In situ autophagy regulation in synergy with phototherapy for breast cancer treatment.

Autophagy is an important factor in reducing the efficacy of tumor phototherapy (including PTT and PDT). Accurate regulation of autophagy in tumor cells is a new strategy to improve the anti-tumor efficiency of PTT/PDT. This project intended to construct a tumor-activated autophagy regulator to efficiently block PTT/PDT-induced autophagy and realize synergistic sensitization to tumor phototherapy. To achieve this goal, we first synthesized TRANSFERRIN (Tf) biomimetic mineralized nano-tellurium (Tf-Te) as photosensitizer and then used disulfide bond reconstruction technology to induce Tf-Te self-assembly. The autophagy inhibitor hydroxychloroquine (HCQ) and iron ions carried by Tf were simultaneously loaded to prepare a tumor-responsive drug reservoir Tf-Te/HCQ. After entering breast cancer cells through the "self-guidance system", Tf-Te/HCQ can generate hyperpyrexia and ROS under NIR laser irradiation, to efficiently induce PTT/PDT effect. Meanwhile, the disulfide bond broke down in response to GSH, and the nanoparticles disintegrated to release Fe2+ and HCQ at fixed points. They simultaneously induce lysosomal alkalinization and increased osmotic pressure, effectively inhibit autophagy, and synergistically enhance the therapeutic effect of phototherapy. In vivo anti-tumor results have proved that the tumor inhibition rate of Tf-Te/HCQ can be as high as 88.6% on 4T1 tumor-bearing mice. This multifunctional drug delivery system might provide a new alternative for more precise and effective tumor phototherapy.

The novel selective TLR7 agonist GY101 suppresses colon cancer growth by stimulating immune cells.

Toll-like receptor (TLR) 7, a transmembrane signal transduction receptor expressed on the surface of endosomes, has become an attractive target for antiviral and cancer immunotherapies. TLR7 can induce signal transduction by recognizing single-stranded RNA or its analogs, leading to the release of cytokines such as IL-6, IL-12, TNF-α and type-I IFN. Activation of TLR7 helps to enhance immunogenicity and immune memory by stimulating immune cells. Herein, we identified a novel selective TLR7 agonist, GY101, and determined its ability to activate TLR7. In summary, in vitro, compound GY101 significantly induced the secretion of IL-6, IL-12, TNF-α and IFN-γ in mouse splenic lymphocytes; in vivo, peritumoral injection of GY101 significantly suppressed colon cancer CT26, as well as poorly immunogenic B16-F10 and 4T1 cancer cell-derived tumor growth by activating the infiltration of lymphocytes and polarization of M2-like macrophages into M1-like macrophages. These results demonstrate that GY101, as a potent TLR7 agonist, holds great potential for cancer immunotherapy.

A "bulldozer" driven by anoxic bacteria for pancreatic cancer chemo-immunotherapy.

Immune evasion is a major obstacle for pancreatic ductal adenocarcinoma (PDAC) therapy. Inhibition of autophagy can improve antigen presentation and enlarge immunogenic cell death (ICD) effect to generate a strong anti-tumor immune response. However, abundant extracellular matrix dominated by hyaluronic acid (HA) hinders the deep penetration of autophagy inhibitors and ICD inducers. Herein, an intelligent autophagy inhibitor hydroxychloroquine (HCQ) and chemotherapeutic drug doxorubicin (DOX) co-loaded "bulldozer" (HD@HH/EcN) driven by anoxic bacteria was constructed for PDAC chemo-immunotherapy. Results demonstrated that probiotic Escherichia coli 1917 (EcN) could carry hyaluronidases (HAases)-hybrided albumin nanoparticles (HD@HH) to reach PDAC tumor tissue quickly and accurately. Thereafter, HAases can efficiently cleave the tumor matrix barrier and promote HD@HH/EcN to accumulate at tumor hypoxic core significantly. After that, high level of glutathione (GSH) in tumor microenvironment (TME) induces intermolecular disulfide bond in HD@HH nanoparticles breakage, to precisely release HCQ and DOX. DOX can induce ICD effect. Meanwhile, HCQ can amplify DOX induced ICD effect by inhibiting tumor autophagy, which further increase cell surface expression of major histocompatibility complex class I (MHC-I) and augment recruitment of CD8+ T cell to improve immunosuppressive TME. This study provides a new strategy for PDAC chemo-immunotherapy.

