Azobenzene-Based Linker Strategy for Selective Activation of Antibody-Drug Conjugates.

Existing antibody-drug conjugate (ADC) linkers, whether cleavable or non-cleavable, are designed to release highly toxic payloads or payload derivatives upon internalisation of the ADCs into cells. However, clinical studies have shown that only <1 % of the dosed ADCs accumulate in tumour cells. The remaining >99 % of ADCs are nonspecifically distributed in healthy tissue cells, thus inevitably leading to off-target toxicity. Herein, we describe an intelligent tumour-specific linker strategy to address these limitations. A tumour-specific linker is constructed by introducing a hypoxia-activated azobenzene group as a toxicity controller. We show that this azobenzene-based linker is non-cleavable in healthy tissues (O2 >10 %), and the corresponding payload derivative, cysteine-appended azobenzene-linker-monomethyl auristatin E (MMAE), can serve as a safe prodrug to mask the toxicity of MMAE (switched off). Upon exposure to the hypoxic tumour microenvironment (O2<1 %), this linker is cleaved to release MMAE and fully restores the high cytotoxicity of the ADC (switched on). Notably, the azobenzene linker-containing ADC exhibits satisfactory antitumour efficacy in vivo and a larger therapeutic window compared with ADCs containing traditional cleavable or non-cleavable linkers. Thus, our azobenzene-based linker sheds new light on the development of next-generation ADC linkers.

Evaluation and mechanism study of Pien Tze Huang against EV-A71 infection.

Hand, foot, and mouth disease (HFMD) caused by enterovirus A71 (EV-A71) infection, currently lacks specific preventive and therapeutic interventions. Here, we demonstrated that Pien Tze Huang (PZH) could dose-dependently inhibit EV-A71 replication at the cellular level, resulting in significant reductions in EV-A71 virus protein 1 (VP1) expression and viral yields in Vero and human rhabdomyosarcoma cells. More importantly, we confirmed that PZH could protect mice from EV-A71 infection for the first time, with Ribavirin serving as a positive control. PZH treatment reduced EV-A71 VP1 protein expression, viral yields in infected muscles, and improved muscle pathology. Additionally, we conducted a preliminary mechanism study using quantitative proteomics. The results suggested that the suppression of the PI3K/AKT/mTOR and NF-κB signaling pathways may contribute to the anti-EV-A71 activity of PZH. These findings provide strong evidence supporting the potential therapeutic application of PZH for EV-A71 infection management.

USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.

The abnormal upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes T-cell mediated cytotoxicity through PD-1 engagement, and further exploring the mechanisms regulation of PD-L1 in cancers may enhance the clinical efficacy of PD-L1 blockade. Here, using single-guide RNAs (sgRNAs) screening system, we identify ubiquitin-specific processing protease 2 (USP2) as a novel regulator of PD-L1 stabilization for tumor immune evasion. USP2 directly interacts with and increases PD-L1 abundance in colorectal and prostate cancer cells. Our results show that Thr288, Arg292 and Asp293 at USP2 control its binding to PD-L1 through deconjugating the K48-linked polyubiquitination at lysine 270 of PD-L1. Depletion of USP2 causes endoplasmic reticulum (ER)-associated degradation of PD-L1, thus attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell-mediated killing. Meanwhile, USP2 ablation-induced PD-L1 clearance enhances antitumor immunity in mice via increasing CD8+ T cells infiltration and reducing immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), whereas PD-L1 overexpression reverses the tumor growth suppression by USP2 silencing. USP2-depletion combination with anti-PD-1 also exhibits a synergistic anti-tumor effect. Furthermore, analysis of clinical tissue samples indicates that USP2 is positively associated with PD-L1 expression in cancer. Collectively, our data reveal a crucial role of USP2 for controlling PD-L1 stabilization in tumor cells, and highlight USP2 as a potential therapeutic target for cancer immunotherapy.

Raddeanin A Enhances Mitochondrial DNA-cGAS/STING Axis-Mediated Antitumor Immunity by Targeting Transactive Responsive DNA-Binding Protein 43.

