Prevalence of metabolic syndrome among patients with hepatocellular carcinoma of different etiologies: a retrospective study.

AIMS: This study compared the prevalences of metabolic syndrome and of cardiac or kidney comorbidities among patients with hepatocellular carcinoma (HCC) associated with metabolic dysfunction-related fatty liver disease (MAFLD), chronic infection with hepatitis B or C virus (HBV or HCV), or the combination of MAFLD and chronic HBV infection. METHODS: Medical records were retrospectively analyzed for patients with HCC who underwent hepatectomy between March 2013 and March 2023. Patients with HCC of different etiologies were compared in terms of their clinicodemographic characteristics and laboratory data before surgery. RESULTS: Of the 2422 patients, 1,822 (75.2%) were chronically infected with HBV without MAFLD and HCV, 415 (17.2%) had concurrent MAFLD and chronic HBV infection but no HCV infection, 121 (5.0%) had MAFLD without hepatitis virus infection, and 64 (2.6%) were chronically infected with HCV in the presence or absence of MAFLD and HBV infection. Compared to patients chronically infected with HBV without MAFLD and HCV, those with MAFLD but no hepatitis virus infection showed significantly lower prevalence of cirrhosis, ascites, portal hypertension, alpha-fetoprotein concentration ≥ 400 ng/mL, tumor size > 5 cm, multinodular tumors and microvascular invasion. Conversely, they showed significantly higher prevalence of metabolic syndrome, hypertension, type 2 diabetes, abdominal obesity, history of cardiovascular disease, T-wave alterations, hypertriglyceridemia and hyperuricemia, as well as higher risk of arteriosclerotic cardiovascular disease. Compared to patients with MAFLD but no hepatitis virus infection, those with concurrent MAFLD and chronic infection with HBV showed significantly higher prevalence of cirrhosis, ascites and portal hypertension, but significantly lower prevalence of hypertension and history of cardiovascular disease. Compared to patients with other etiologies, those chronically infected with HCV in the presence or absence of MAFLD and HBV infection, showed significantly higher prevalence of cirrhosis, portal hypertension, ascites, and esophagogastric varices. CONCLUSION: Patients with HCC associated with MAFLD tend to have a background of less severe liver disease than those with HCC of other etiologies, but they may be more likely to suffer metabolic syndrome or comorbidities affecting the heart or kidneys.

Deep learning of pretreatment multiphase CT images for predicting response to lenvatinib and immune checkpoint inhibitors in unresectable hepatocellular carcinoma.

Objectives: Combination therapy of lenvatinib and immune checkpoint inhibitors (CLICI) has emerged as a promising approach for managing unresectable hepatocellular carcinoma (HCC). However, the response to such treatment is observed in only a subset of patients, underscoring the pressing need for reliable methods to identify potential responders. Materials & methods: This was a retrospective analysis involving 120 patients with unresectable HCC. They were divided into training (n = 72) and validation (n = 48) cohorts. We developed an interpretable deep learning model using multiphase computed tomography (CT) images to predict whether patients will respond or not to CLICI treatment, based on the Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST v1.1). We evaluated the models' performance and analyzed the impact of each CT phase. Critical regions influencing predictions were identified and visualized through heatmaps. Results: The multiphase model outperformed the best biphase and uniphase models, achieving an area under the curve (AUC) of 0.802 (95% CI = 0.780-0.824). The portal phase images were found to significantly enhance the model's predictive accuracy. Heatmaps identified six critical features influencing treatment response, offering valuable insights to clinicians. Additionally, we have made this model accessible via a web server at http://uhccnet.com/ for ease of use. Conclusions: The integration of multiphase CT images with deep learning-generated heatmaps for predicting treatment response provides a robust and practical tool for guiding CLICI therapy in patients with unresectable HCC.

Integrating single-cell and bulk RNA sequencing reveals CK19 + cancer stem cells and their specific SPP1 + tumor-associated macrophage niche in HBV-related hepatocellular carcinoma.