Advanced prodrug strategies in nucleoside analogues targeting the treatment of gastrointestinal malignancies.

Gastrointestinal malignancies are common digestive system tumor worldwide. Nucleoside analogues have been widely used as anticancer drugs for the treatment of a variety of conditions, including gastrointestinal malignancies. However, low permeability, enzymatic deamination, inefficiently phosphorylation, the emergence of chemoresistance and some other issues have limited its efficacy. The prodrug strategies have been widely applied in drug design to improve pharmacokinetic properties and address safety and drug-resistance issues. This review will provide an overview of the recent developments of prodrug strategies in nucleoside analogues for the treatment of gastrointestinal malignancies.

Synthesis of novel oxazol-5-one derivatives containing chiral trifluoromethyl and isoxazole moieties as potent antitumor agents and the mechanism investigation.

In this study, a series of novel oxazol-5-one derivatives containing a chiral trifluoromethyl and isoxazole moiety were synthesized and evaluated for cytotoxic activities. Among them, 5t was the most effective compound against HepG2 liver cancer cells with an IC50 of 1.8 µM. 5t inhibited cell proliferation, migration, invasion, and induced cell cycle arrest and apoptosis in vitro. Nevertheless, the potential anti-hepatocellular carcinoma (HCC) target and mechanism of 5t were unclear. This work aimed to seek the molecular target of 5t against HCC and investigate its mechanism. Liquid chromatography tandem-mass spectrometry was used to identify peroxiredoxin 1(PRDX1) as a possible target of 5t. Cellular thermal shift assay, drug affinity responsive target stability, and molecular docking provided conclusive evidence that 5t targeted PRDX1 and inhibited its enzymatic activity. 5t augmented the level of reactive oxygen species (ROS) and led to ROS-dependent DNA damage, endoplasmic reticulum stress, mitochondrial dysfunction, and apoptosis in HepG2 cells. Silencing PRDX1 also resulted in ROS-mediated apoptosis in HepG2 cells. In vivo, 5t inhibited mouse tumor growth by increasing oxidative stress. Briefly, our studies revealed that compound 5t targeted PRDX1 through a ROS-dependent mechanism, highlighting the future development of compound 5t as a novel therapeutic drug for HCC.

A metabolic intervention strategy to break evolutionary adaptability of tumor for reinforced immunotherapy.

The typical hallmark of tumor evolution is metabolic dysregulation. In addition to secreting immunoregulatory metabolites, tumor cells and various immune cells display different metabolic pathways and plasticity. Harnessing the metabolic differences to reduce the tumor and immunosuppressive cells while enhancing the activity of positive immunoregulatory cells is a promising strategy. We develop a nanoplatform (CLCeMOF) based on cerium metal-organic framework (CeMOF) by lactate oxidase (LOX) modification and glutaminase inhibitor (CB839) loading. The cascade catalytic reactions induced by CLCeMOF generate reactive oxygen species "storm" to elicit immune responses. Meanwhile, LOX-mediated metabolite lactate exhaustion relieves the immunosuppressive tumor microenvironment, preparing the ground for intracellular regulation. Most noticeably, the immunometabolic checkpoint blockade therapy, as a result of glutamine antagonism, is exploited for overall cell mobilization. It is found that CLCeMOF inhibited glutamine metabolism-dependent cells (tumor cells, immunosuppressive cells, etc.), increased infiltration of dendritic cells, and especially reprogrammed CD8+ T lymphocytes with considerable metabolic flexibility toward a highly activated, long-lived, and memory-like phenotype. Such an idea intervenes both metabolite (lactate) and cellular metabolic pathway, which essentially alters overall cell fates toward the desired situation. Collectively, the metabolic intervention strategy is bound to break the evolutionary adaptability of tumors for reinforced immunotherapy.