Immune checkpoint therapies (ICT) have achieved unprecedented efficacy in multiple cancer treatments, but are still limited by low clinical response rates. Identification of immunogenic cell death (ICD)-inducing drugs that can induce tumor cell immunogenicity and reprogram the tumor microenvironment is an attractive approach to enhance antitumor immunity. In the present study, Raddeanin A (RA), an oleanane class triterpenoid saponin isolated from Anemone raddeana Regel, is uncovered as a potent ICD inducer through an ICD reporter assay combined with a T cell activation assay. RA significantly increases high-mobility group box 1 release in tumor cells and promotes dendritic cell (DC) maturation and CD8+ T cell activation for tumor control. Mechanistically, RA directly binds to transactive responsive DNA-binding protein 43 (TDP-43) and induces TDP-43 localization to mitochondria and mtDNA leakage, leading to cyclic GMP-AMP synthase/stimulator of interferon gene-dependent upregulation of nuclear factor κB and type I interferon signaling, thereby potentiating the DC-mediated antigen cross-presentation and T cell activation. Moreover, combining RA with anti-programmed death 1 antibody effectively enhances the efficacy of ICT in animals. These findings highlight the importance of TDP-43 in ICD drug-induced antitumor immunity and reveal a potential chemo-immunotherapeutic role of RA in enhancing the efficacy of cancer immunotherapy.

Novel Small-Molecule PD-L1 Inhibitor Induces PD-L1 Internalization and Optimizes the Immune Microenvironment.

Blocking the PD-1/PD-L1 interaction has become an important strategy for tumor therapy, which has shown outstanding therapeutic effects in clinical settings. However, unsatisfactory response rates and immune-related adverse effects limit the use of anti-PD1/PD-L1 antibodies. Here, we report the discovery and identification of S4-1, an innovative small-molecule inhibitor of PD-L1. In vitro, S4-1 effectively altered the PD-L1/PD-1 interaction, induced PD-L1 dimerization and internalization, improved its localization to endoplasmic reticulum, and thus enhanced the cytotoxicity of peripheral blood mononuclear cells toward tumor cells. In vivo, S4-1 significantly inhibited tumor growth in both lung and colorectal cancer models, particularly in colorectal cancer, where it led to complete clearance of a portion of the tumor cells. Furthermore, S4-1 induced T-cell activation and inversed the inhibitory tumor microenvironment, consistent with the PD-L1/PD-1 pathway blockade. These data support the continued evaluation of S4-1 as an alternative ICB therapeutic strategy.

Novel phthalimides regulating PD-1/PD-L1 interaction as potential immunotherapy agents.

Programmed cell death 1(PD-1)/programmed cell death ligand 1(PD-L1) have emerged as one of the most promising immune checkpoint targets for cancer immunotherapy. Despite the inherent advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has fallen behind that of antibody drugs. Based on docking studies between small molecule inhibitor and PD-L1 protein, changing the chemical linker of inhibitor from a flexible chain to an aromatic ring may improve its binding capacity to PD-L1 protein, which was not reported before. A series of novel phthalimide derivatives from structure-based rational design was synthesized. P39 was identified as the best inhibitor with promising activity, which not only inhibited PD-1/PD-L1 interaction (IC50 = 8.9 nmol/L), but also enhanced killing efficacy of immune cells on cancer cells. Co-crystal data demonstrated that P39 induced the dimerization of PD-L1 proteins, thereby blocking the binding of PD-1/PD-L1. Moreover, P39 exhibited a favorable safety profile with a LD50 > 5000 mg/kg and showed significant in vivo antitumor activity through promoting CD8+ T cell activation. All these data suggest that P39 acts as a promising small chemical inhibitor against the PD-1/PD-L1 axis and has the potential to improve the immunotherapy efficacy of T-cells.

Reirradiation of Whole Brain for Recurrent Brain Metastases: A Case Report of Lung Cancer With 12-Year Survival.