PURPOSE: Cytokeratin 19-positive cancer stem cells (CK19 + CSCs) and their tumor-associated macrophages (TAMs) have not been fully explored yet in the hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Single-cell RNA sequencing was performed on the viable cells obtained from 11 treatment-naïve HBV-associated HCC patients, including 8 CK19 + patients, to elucidate their transcriptomic landscape, CK19 + CSC heterogeneity, and immune microenvironment. Two in-house primary HCC cohorts (96 cases-related HBV and 89 cases with recurrence), TCGA external cohort, and in vitro and in vivo experiments were used to validate the results. RESULTS: A total of 64,581 single cells derived from the human HCC and adjacent normal tissues were sequenced, and 11 cell types were identified. The result showed that CK19 + CSCs were phenotypically and transcriptionally heterogeneous, co-expressed multiple hepatics CSC markers, and were positively correlated with worse prognosis. Moreover, the SPP1 + TAMs (TAM_SPP1) with strong M2-like features and worse prognosis were specifically enriched in the CK19 + HCC and promoted tumor invasion and metastasis by activating angiogenesis. Importantly, matrix metalloproteinase 9 (MMP9) derived from TAM_SPP1, as the hub gene of CK19 + HCC, was activated by the VEGFA signal. CONCLUSIONS: This study revealed the heterogeneity and stemness characteristics of CK19 + CSCs and specific immunosuppressive TAM_SPP1 in CK19 + HCC. The VEGFA signal can activate TAM_SPP1-derived MMP9 to promote the invasion and metastasis of CK19 + HCC tumors. This might provide novel insights into the clinical treatment of HCC patients.

Development and Validation of a Prediction Model for Hepatitis B Virus-Related Hepatocellular Carcinoma Patients Receiving Postoperative Adjuvant Transarterial Chemoembolization.

Background: Hepatocellular carcinoma (HCC) patients who are at significant risk of tumor recurrence and mortality can benefit from postoperative adjuvant transarterial chemoembolization (PA-TACE). However, the benefits of PA-TACE remain unclear. Herein, we aimed to develop a model for predicting the prognosis of HBV-related patients who undergo PA-TACE and endeavored to guide individualized clinical treatment. Methods: We included 432 HBV-related patients who underwent PA-TACE after curative resection were included. The dataset was divided into a training set (n=216) and an internal validation set (n=216). For identifying independent risk factors, the least absolute shrinkage and selection operator and univariate and multivariate Cox analyses were performed. We derived a prognostic model from the training set that was internally validated. The concordance index (C-index), receiver operating characteristic (ROC) curve, calibration curve, and risk stratification were used to evaluate the performance of the nomogram. Results: Patients undergoing PA-TACE had significantly longer overall survival (OS) than those who did not undergo PA-TACE. Age, albumin levels, macrovascular invasion, tumor size, and, stages of Barcelona Clinic Liver Cancer were identified as independent risk variables and concluded into the nomogram to predict the OS of HBV-related patients who received PA-TACE. The nomogram's C-index values OS were 0.710 and 0.652 in the training and internal validation sets, respectively. Both time-dependent AUC and the calibration curve showed good discrimination and model fitness. The risk score -0.12 was kept as the cut-off value that would accurately divide patients into high-risk and low-risk groups; furthermore, the Kaplan-Meier curve showed a high discriminative ability of the model. Conclusion: We developed a predictive model. comprising a formula and nomogram to predict the OS and provide risk stratification for HBV-related patients undergoing PA-TACE, which could contribute to suitable treatment options for this patient population.

The Novel-Natural-Killer-Cell-Related Gene Signature Predicts the Prognosis and Immune Status of Patients with Hepatocellular Carcinoma.