Synthesis of Quinazolinone-Fused Tetrahydroisoquinolines and Related Polycyclic Scaffolds by Iodine-Mediated sp3 C-H Amination.

An iodine-mediated intramolecular sp3 C-H amination reaction producing quinazolinone-fused polycyclic skeletons from 2-aminobenzamide precursors is reported. This reaction does not use transition metals, has a broad substrate scope, and can be used on a gram scale. Under the optimal reaction conditions, a variety of quinazolinone-fused tetrahydroisoquinolines and derivatives of Rutaecarpine were synthesized from readily accessible compounds. The reaction proceeds well with crude 2-aminobenzamide derivatives, allowing for the synthesis of the products from simple 2-aminobenzoic acids and tetrahydroisoquinolines without purification of the 2-aminobenzamide intermediates. Preliminary biological experiments have identified Cereblon (CRBN) inhibitory activity and relevant anti-myeloma medicinal properties in some of these polycyclic products.

TIR-catalyzed ADP-ribosylation reactions produce signaling molecules for plant immunity.

Plant pathogen-activated immune signaling by nucleotide-binding leucine-rich repeat (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain converges on Enhanced Disease Susceptibility 1 (EDS1) and its direct partners, Phytoalexin Deficient 4 (PAD4) or Senescence-Associated Gene 101 (SAG101). TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) produces signaling molecules to promote exclusive EDS1-PAD4 and EDS1-SAG101 interactions with helper NLR subclasses. In this work, we show that TIR-containing proteins catalyze adenosine diphosphate (ADP)-ribosylation of adenosine triphosphate (ATP) and ADP ribose (ADPR) through ADPR polymerase-like and NADase activity, forming ADP-ribosylated ATP (ADPr-ATP) and ADPr-ADPR (di-ADPR), respectively. Specific binding of ADPr-ATP or di-ADPR allosterically promotes EDS1-SAG101 interaction with helper NLR N requirement gene 1A (NRG1A) in vitro and in planta. Our data reveal an enzymatic activity of TIRs that enables specific activation of the EDS1-SAG101-NRG1 immunity branch.

Identification and receptor mechanism of TIR-catalyzed small molecules in plant immunity.

Plant nucleotide-binding leucine-rich repeat-containing (NLR) receptors with an N-terminal Toll/interleukin-1 receptor (TIR) domain sense pathogen effectors to enable TIR-encoded nicotinamide adenine dinucleotide hydrolase (NADase) activity for immune signaling. TIR-NLR signaling requires the helper NLRs N requirement gene 1 (NRG1), Activated Disease Resistance 1 (ADR1), and Enhanced Disease Susceptibility 1 (EDS1), which forms a heterodimer with each of its paralogs Phytoalexin Deficient 4 (PAD4) and Senescence-Associated Gene 101 (SAG101). Here, we show that TIR-containing proteins catalyze the production of 2'-(5''-phosphoribosyl)-5'-adenosine monophosphate (pRib-AMP) and diphosphate (pRib-ADP) in vitro and in planta. Biochemical and structural data demonstrate that EDS1-PAD4 is a receptor complex for pRib-AMP and pRib-ADP, which allosterically promote EDS1-PAD4 interaction with ADR1-L1 but not NRG1A. Our study identifies TIR-catalyzed pRib-AMP and pRib-ADP as a missing link in TIR signaling through EDS1-PAD4 and as likely second messengers for plant immunity.

Comparative study of the binding between chlorogenic acid and four proteins by isothermal titration calorimetry, spectroscopy and docking methods.