Whole brain radiotherapy (WBRT) for brain metastases (BMs) was considered to be dose limited. Reirradiation of WBRT for recurrent BM has always been challenged. Here, we report a patient with multiple BMs of non-small-cell lung cancer (NSCLC), who received two courses of WBRT at the interval of 5 years with the cumulative administration dose for whole brain as 70 Gy and a boost for the local site as 30 Gy. Furthermore, after experiencing relapse in the brain, he underwent extra gamma knife (GK) radiotherapy for local brain metastasis for the third time after 5 years. The overall survival was 12 years since he was initially diagnosed with NSCLC with multiple brain metastases. Meanwhile, each time of radiotherapy brought a good tumor response to brain metastasis. Outstandingly, during the whole survival, he had a good quality of life (QoL) with Karnofsky Performance Score (KPS) above 80. Even after the last GK was executed, he had just a mild neurocognitive defect. In conclusion, with the cautious evaluation of a patient, we suggest that reirradiation of WBRT could be a choice, and the cumulative radiation dose of the brain may be individually modified.

Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR.

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.

Structure-activity relationship and biological evaluation of 12 N-substituted aloperine derivatives as PD-L1 down-regulatory agents through proteasome pathway.

Twenty-nine 12 N-substituted aloperine derivatives were synthesized and screened for suppression on PD-L1 expression in H460 cells, as a continuation of our work. Systematic structural modifications led to the identification of compound 6b as the most active PD-L1 modulator. Compound 6b could significantly down-regulate both constitutive and inductive PD-L1 expression in NSCLC cells, and successively enhance the cytotoxicity of co-cultured T cells against tumor cells at the concentration of 20 µM. Also, it exhibited a moderate in vivo anticancer efficacy against Lewis tumor xenograft with a stable PK and safety profile. The mechanism study indicated that 6b mediated the degradation of PD-L1 through a proteasome pathway, rather than a lysosome route. These results provided the powerful information for cancer immunotherapy of aloperine derivatives with unique endocyclic skeleton by targeting PD-L1 to activate immune cells to kill cancer cells.

Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5.

Programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1) blocking therapy has become a major pillar of cancer immunotherapy. Compared with antibodies targeting, small-molecule checkpoint inhibitors which have favorable pharmacokinetics are urgently needed. Here we identified berberine (BBR), a proven anti-inflammation drug, as a negative regulator of PD-L1 from a set of traditional Chinese medicine (TCM) chemical monomers. BBR enhanced the sensitivity of tumour cells to co-cultured T-cells by decreasing the level of PD-L1 in cancer cells. In addition, BBR exerted its antitumor effect in Lewis tumor xenograft mice through enhancing tumor-infiltrating T-cell immunity and attenuating the activation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs). BBR triggered PD-L1 degradation through ubiquitin (Ub)/proteasome-dependent pathway. Remarkably, BBR selectively bound to the glutamic acid 76 of constitutive photomorphogenic-9 signalosome 5 (CSN5) and inhibited PD-1/PD-L1 axis through its deubiquitination activity, resulting in ubiquitination and degradation of PD-L1. Our data reveals a previously unrecognized antitumor mechanism of BBR, suggesting BBR is small-molecule immune checkpoint inhibitor for cancer treatment.

Development and Validation of a Radiomics Nomogram Model for Predicting Postoperative Recurrence in Patients With Esophageal Squamous Cell Cancer Who Achieved pCR After Neoadjuvant Chemoradiotherapy Followed by Surgery.