The current understanding of the prognostic significance of natural killer (NK) cells and their tumor microenvironment (TME) in hepatocellular carcinoma (HCC) is limited. Thus, we screened for NK-cell-related genes by single-cell transcriptome data analysis and developed an NK-cell-related gene signature (NKRGS) using multi-regression analyses. Patients in the Cancer Genome Atlas cohort were stratified into high- and low-risk groups according to their median NKRGS risk scores. Overall survival between the risk groups was estimated using the Kaplan-Meier method, and a NKRGS-based nomogram was constructed. Immune infiltration profiles were compared between the risk groups. The NKRGS risk model suggests significantly worse prognoses in patients with high NKRGS risk (p < 0.05). The NKRGS-based nomogram showed good prognostic performance. The immune infiltration analysis revealed that the high-NKRGS-risk patients had significantly lower immune cell infiltration levels (p < 0.05) and were more likely to be in an immunosuppressive state. The enrichment analysis revealed that immune-related and tumor metabolism pathways highly correlated with the prognostic gene signature. In this study, a novel NKRGS was developed to stratify the prognosis of HCC patients. An immunosuppressive TME coincided with the high NKRGS risk among the HCC patients. The higher KLRB1 and DUSP10 expression levels correlated with the patients' favorable survival.

Structure-Based Design of Potent Peptidomimetic Inhibitors Covalently Targeting SARS-CoV-2 Papain-like Protease.

The papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a critical role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. Here, we report the structure-guide design of novel peptidomimetic inhibitors covalently targeting SARS-CoV-2 PLpro. The resulting inhibitors demonstrate submicromolar potency in the enzymatic assay (IC50 = 0.23 µM) and significant inhibition of SARS-CoV-2 PLpro in the HEK293T cells using a cell-based protease assay (EC50 = 3.61 µM). Moreover, an X-ray crystal structure of SARS-CoV-2 PLpro in complex with compound 2 confirms the covalent binding of the inhibitor to the catalytic residue cysteine 111 (C111) and emphasizes the importance of interactions with tyrosine 268 (Y268). Together, our findings reveal a new scaffold of SARS-CoV-2 PLpro inhibitors and provide an attractive starting point for further optimization.

Comparison of survival rates as predicted by total tumor volume or tumor burden score in patients with hepatocellular carcinoma concurrent with fatty liver disease and hepatitis B virus.

OBJECTIVES: To analyze prognostic value of total tumor volume (TTV) and tumor burden score (TBS) in surgically treated patients with hepatocellular carcinoma and concurrent fatty liver disease and hepatitis B virus (FLD-HCC). METHODS: FLD-HCC patients who treated with hepatectomy from 2010 to 2018 were analyzed. Prognostic performance of TTV and TBS was determined by ROC analysis. Patients were stratified into low and high tumor burden by optimal cutoff value of 113.4 cm3 for TTV or 6.3 points for TBS. Survival rates were compared between subgroups and independent risk factors were identified by Cox regression. Correlation between TTV and TBS was evaluated. RESULTS: This study enrolled 342 FLD-HCC patients. Survival was significantly higher among patients with low tumor burden than among those with high tumor burden (p < 0.001). High TTV and TBS were independent risk factors for poor survival of FLD-HCC (HR: 3.27 (2.17-4.93) and 3.48 (2.31-5.26), respectively, all p < 0.001). ROC analyses revealed that TTV and TBS had comparable discriminative ability in stratifying overall and recurrence-free survival of FLD-HCC. Correlation analysis revealed a strong correlation between TTV and TBS. CONCLUSIONS: Both TTV and TBS have comparable prognostic value and high TTV/TBS predicts poor survival of patients with FLD-HCC.

Effects of Clonorchis sinensis combined with Hepatitis B virus infection on the prognosis of patients with Hepatocellular Carcinoma following Hepatectomy.

