGS2, HSP90AA1, etc.) were authenticated. HJD potentially participates in response to stimuli, biological regulation, and reproduction through the PI3K-Akt signaling pathway, MAPK signaling pathway, Ras signaling pathway, and Fc epsilon RI signaling pathway, which are interrelated to the pathogenesis of AD. Compared with the control group, the thickening of the epidermis in the model group was obvious with inflammatory cells infiltrating, the levels of PI3K, AKT, JNK, ERK, IL-4 and TNF-α were up-regulated; and 6.4g/kg and 12.8g/kg HJD could significantly reduce the thickening of the epidermis and infiltration of inflammatory cells, down-regulate the levels of PI3K, AKT, JNK, ERK, IL-4 and TNF-α in the AD mice. HJD might exert its anti-AD effects by downregulating key indicators (PI3K, AKT, JNK, ERK, IL-4, and TNF-α) in the PI3K/AKT and MAPK pathways. Conclusions: Our study could help us understand the compound and mechanism of HJD for AD. Moreover, it had a guidance function to change the traditional arrangement of formula for HJD.

Background: Gastric cancer (GC) remains a common cause of cancer death in East Asia. Current treatment strategies for GC, including medical and surgical interventions, are suboptimal. Butyrate, a short-chain fatty acid produced by the intestinal flora, has been reported to be able to inhibit gastric carcinogenesis. This study aimed to investigate the effects of butyrate on human GC and its underlying mechanisms. Materials and Methods: Human GC cell lines BGC-823 and SGC-7901, human GC tissues and adjacent normal tissues were used for this study. Cell proliferation was assessed using CCK-8 and EdU staining. TUNEL fluorescence and Annexin V/PI staining were adopted for qualitative and quantitative evaluation of cell apoptosis, respectively. Reactive oxygen species (ROS) assay was performed to analyse mitochondrial function. Real-time q-PCR and western blot were carried out to examine the expression of apoptosis-related genes and the synthesis of apoptosis-related proteins. The association between G protein-coupled receptor 109a (GPR109a) and GC prognosis was analyzed using data from The Cancer Genome Atlas (TCGA). Results: CCK-8 and EdU staining confirmed inhibitory activities of butyrate against human GC cells. Annexin V/PI staining and TUNEL fluorescence microscopy showed that butyrate promoted GC cell apoptosis. No difference in the expression of GPR109a was found between GC tissues and adjacent normal tissues, and no direct association between GPR109a and GC prognosis was discovered, suggesting that GPR109a may not be a key factor mediating the apoptosis of GC cells. Butyrate increased the synthesis of caspase 9 and decreased BCL-2, the well-known effector and regulator of mitochondria-mediated apoptosis, and significantly induced mitochondrial ROS. Conclusion: Collectively, our results suggest that butyrate is able to inhibit the proliferation of GC cells and induce GC apoptosis, possibly via a mitochondrial pathway.

Background: To assess the levels and potential therapeutic and prognostic significance of TIGIT in invasive breast cancer. Methods: The Cancer Genome Atlas database was used to evaluate TIGIT levels in invasive breast cancer and its association with clinicopathological features. Immunohistochemistry (IHC) was performed to validate it. Further, the Kaplan-Meier survival curve, univariate and multivariate Cox regression models were applied in analyzing the role of TIGIT in the prognosis of invasive breast cancer. Go / KEGG enrichment analyses techniques were used to investigate the possible cellular mechanism, and string database was used to explore TIGIT-related proteins. Finally, the TIMER database was used to determine the association between TIGIT and immune cell infiltrations. Results: TIGIT was differentially expressed in Pan cancer tissues compared with normal tissues. Relative to normal tissues, TIGIT levels in invasive breast cancer were elevated (p<0.05). TIGIT mRNA level was significantly different from T stage, age, ER and PR level (p<0.05). The high levels of TIGIT exhibited positive correlations with PFI and OS (p<0.05). Univariate analysis revealed that age, clinical stage, high TNM stage, menopausal status and radiotherapy were the factors affecting OS (p< 0.05). Multivariate analysis revealed that age, high clinical stage and menopausal status were independent risk factors for tumor progression (p<0.05). CD226, INPP5D, PVR, PVRL2 and PVRL3 proteins interact with TIGIT. The TIGIT levels were significantly correlated with infiltrations of immune cells (such as CD8+ T cells) (r=0.917, p<0.05). Conclusion: TIGIT is elevated in invasive breast tumor and is closely associated with the prognosis of invasive breast cancer. TIGIT may be the target of immunotherapy for invasive breast cancer.