Background and purpose: Although patients with esophageal squamous cell carcinoma (ESCC) can achieve a pathological complete response (pCR) after neoadjuvant chemoradiotherapy (nCRT) followed by surgery, one-third of these patients with a pCR may still experience recurrence. The aim of this study is to develop and validate a predictive model to estimate recurrence-free survival (RFS) in those patients who achieved pCR. Materials and methods: Two hundred six patients with ESCC were enrolled and divided into a training cohort (n = 146) and a validation cohort (n = 60). Radiomic features were extracted from contrast-enhanced computed tomography (CT) images of each patient. Feature reduction was then implemented in two steps, including a multiple segmentation test and least absolute shrinkage and selection operator (LASSO) Cox proportional hazards regression method. A radiomics signature was subsequently constructed and evaluated. For better prediction performance, a clinical nomogram based on clinical risk factors and a nomogram incorporating the radiomics signature and clinical risk factors was built. Finally, the prediction models were further validated by calibration and the clinical usefulness was examined in the validation cohort to determine the optimal prediction model. Results: The radiomics signature was constructed using eight radiomic features and displayed a significant correlation with RFS. The nomogram incorporating the radiomics signature with clinical risk factors achieved optimal performance compared with the radiomics signature (P < 0.001) and clinical nomogram (P < 0.001) in both the training cohort [C-index (95% confidence interval [CI]), 0.746 (0.680-0.812) vs. 0.685 (0.620-0.750) vs. 0.614 (0.538-0.690), respectively] and validation cohort [C-index (95% CI), 0.724 (0.696-0.752) vs. 0.671 (0.624-0.718) vs. 0.629 (0.597-0.661), respectively]. The calibration curve and decision curve analysis revealed that the radiomics nomogram outperformed the other two models. Conclusions: A radiomics nomogram model incorporating radiomics features and clinical factors has been developed and has the improved ability to predict the postoperative recurrence risk in patients with ESCC who achieved pCR after nCRT followed by surgery.

Prognostic value of TP53 co-mutation status combined with EGFR mutation in patients with lung adenocarcinoma.

PURPOSE: TP53/EGFR co-mutation has been reported to affect the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in lung adenocarcinoma (LUAD). However, its impact on survival is unclear. In this analysis, we explored the prognostic effect of TP53/EGFR co-mutation in LUAD. METHODS: Clinical data and transcriptome sequencing of LUAD patients with matched genomic data were downloaded from the Cancer Genome Atlas (TCGA) database for overall survival (OS) analysis. Differential expression genes (DEGs) were recognized by R software and bioconductor package. Clusterprofiler was used for functional analysis. STRING was used for estimating PPI information and plug-in CytoHubba to screen hub modules in Cytoscape. The association between tumor mutation burden (TMB) and survival was also analyzed. RESULTS: OS was shorter for patients carrying TP53 mutation (MUT) than that of wild type (WT) (37.7 m vs 52.8 m; p = 0.040, HR = 1.38, 95% CI 1.01-1.89). Dual TP53/EGFR-MUT was associated with inferior OS compared with the dual WT/WT cohort (38.4 m vs 51.9 m; p = 0.023, HR 1.83, 95% CI 0.95-3.52). 316 DEGs between dual TP53/EGFR-MUT and dual WT/WT samples were obtained and functional analysis made known that DEGs were strikingly enriched in regulating the metabolism of important amino acids, cell division, cell cycle regulation, cell adhesion, and extracellular matrix composition. KEGG analysis discovered that DEGs were mainly enriched in signaling pathways such as PI3K-Akt, cytokine-cytokine receptor interaction, focal adhesions, and extracellular matrix receptor interaction. PPI network suggested that GPC3, CCL28, GPR37, and NPY genes were up-regulated in dual mutation samples. OS in the high TMB cohort was significantly better than that in the low TMB in patients with TP53 MUT(43.2 m vs 32.4 m; P = 0.007, HR = 0.52, 95% CI: 0.34-0.81), as well as in the combination of TP53 MUT and EGFR WT group (44.4 m vs 31.2 m; P = 0.021, HR = 0.55, 95% CI 0.34 - 0.89). CONCLUSIONS: TP53 MUT is a poor prognostic factor in LUAD patients, and the prognosis of TP53/EGFR co-mutation is worse. GPC3, CCL28, GPR37, and NPY may be novel prognostic markers and potential therapeutic targets for patients with dual TP53/EGFR mutation LUAD.

Co-occurring genetic alterations predict distant metastasis and poor efficacy of first-line EGFR-TKIs in EGFR-mutant NSCLC.