BACKGROUND: This study aimed to determine the impact of co-infection of Clonorchis sinensis (CS) and hepatitis B virus (HBV) on the prognosis of patients with hepatocellular carcinoma (HCC) following hepatectomy. METHODS: The clinicopathological information of 946 patients with HCC following hepatectomy was retrospectively analyzed. The patients were divided into four groups depending on whether they had CS infection and/or HBV infection: double-negative group (infected with neither CS nor HBV), simple CS group (infected with only CS), simple HBV group (infected with only HBV), and double-positive group (co-infected with CS and HBV). Kaplan-Meier curves were used to evaluate the overall survival (OS) and recurrence-free survival (RFS), while log-rank tests were used to compare survival rates. Further, Cox regression was used to perform both univariate and multivariate survival analyses to identify variables linked to the prognosis of HCC. RESULTS: The median overall survival (OS) and recurrence-free survival (RFS) in the double-positive, simple CS, simple HBV, and double-negative groups were 27 months and 9 months, 20 months and 7 months, 44 months and 12 months, and 42 months and 17 months, respectively. The double-positive group's 1-year, 3-year, and 5-year OS and RFS rates were 79.2% and 46.9%, 62.6% and 28.4%, 47.8%, and 12.2%, respectively. The simple CS group's 1-year, 3-year, and 5-year OS and RFS rates were 86.3% and 41.5%, 56.5% and 27.7%, 50.2%, and 18.5%, respectively. The simple HBV group's 1-year, 3-year, and 5-year OS and RFS rates were 89.8% and 56.0%, 72.5% and 30.5%, 63.8%, and 19.9%, respectively. The double-negative group's 1-year, 3-year, and 5-year OS and RFS rates were 91.5% and 62.3%, 76.1% and 32.9%, 64.0%, and 22.4%, respectively. Further, according to a Cox multivariate analysis, tumor size (> 5cm), Edmonson grade (III-IV), BCLC-C stage, and tumor satellite focus were independent risk factors for RFS and OS in patients with HCC. CONCLUSION: Patients with HCC and Clonorchis sinensis infection experience a poor prognosis after hepatectomy, regardless of whether they are co-infected with HBV.

Dipyridamole interacts with the N-terminal domain of HSP90 and antagonizes the function of the chaperone in multiple cancer cell lines.

Molecular chaperone HSP90 has been considered as a promising target for anti-cancer drug development for years. However, due to the heat shock response induced by the ATP competitive inhibitors against HSP90, the therapeutic efficacies of the compounds are compromised, which consequently restricts the clinical use of HSP90-targeted inhibitors. Therefore, there is a need to discover novel HSP90-targeted modulators which exhibit acceptable inhibition activity against the chaperone and do not induce significant heat shock response in the meantime. Here in this study, we firstly developed a tip-based affinity selection-mass spectrometry platform with optimized experimental conditions/parameters for HSP90-targeted active compound screening, and then applied it to fish out inhibitors against HSP90 from a collection of 2,395 compounds composed of FDA-approved drugs and drug candidates. Dipyridamole, which acts as an anti-thrombotic agent by modulating multiple targets and has a long history of safe use, was identified to interact with HSP90's N-terminal domain. The following conducted biophysical and biochemical experiments demonstrated that Dipyridamole could bind to HSP90's ATP binding pocket and function as an ATP competitive inhibitor of the chaperone. Finally, cellular-based assays including CESTA, cell viability assessment and proteomic analysis etc. were performed to evaluate whether the interaction between HSP90 and Dipyridamole contributes to the anti-tumor effects of the compound. We then found that Dipyridamole inhibits the growth and proliferation of human cancer cells by downregulating cell cycle regulators and upregulating apoptotic cell signaling, which are potentially mediated by the binding of Dipyridamole to HSP90 and to PDEs (phosphodiesterases), respectively.

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS-CoV-2 Papain-Like Protease.

The coronavirus papain-like protease (PLpro ) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS-CoV-2 PLpro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS-CoV-2 PLpro and provid a novel site for allosteric inhibitors design.

Structure-based design of a novel inhibitor of the ZIKA virus NS2B/NS3 protease.

Zika virus (ZIKV) has been a serious public health problem, and there is no vaccine or drug approved for the prevention or treatment of ZIKV yet. The ZIKV NS2B/NS3 protease plays an important role in processing the virus precursor polyprotein and is thus a promising target for antiviral drugs development. In order to discover novel inhibitors of this protease, we carried out a fragment-based hit screening and characterized protein-inhibitor interactions using the X-ray crystallography together with isothermal titration calorimetry. We reported two high-resolution crystal structures of the protease (bZiProC143S) in complex with an active fragment as well as a tetrapeptide, revealing that there is domain swapping in the protein structures and two ligands only occupy the substrate-binding pocket of one copy in a symmetric unit. Based on the detailed binding modes of two ligands revealed by crystal structures, we designed a novel inhibitor which inhibits the NS2B/NS3 protease with a higher potency than the fragment and possesses a higher ligand-binding efficiency and a comparable IC50 compared to the tetrapeptide. These results thus provide a structural basis and valuable hint for development of more potent inhibitors of the ZIKV NS2B/NS3 protease.