As a polyphenolic compound, chlorogenic acid has antioxidant, anti-inflammatory, antiviral, anti-obesity and other effects. Based on the interactions between chlorogenic acid and the proteins (human serum albumin (HSA), pepsin (Pep), trypsin (Try), fat mass and obesity-associated protein (FTO)), results will provide clues for screening effective inhibitors. The interaction between chlorogenic acid and the four proteins (HSA, Pep, Try, FTO) was analyzed by the aid of fluorescence quenching, synchronous fluorescence, three-dimensional fluorescence, isothermal titration calorimetry, and molecular docking. It can be concluded that there is no obvious interaction between chlorogenic acid and FTO. The binding affinity between chlorogenic acid and three proteins is HSA > Try > Pep. The binding process is spontaneous, and the quenching type is static quenching. Hydrophobic interaction and hydrogen bonding is observed in the binding process. This study provides valuable information for understanding the interaction mechanism between chlorogenic acid and proteins, and provides clues for screening inhibitors.

Pharmacological suppression of glycogen synthase kinase-3 reactivates HIV-1 from latency via activating Wnt/ß-catenin/TCF1 axis in CD4+ T cells.

ABSTRACTHIV-1 latency posts a major obstacle for HIV-1 eradication. Currently, no desirable latency reversing agents (LRAs) have been implicated in the "Shock and Kill" strategy to mobilize the latently infected cells to be susceptible for clearance by immune responses. Identification of key cellular pathways that modulate HIV-1 latency helps to develop efficient LRAs. In this study, we demonstrate that the Wnt downstream ß-catenin/TCF1 pathway is a crucial modulator for HIV-1 latency. The pharmacological activation of the ß-catenin/TCF1 pathway with glycogen synthase kinase-3 (GSK3) inhibitors promoted transcription of HIV-1 proviral DNA and reactivated latency in CD4+ T cells; the GSK3 kinase inhibitor 6-bromoindirubin-3'-oxime (6-BIO)-induced HIV-1 reactivation was subsequently confirmed in resting CD4+ T cells from cART-suppressed patients and SIV-infected rhesus macaques. These findings advance our understanding of the mechanisms responsible for viral latency, and provide the potent LRA that can be further used in conjunction of immunotherapies to eradicate viral reservoirs.

Highly Regio- and Enantioselective Hydrosilylation of gem-Difluoroalkenes by Nickel Catalysis.

The synthesis of small organic molecules with a difluoromethylated stereocenter is particularly attractive in drug discovery. Herein, we have developed an efficient method for the direct generation of difluoromethylated stereocenters through Ni0 -catalyzed regio- and enantioselective hydrosilylation of gem-difluoroalkenes. The reaction also represents the enantioselective construction of carbon(sp3 )-silicon bonds with nickel catalysis, which provides an atom- and step-economical synthesis route of high-value optically active α-difluoromethylsilanes. This protocol features readily available starting materials and commercial chiral catalysis, broad substrates spanning a range of functional groups with high yield (up to 99 % yield) and excellent enantioselectivity (up to 96 % ee). The enantioenriched products undergo a variety of stereospecific transformations. Preliminary mechanistic studies were performed.

FNC inhibits non-small cell lung cancer by activating the mitochondrial apoptosis pathway.

BACKGROUND: Previously, we published that 4'-azid-2'-deoxy-2'-fluorarabinoside (FNC), a novel cytosine nucleoside analog, has good anti-viral and anti-tumor activity. OBJECTIVE: This study aimed to further explore the role and molecular mechanism of FNC in non-small cell lung cancer (NSCLC). METHODS: FNC was tested in the NSCLC H460 cell line, the Lewis mouse model, and the H460 cell xenograft model. The effects of FNC were assessed by cell viability, transwell migration, and wound scratch analyses of cell migration and invasion. Apoptosis was assessed by flow cytometry. Proteins expression was assessed by western blot and immunohistochemistry staining (IHC). RESULTS: FNC inhibits the proliferation and metastasis of H460 cells in a time- and dose-dependent manner. FNC treatment showed efficacy and low toxicity in the Lewis mouse lung cancer model as well as in the H460 cell xenograft model. Further, FNC induced H460 cell apoptosis through the activation of the mitochondrial pathway. Notably, FNC inhibited invasion by increasing E-cadherin protein and reducing the protein expression of VEGF, MMP-2, MMP-9, and CD31. CONCLUSION: FNC inhibits NSCLC by activating the mitochondrial apoptosis pathway and regulating the expressions of multiple proteins related to cell adhesion and invasion, highlighting its potential as an NSCLC therapeutic.