PURPOSE: To determine the frequency of co-occurring genes in non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation and the predictive effect of co-mutations on the efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). METHODS: 54 patients with advanced NSCLC were tested for 422 clinically relevant genes by next-generation sequencing (NGS) before treatment. Among them, patients with EGFR mutation received first-line treatment of EGFR-TKIs. Progression-free survival (PFS) and objective response rate (ORR) were evaluated using Kaplan-Meier methods and compared between two groups using log-rank test. RESULTS: Among 24 EGFR mutant and 30 EGFR wild-type patients, co-mutation rate was lower in patients with EGFR mutation (62.5% [15/24] vs 93.3% [28/30], p = 0.005). There was lower frequency for co-alterations in BRAF (0% [0/24] vs 20% [7/30], p = 0.033), NF1 (4.2% [1/24] vs 30% [9/30], p = 0.038) and RAS-RAF-MAPK pathway genes (16.6% [4/24] vs 56.7% [17/30], p = 0.003) in EGFR mutation group. 24 patients with EGFR mutation received first-line treatment of gefitinib or erlotinib, with an ORR of 83.3% and a median PFS of 12.3 months (95% CI 10.00-14.60). Co-mutation was associated with shorter median PFS (10.2 months [95% CI 5.20-15.20] vs 15.3 months [95% CI 12.09-15.81]; HR 0.29 [95% CI 0.10-0.82]; p = 0.014) in EGFR mutation cohort. Among patients with EGFR mutation and distant metastasis, median PFS was decreased in those with co-mutations (6.3 months [95% CI 3.25-9.35] vs 22.0 months[95% CI 12.10-31.90]; HR 0.12 [95% CI 0.00-5.87]; p = 0.007) and frequency of PIK3CA (0% [0/12] vs 41.7% [5/12], p = 0.037) and PI3K/AKT/mTOR pathway genes (0% [0/12] vs 50% [6/12], p = 0.014) was lower. CONCLUSION: The presence of co-mutations was lower in the EGFR mutation patients and reduces the efficacy of EGFR-TKI, especially in patients with distant metastases. Lower frequency of co-mutation in PIK3CA and PI3K/AKT/mTOR pathway genes may be responsible for promoting metastasis and limiting the efficacy of EGFR-TKIs.

Synthesis and Biological Evaluation of Fangchinoline Derivatives as Anti-Inflammatory Agents through Inactivation of Inflammasome.

Twenty eight 7-substitued fangchinoline analogues, of which twenty two were novel, were synthesized and evaluated for their effect to inhibit lipopolysaccharide/nigericin (LPS/NIG)-induced IL-1ß release at both cell and protein levels at the concentration of 5 µM. Among them, compound 6 exhibited promising inhibitory potency against IL-ß activation with an IC50 value of 3.7 µM. Preliminary mechanism study revealed that 6 might target NLRP3 protein, and then block ASC pyroptosome formation with-NLRP3, rather than acting on the activation of the NLRP3 inflammasome (NF-κB and MAPK pathways) or caspase-1 protein. Our current study supported the potential role of compound 6 against IL-ß activation, and provided powerful information for developing fangchinoline derivatives into a novel class of anti-inflammatory agents.

SA-49, a novel aloperine derivative, induces MITF-dependent lysosomal degradation of PD-L1.

BACKGROUND: Programmed death-ligand 1 (PD-L1) is a T-cell inhibitory checkpoint molecule that suppresses antitumor immunity. Anti-PD-L1 antibodies have shown remarkable promise in treating tumors, but the patient response rate is low. Therefore, small-molecule checkpoint inhibitors blocking PD-L1 function are urgently needed. METHODS: Changes of protein expression and phosphorylation levels were determined by immunoblotting. The level of Membrane PD-L1 was examined by flow cytometer. Cytotoxicity of T cells and NK cells toward tumor cells were detected using LDH and cell index assays. Lysosome function was investigated by NAG assay. Changes in lysosomal-related genes were measured by RT-PCR. In vivo anti-NSCLC cancer effects were assessed using C57BL/6 mice bearing Lewis tumor xenografts. FINDINGS: We identified SA-49 as a new regulator of PD-L1 expression from a series of novel aloperine derivatives. SA-49 decreased the expression of PD-L1 in NSCLC cells and enhanced the cytotoxicity of co-cultured T and NK cells toward tumor cells. Importantly, lysosomal pathway contributed to SA-49-mediated down-regulation of PD-L1. SA-49 increased the biogenesis of lysosome and promoted translocation of PD-L1 to lysosome for proteolysis, which was associated with nuclear translocation of MITF. SA-49-induced MITF translocation acted through activation of PKCα and subsequently suppression of GSK3ß activity. Furthermore, SA-49 suppressed Lewis tumor xenograft growth by activating immune microenvironment in C57BL/6 mice. INTERPRETATION: Our data demonstrate that SA-49 can be used to regulate PD-L1 in cancer cells and trigger its degradation by activating lysosome function.