A Novel Multicolor Flow Cytometry Scoring System for the Diagnosis and Prognostic Stratification of Myelodysplastic Syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a class of myeloid neoplasms featuring inefficient maturation and differentiation of hematopoietic cells, blood cytopenia, and a high risk of leukemia onset. The diagnosis of MDS remains a challenging task owing to its complexity, heterogeneity, and the lack of specific characteristics. METHODS: To look for an easy and inexpensive diagnostic method for MDS, we tried to establish an FCM scoring systems (FCSS) with a combination of antibodies for diagnosis and prognostic stratification of MDS. This FCSS adopted four parameters; i.e., the frequency of myeloblasts in nucleated cells, the ratio between pro-B cells and CD117+ cells, the ratio of CD45 mean fluorescence intensity between lymphocytes and myeloblasts, and the ratio of SSC peak values between mature granulocytes and lymphocytes. RESULTS: We tested the correlation between the total FCSS score with conventional IPSS-R. Additionally, the correlation between the score of each FCSS parameter and IPSS-R was also evaluated. We found that total FCSS score had a positive correlation with IPSS-R, while FCSS parameter 1 and 4 were also correlated with IPSS-R. Furthermore, this FCSS had a sound sensitivity and specificity in the diagnosis of MDS. CONCLUSIONS: The FCSS represents a convenient and affordable approach for the diagnosis and prognostic stratification of MDS.

Propofol Upregulates MicroRNA-30b to Inhibit Excessive Autophagy and Apoptosis and Attenuates Ischemia/Reperfusion Injury In Vitro and in Patients.

Evidence reveals that propofol protects cells via suppressing excessive autophagy induced by hypoxia/reoxygenation (H/R). Previously, we found in a genome-wide microRNA profile analysis that several autophagy-related microRNAs were significantly altered during the process of H/R in the presence or absence of propofol posthypoxia treatment (P-PostH), but how these microRNAs work in P-PostH is still largely unknown. Here, we found that one of these microRNAs, microRNA-30b (miR-30b), in human umbilical vein endothelial cells (HUVECs) was downregulated by H/R treatment but significantly upregulated by 100 M propofol after H/R treatment. miR-30b showed similar changes in open heart surgery patients. By dual-luciferase assay, we found that Beclin-1 is the direct target of miR-30b. This conclusion was also supported by knockdown or overexpression of miR-30b. Further studies showed that miR-30b inhibited H/R-induced autophagy activation. Overexpression or knockdown of miR-30b regulated autophagy-related protein gene expression in vitro. To clarify the specific role of propofol in the inhibition of autophagy and distinguish the induction of autophagy from the damage of autophagy flux, we used bafilomycin A1. LC3-II levels were decreased in the group treated with propofol combined with bafilomycin A1 compared with the group treated with bafilomycin A1 alone after hypoxia and reoxygenation. Moreover, HUVECs transfected with Ad-mCherry-GFP-LC3b confirmed the inhibitory effect of miR-30b on autophagy flux. Finally, we found that miR-30b is able to increase the cellular viability under the H/R condition, partially mimicking the protective effect of propofol which suppressed autophagy via enhancing miR-30b and targeting Beclin-1. Therefore, we concluded that propofol upregulates miR-30b to repress excessive autophagy via targeting Beclin-1 under H/R condition. Thus, our results revealed a novel mechanism of the protective role of propofol during anesthesia. Clinical Trial Registration Number. This trial is registered with ChiCTR-IPR-14005470. The name of the trial register: Propofol Upregulates MicroRNA-30b to Repress Beclin-1 and Inhibits Excessive Autophagy and Apoptosis.