SARS-CoV-2-triggered mast cell rapid degranulation induces alveolar epithelial inflammation and lung injury.

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. In this study, we showed that SARS-CoV-2-triggered MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation altered various signaling pathways in alveolar epithelial cells, particularly, the induction of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.

Evolution of the protein corona affects macrophage polarization.

When nanoparticles (NPs) come into contact with bioenvironments, a protein corona forms on the NP surface. Previous reports showed that the constituents of the corona change with time. However, how different protein corona compositions influence cells, especially immune cells, has received less attention. Macrophages are important immune cells that can be polarized into a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype. In this study, AuNPs were incubated with human plasma for different periods to obtain time-related AuNP-coronas, and the influences of time-related AuNP-coronas on macrophage polarization were investigated. The macrophage morphology, biomarkers, cytokine secretion studies show that the pristine AuNPs and 4 h-AuNP-corona induced macrophage cells into M2 phenotype, while the co-incubation of 12 h-AuNP-corona and macrophage cells result in M1 phenotype. Further proteomic analysis showed that the compositions of protein corona were changing constantly after AuNPs contacted with plasma. When the incubation time increased to 12 h, the immune proteins in protein corona were increased significantly, which play a key role in modulation of the different macrophages polarization. Our findings demonstrated that plasma incubation time is an important parameter that needs to be taken into account in the study of nano-immune interactions and safe use of NPs in biological systems. Moreover, our finding can be a new efficient strategy for activating inflammatory or anti-inflammatory in medical treatment.

Design, synthesis and biological evaluation of tanshinone IIA-based analogues: Potent inhibitors of microtubule formation and angiogenesis.

We report the structural optimization of tanshinone IIA, a natural product which possesses anti-tumor properties but low water-solubility, weak antiproliferative activity and poor PK properties. A new series of ring A/C/D modified tanshinone analogues were synthesized and studied for their antiproliferative capacities against six human cancer cell lines. SAR study revealed that ring A cleavage of tanshinone IIA led to improved anti-cancer activity. Introduction of a methoxy group to the phenyl ring could enhance the anti-cancer activity even further. Compound 2f with methoxy group at C-8 position was selected as an early lead with IC50 values of 0.28-3.16 µM against six tested cell lines. 2f could bind to tubulin colchicine site, inhibit tubulin assembly and disrupt the normal formation of microtubule networks. Cellular mechanistic studies revealed that 2f induced apoptotic cell death of A549 cells in a dose-dependent manner. In vitro investigations showed that 2f impeded the tubule-formation of HUVECs and potently inhibited the proliferation, migration and invasion of A549 cells as well as HUVECs. Furthermore, the in vivo anti-angiogenic effect of 2f was confirmed via a zebrafish model test. The satisfactory physicochemical property and metabolic stability of 2f, as well as improved water-solubility, further suggested that 2f could serve as a promising tubulin inhibitor and anti-angiogenic agent.

Structural characteristics of small-molecule inhibitors targeting FTO demethylase.

Studies have shown that the FTO gene is closely related to obesity and weight gain in humans. FTO is an N6-methyladenosine demethylase and is linked to an increased risk of obesity and a variety of diseases, such as acute myeloid leukemia, type 2 diabetes, breast cancer, glioblastoma and cervical squamous cell carcinoma. In light of the significant role of FTO, the development of small-molecule inhibitors targeting the FTO protein provides not only a powerful tool for grasping the active site of FTO but also a theoretical basis for the design and synthesis of drugs targeting the FTO protein. This review focuses on the structural characteristics of FTO inhibitors and discusses the occurrence of obesity and cancer caused by FTO gene overexpression.