Low-density lipoprotein promotes lymphatic metastasis of esophageal squamous cell carcinoma and is an adverse prognostic factor.

The purpose of the current study was to investigate the prognostic role of preoperative serum lipid levels in patients with esophageal squamous cell carcinoma (ESCC) and to preliminarily explore the mechanism of serum lipids in this disease. Preoperative lipids, including total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol and triglyceride levels, were assessed in 242 patients with ESCC. To eliminate the influence of nutritional status, all patients had previously undergone esophagectomy. Univariate and multivariate Cox regression analyses were performed to identify predictors of overall survival (OS). Associations between significant lipid targets and clinical features were then analyzed and the results were validated using TE-1 and ECa109 esophageal cancer cell lines. The cell proliferation was evaluated with a Cell Counting Kit-8 (CCK8) assay and the cell cycle was assessed with propidium iodide staining and flow cytometry. Univariate analysis revealed that HDL (P=0.048), LDL (P=0.020), Pathological T-staging status (pT status) (P=0.001), Pathological N-staging status (pN status) (P=0.001) and histological differentiation (P=0.002) were significantly associated with OS. Based on multivariate analysis, LDL [hazard ratio (HR)=2.164, P=0.005], pT status (HR=1.714, P=0.001), pN status (HR=1.966, P=0.001) and histological differentiation (HR=4.083, P=0.002) were risk factors in patients with ESCC. A high LDL level (>3.12 mmol/l) was associated with sex (P=0.001), tumor location (P=0.004) and a higher susceptibility to lymphatic metastasis (P=0.007). A CCK8 assay demonstrated that LDL promoted TE-1 and ECa109 cell proliferation, and flow cytometry analysis revealed that treatment with LDL at an appropriate concentration resulted in an accumulation of cells in G2 phase and decreased the number of cells in G1 phase. In summary, the current study identified that preoperative LDL serum level serves an important role in predicting ESCC outcome as LDL promotes lymphatic metastasis. Furthermore, a preliminary mechanism for this association has been validated in vitro.

MPB, a novel berberine derivative, enhances lysosomal and bactericidal properties via TGF-ß-activated kinase 1-dependent activation of the transcription factor EB.

Lysosome has a crucial role in clearance of endocytosed pathogens from the cell. Small molecules that can boost lysosome function and bactericidal ability to cope with subsequent infection are urgently needed. Here, we report that MPB, a novel berberine derivative, induced lysosome-based degradation and clearance of methicillin-resistant Staphylococcus aureus and enteroinvasive Escherichia coli in macrophages. MPB caused nuclear translocation of transcription factor EB (TFEB), which boosted expression of lysosome genes. TFEB silencing repressed the MPB-mediated enhancements in degradation and bacterial eradication. MPB switched on TFEB nuclear translocation by coupling 2 parallel signaling pathways. MPB-triggered JNK activation led to 14-3-3δ being released from TFEB, which, in turn, caused TFEB nuclear translocation. In addition, MPB induced AMPK activation and subsequent inhibition of mechanistic target of rapamycin activity, which also contributed to TFEB nuclear translocation. Importantly, genetical or pharmaceutical inhibition of TGF-ß-activated kinase 1 (TAK1) reduced MPB action remarkably. MPB acted through TAK1 at lysine 158 to activate JNK and AMPK and, thus, induced TFEB-dependent bactericidal activity in macrophages. Therefore, our study reveals a novel mechanism by which MPB controls JNK and AMPK phosphorylation cascades to activate lysosomal function and bactericidal activity via TAK1 K158-dependent manner, which may offer insight into novel therapeutic strategies to control bacterial infection.-Liu, X., Zhang, N., Liu, Y., Liu, L., Zeng, Q., Yin, M., Wang, Y., Song, D., Deng, H. MPB, a novel berberine derivative, enhances lysosomal and bactericidal properties via TGF-ß-activated kinase 1-dependent activation of the transcription factor EB.