Lower risk of hepatocellular carcinoma with tenofovir than entecavir treatment in subsets of chronic hepatitis B patients: an updated meta-analysis.

BACKGROUND AND AIM: Previous smaller meta-analyses comparing the incidence of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB) patients treated with tenofovir disoproxil fumarate (TDF) versus entecavir (ETV) provided controversial results. This updated meta-analysis aimed to reliably identify any difference in the HCC incidence between TDF-treated or ETV-treated CHB patients in general or in specific subgroups. METHODS: PubMed, EMBASE, Web of Science, and Cochrane Library were systematically searched for relevant studies with hazard ratios (HRs) for HCC between TDF-treated and ETV-treated CHB patients. Retrieved dates ranged from January 2009 to October 2021. HRs with or without adjustment were pooled with random-effects model. RESULTS: Twenty-four comparative studies involving 37 771 CHB patients treated with TDF and 72 094 treated with ETV were included. TDF was associated with lower risk of HCC compared with ETV, with pooled unadjusted HR of 0.76 (95% confidence interval [CI]: 0.67-0.86) (24 studies) and adjusted HR of 0.81 (95% CI: 0.72-0.91) (21 studies). In propensity score matching cohorts, the TDF superiority was confirmed for unadjusted HR 0.83 (95% CI: 0.71-0.97) (14 studies) and was close to significance for adjusted HR (0.78, 95% CI: 0.58-1.04) (8 studies). Subgroup analyses showed that TDF was associated with lower HCC risk than ETV treatment in CHB patients who were from Asia (adjusted HR: 0.76, 95% CI: 0.66-0.87; 15 studies) or nucleos(t)ide naïve (adjusted HR:0.74, 95% CI: 0.65-0.84; 18 studies). CONCLUSION: Current evidence from a sizable population suggests that TDF is associated with significantly lower HCC risk compared with ETV treatment in patients who are from Asia and/or nucleos(t)ide naïve.

Identification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urgently needs an effective cure. 3CL protease (3CLpro) is a highly conserved cysteine proteinase that is indispensable for coronavirus replication, providing an attractive target for developing broad-spectrum antiviral drugs. Here we describe the discovery of myricetin, a flavonoid found in many food sources, as a non-peptidomimetic and covalent inhibitor of the SARS-CoV-2 3CLpro. Crystal structures of the protease bound with myricetin and its derivatives unexpectedly revealed that the pyrogallol group worked as an electrophile to covalently modify the catalytic cysteine. Kinetic and selectivity characterization together with theoretical calculations comprehensively illustrated the covalent binding mechanism of myricetin with the protease and demonstrated that the pyrogallol can serve as an electrophile warhead. Structure-based optimization of myricetin led to the discovery of derivatives with good antiviral activity and the potential of oral administration. These results provide detailed mechanistic insights into the covalent mode of action by pyrogallol-containing natural products and a template for design of non-peptidomimetic covalent inhibitors against 3CLpros, highlighting the potential of pyrogallol as an alternative warhead in design of targeted covalent ligands.

In Silico Screening-Based Discovery of Novel Inhibitors of Human Cyclic GMP-AMP Synthase: A Cross-Validation Study of Molecular Docking and Experimental Testing.

Cyclic GMP-AMP synthase (cGAS) has been recently uncovered to be a promising therapeutic target for immune-associated diseases. Until now, only a few inhibitors have been identified through high-throughput screening campaigns. Here, we reported the discovery of novel inhibitors for the catalytic domain of human cGAS (h-cGASCD) by virtual screening for the first time. To generate a reliable docking mode, we first obtained a high-resolution crystal structure of h-cGASCD in complex with PF-06928215, a known inhibitor of h-cGAS, followed by molecular dynamics simulations on this complex structure. Four fragment hits were identified by the virtual screening together with a thermal shift assay. The crystal structures of these four compounds in complex with h-cGASCD were subsequently determined, and the binding modes of the compounds were similar to those predicted by molecular docking, supporting the reliability of the docking model. In addition, an enzyme activity assay identified compound 18 (IC50 = 29.88 ± 3.20 µM) from the compounds predicted by the virtual screening. A similarity search of compound 18 followed by a second virtual screening led to the discovery of compounds S2 (IC50 = 13.1 ± 0.09 µM) and S3 (IC50 = 4.9 ± 0.26 µM) as h-cGAS inhibitors with improved potency. Therefore, the present study not only provides the validated hit compounds for further development of h-cGAS inhibitors but also demonstrates a cross-validation study of virtual screening, in vitro experimental assays, and crystal structure determination.