Discovery of Dosimertinib, a Highly Potent, Selective, and Orally Efficacious Deuterated EGFR Targeting Clinical Candidate for the Treatment of Non-Small-Cell Lung Cancer.

Osimertinib is a highly potent and selective third-generation epidermal growth factor receptor (EGFR) inhibitor, which provides excellent clinical benefits and is now a standard-of-care therapy for advanced EGFR mutation-positive non-small-cell lung cancer (NSCLC). However, AZ5104, a primary toxic metabolite of osimertinib, has caused unwanted toxicities. To address this unmet medical need, we initiated an iterative program focusing on structural optimizations of osimertinib and preclinical characterization, leading to the discovery of a highly potent, selective, and orally efficacious deuterated EGFR-targeting clinical candidate, dosimertinib. Preclinical studies revealed that dosimertinib demonstrated robust in vivo antitumor efficacy and favorable PK profiles, but with lower toxicity than osimertinib. These preclinical data support further clinical development of dosimertinib for the treatment of NSCLC. Dosimertinib has received official approval in China to initiate the phase I clinical trial (registration numbers: CXHL2000060 and CXHL2000061).

Cascade Catalytic Nanoplatform Based on "Butterfly Effect" for Enhanced Immunotherapy.

The unique tumor microenvironment (TME) characteristics such as immunosuppression impeded traditional cancer treatments. In contrast, developing cascade catalytic nanoplatforms by fully making use of substances in TME for cancer therapy may deserve full credit. Herein, a cascade catalytic nanoplatform based on glucose oxidase (GOD) modified mesoporous iron oxide nanoparticles (IONP) loaded with Artemisinin (ART) is developed, which is designed as IONP-GOD@ART. GOD can catalyze the oxidization of glucose into gluconic acid and H2 O2 , which not only realizes tumor starvation therapy, but also provides H2 O2 for IONP mediated Fenton reaction. Simultaneously, mesoporous IONP releases Fe2+ and Fe3+ ions in acidic TME. On the one hand, iron ions undergo Fenton reaction to generate hydroxyl radicals for chemodynamic therapy. On the other hand, the endoperoxide bridge in ART is broken in presence of Fe2+ and further generates reactive oxygen species (ROS) to achieve therapeutic purpose. In this sense, IONP-GOD@ART manipulates TME characteristics and leads to "butterfly effect", which brings out a large amount of ROS for eliciting immunogenic cell death, inducing M1-TAMs polarization, and further reprogramming immunosuppressive TME for enhanced immunotherapy. By this delicate design, the cascade catalytic nanoplatform of IONP-GOD@ART realizes potent cancer immunotherapy for tumor regression and metastasis prevention.

The role of chlorine atom on the binding between acrylonitrile derivatives and fat mass and obesity-associated protein.

In this work, seven acrylonitrile derivatives were selected as potential inhibitors of fat and obesity-related proteins (FTO) by the aid of fluorescence spectroscopy, ultraviolet visible spectroscopy, molecular docking, and cytotoxicity methods. Results show that the interaction between 3-amino-2-(4-chlorophenyl)-3-phenylacrylonitrile (1a) and FTO was the strongest among these derivatives. Thermodynamic analysis and molecular modeling show that the main force between 1a and FTO is hydrophobic interaction. The cytotoxicity test showed that the IC50 value of 1a was 46.64 µmol/L, which indicated 1a had the smallest IC50 value and had the best inhibitory effect on the proliferation of leukemia K562 cells among the seven derivatives. Both our previous results and this work show that chlorine atoms play important role in the binding of small molecules and FTO. This work brings new information for the study of FTO inhibitors.