Resibufogenin suppresses transforming growth factor-ß-activated kinase 1-mediated nuclear factor-κB activity through protein kinase C-dependent inhibition of glycogen synthase kinase 3.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has received considerable attention for its potency in cancer therapy. However, the anticancer effects and the underlying mechanisms of RB on pancreatic cancer remain elusive. Here, we found that RB inhibited the viability and induces caspase-dependent apoptosis in human pancreatic cancer cells Panc-1 and Aspc. Resibufogenin-induced apoptosis was through inhibition of constitutive nuclear factor-κB (NF-κB) activity and its target genes' expression, which was caused by downregulation of transforming growth factor-ß-activated kinase 1 (TAK1) levels and suppression of IκB kinase activity in Panc-1 and Aspc cells. This induction of TAK1-mediated NF-κB inactivation by RB was associated with increased glycogen synthase kinase-3 (GSK-3) phosphorylation and subsequent suppression of its activity. Moreover, RB-induced GSK-3 phosphorylation/inactivation acted through activation of protein kinase C but not Akt. Finally, RB suppressed human pancreatic tumor xenograft growth in athymic nude mice. Thus, our findings reveal a novel mechanism by which RB suppresses TAK1-mediated NF-κB activity through protein kinase C-dependent inhibition of GSK-3. Our findings provide a rationale for the potential application of RB in pancreatic cancer therapy.

Discovery and evolution of aloperine derivatives as novel anti-filovirus agents through targeting entry stage.

Preventing filoviruses in the entry stage is an attractive antiviral strategy. Taking aloperine, a Chinese natural herb with an endocyclic skeleton, as the lead, 23 new aloperine derivatives were synthesized and evaluated for their anti-filovirus activities including ebola virus (EBOV) and marburg virus (MARV) using pseudotyped virus model. Structure-activity relationship (SAR) analysis indicated that the introduction of a 12N-dichlorobenzyl group was beneficial for the potency. Compound 2e exhibited the most potent anti-EBOV and anti-MARV effects both in vitro and in vivo. It also displayed a good pharmacokinetic and safety profile in vivo, indicating an ideal druglike feature. The primary mechanism study showed that 2e could block a late stage of viral entry, mainly through inhibiting cysteine cathepsin B activity of host components. We consider compound 2e to be a promising broad-spectrum anti-filovirus agent with the advantages of a unique chemical scaffold and a specific biological mechanism.

Glycogen synthase kinase-3ß inhibition promotes lysosome-dependent degradation of c-FLIPL in hepatocellular carcinoma.

Glycogen synthase kinase-3ß (GSK-3ß) is a ubiquitously expressed serine/threonine kinase involved in a variety of functions ranging from the control of glycogen metabolism to transcriptional regulation. We recently demonstrated that GSK-3ß inhibition triggered ASK1-JNK-dependent apoptosis in human hepatocellular carcinoma (HCC) cells. However, the comprehensive picture of downstream GSK-3ß-regulated pathways/functions remains elusive. In this study, we showed that GSK-3ß was aberrantly activated in HCC. Pharmacological inhibition and genetic depletion of GSK-3ß suppressed the growth and induced caspase-dependent apoptosis in HCC cells. In addition, GSK-3ß inhibition-induced apoptosis through downregulation of c-FLIPL in HCC, which was caused by biogenesis of functional lysosomes and subsequently c-FLIPL translocated to lysosome for degradation. This induction of the lysosome-dependent c-FLIPL degradation was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Moreover, GSK-3ß inhibition-induced TFEB translocation acts through activation of AMPK and subsequently suppression of mTOR activity. Thus our findings reveal a novel mechanism by which inhibition of GSK-3ß promotes lysosome-dependent degradation of c-FLIPL. Our study shows that GSK-3ß may become a promising therapeutic target for HCC.