Identification of Highly Selective Lipoprotein-Associated Phospholipase A2 (Lp-PLA2) Inhibitors by a Covalent Fragment-Based Approach.

Covalent ligands are of great interest as therapeutic drugs or biochemical tools. Here, we reported the discovery of highly selective and irreversible inhibitors of lipoprotein-associated phospholipase A2 (Lp-PLA2) using a covalent fragment-based approach. The crystal structure of Lp-PLA2 in complex with a covalent fragment not only reveals the covalent reaction mechanism but also provides a good starting point to design compound 8, which has a more than 130,000-fold and 3900-fold increase in potency and selectivity, respectively, compared to those of the covalent fragment. Furthermore, fluorescent probes with high selectivity and sensitivity are developed to characterize Lp-PLA2 and its enzymatic activity in vitro or even in living cells in a way more convenient than immunoblotting tests or immunofluorescence imaging. Overall, we provide a paradigm for application of the covalent fragment-based strategy in covalent ligand discovery and the advantage of enol-cyclocarbamate as a new warhead in designing covalent inhibitors of serine hydrolases.

Large conformation shifts of Vibrio cholerae VqmA dimer in the absence of target DNA provide insight into DNA-binding mechanisms of LuxR-type receptors.

Quorum sensing regulates the biofilm formation and expression of virulence factors in Vibrio cholerae, an obligate human pathogen that continues to imperil human health. Cytoplasmic transcription factor VqmA is a LuxR-type receptor ubiquitous in the Vibrio genus and one vibriophage VP882 and plays an important role in V. cholerae pathogenicity. Here we presented the X-ray crystal structure of V. cholerae VqmA-DPO complex and compared it with the previously determined VqmA-DPO-DNA complex. To our knowledge, this is the first report on the crystal structures of the same LuxR-type receptor with two conformations of binding to DNA and not binding to DNA. Based on the results of structural analysis and biochemical assays, we revealed the secondary structure of the linker region between two function domains changed significantly, and DNA binding domains were covalently linked by a disulfide bond formed by the highly conserved Cys134. Besides, the distance between two DBD monomers became longer than that in DNA-binding conformation, and two α8 helixes underwent a large conformation shift. The results of the structure-function analyses presented here improve our understanding of the complex mechanisms in the conformational changes of LuxR-type receptors caused by DNA binding.

Crystal structures of phage NrS-1 N300-dNTPs-Mg2+ complex provide molecular mechanisms for substrate specificity.

A novel DNA polymerase from the deep-sea vent phage NrS-1, was characterized as a primase-polymerase (referred to as prim-pol), which works as a self-priming DNA polymerase to synthesize de novo long DNA strands. Functional research on the NrS-1 prim-pol illustrated that the N-terminal 300 residues (referred to as N300) have de novo synthesis activity similar to that of the full-length enzyme. Just like other prim-pols, NrS-1 prim-pol was able to initiate DNA synthesis, proficiently discriminating against ribonucleotides (NTPs), exclusively using deoxynucleotides (dNTPs). However, the structural basis for this discrimination is not well understood. Here, the three kinds of crystal structures of N300-dNTPs-Mg2+ complex were determined. These complex structures shared the identical steric architecture and hydrogen-bond interactions in the catalytic center. The results of biochemical studies indicated that R145 possibly plays an indispensable role in the primer extension. Mutagenesis and structural simulation showed that the backbone carboxyl group of Y146, as a potential sugar selector, was involved in steric clashing with the incoming 2'-OH group of NTPs. However, the mechanism of substrate discrimination probably was different from that of other prim-pols, according to the structural analyses and sequence comparison.