Feedback activation of CD73-Adenosine axis attenuates the antitumor immunity of STING pathway.

The cGAS-STING pathway, a crucial component of innate immunity, has garnered attention as a potential therapeutic target for tumor treatment, but targeting this pathway is complicated by diverse feedback mechanisms of the cGAS-STING pathway. In this study, we demonstrated that STING activation enhanced the expression of CD73 and the subsequent production of adenosine in immune cells and cancer cells. Mechanistically, the feedback activation of CD73 depended on the type I IFN/IFNAR axis induced by STING activation. Furthermore, the combination of STING agonist and anti-CD73 mAb markedly blocked tumor growth in vivo by promoting the infiltration of CD8+ T cells and reducing the accumulation of Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment. Our work provides a rationale for the combination of STING agonists and CD73 inhibitors in cancer immunotherapy.

S-acylthioalkyl ester (SATE)-based prodrugs of deoxyribose cyclic dinucleotides (dCDNs) as the STING agonist for antitumor immunotherapy.

Cancer immunotherapy is a powerful weapon in the fight against cancers. Cyclic dinucleotides (CDNs) have demonstrated the great potential by evoking the immune system to fight cancers. There are still a lot of unmet needs for highly active CDNs in clinical applications due to low cell permeation and serum stability. Here we reported S-acylthioalkyl ester (SATE)-based prodrugs of deoxyribose cyclic dinucleotides (dCDNs) with three different types of internucleotide linkages (3',3':11a; 2',3':11b; 2',2':11c). The parent dCDNs could be efficiently released from SATE-dCDNs by cellular esterases. Compared to 2',3'-cGAMP and ADU-S100, 11a exhibited much higher potency of activating STING pathway and higher serum stability. In a CT26-Luc tumor-bearing animal model, 11a showed the efficient antitumor activity in eliminating the established tumor and induced significant increase of mRNA expression of IFN-ß and other related inflammatory cytokines. Hence, SATE-dCDN prodrugs demonstrated their benefits in promoting cell penetration, improving serum stability, and thus enhancing bioactivity, suggesting their potential application as immunotherapy in a variety of malignancies.

Blockade of USP14 potentiates type I interferon signaling and radiation-induced antitumor immunity via preventing IRF3 deubiquitination.

PURPOSE: The adaptive immune responses induced by radiotherapy has been demonstrated to largely rely on STING-dependent type I interferons (IFNs) production. However, irradiated tumor cells often fail to induce dendritic cells (DCs) to produce type I IFNs. Hence, we aim to uncover the limitation of STING-mediated innate immune sensing following radiation, and identify efficient reagents capable to rescue the failure of type I IFNs induction for facilitating radiotherapy. METHODS: A targeted cell-based phenotypic screening was performed to search for active molecules that could elevate the production of type I IFNs. USP14 knockout or inhibition was assayed for IFN production and the activation of STING signaling in vitro. The mechanisms of USP14 were investigated by western blot and co-immunoprecipitation in vitro. Additionally, combinational treatments with PT33 and radiation in vivo and in vitro models were performed to evaluate type I IFNs responses to radiation. RESULTS: PT33 was identified as an enhancer of STING agonist elicited type I IFNs production to generate an elevated and durable STING activation profile in vitro. Mechanistically, USP14 inhibition or deletion impairs the deubiquitylation of K63-linked IRF3. Furthermore, blockade of USP14 with PT33 enhances DC sensing of irradiated-tumor cells in vitro, and synergizes with radiation to promote systemic antitumor immunity in vivo. CONCLUSION: Our findings reveal that USP14 is one of the major IFN production suppressors and impairs the activation of IRF3 by removing the K63-linked ubiquitination of IRF3. Therefore, blockage of USP14 results in the gain of STING signaling activation and radiation-induced adaptive immune responses.

N-cystaminylbiguanide MC001 prevents neuron cell death and alleviates motor deficits in the MPTP-model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra (SN), which is highly associated with oxidative stress. Antioxidants are therefore considered as potential therapies in PD treatment. In this study, we examined the neuroprotective effect of a cysteamine-based biguanide N-cystaminylbiguanide (MC001) in the MPTP mouse model of PD. The results showed that MC001 prevented neuron cell death and alleviated motor deficits in the MPTP mouse model of PD. Both in vitro and in vivo data indicated that MC001 may exert its neuroprotective effect by alleviating ROS production, suppressing neuroinflammation, and upregulating BDNF expression. Further mechanistic studies revealed that MC001 promoted GSH synthesis by inducing the expression of Glutamate-cysteine ligase catalytic subunit (Gclc) and enhancing the activity of Glutamate-cysteine ligase (Gcl). Our results suggest that MC001 warrants further investigation as a potential candidate for the treatment of PD.

Identification of DNA aptamers that specifically targets EBV+ nasopharyngeal carcinoma via binding with EphA2/CD98hc complex.

Nasopharyngeal carcinoma (NPC) is one of the Epstein-Barr virus (EBV)-associated malignancies and has a distinct geographical distribution. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for biomarkers for early diagnosis and effective treatments. In this study, we developed DNA aptamers that specifically bind to EBV positive NPC cells by the Cell-SELEX procedure. We further identified the EphA2 (ephrin type-A receptor 2)/CD98hc (CD98 heavy chain) complex as the potential target of the aptamer EA-3 by combining aptamer-based separation and mass spectrometry analysis. Our results revealed for the first time that EphA2 colocalized with CD98hc at the plasma membrane and EphA2 coimmunoprecipitated with CD98hc, which may serve as a starting point for exploring the potential functions of the complex of EphA2 and CD98hc in NPCs. Here, we demonstrated that aptamers can be useful for the identification of protein complexes on the surface of cancer cells.

Biguanide MC001, a Dual Inhibitor of OXPHOS and Glycolysis, Shows Enhanced Antitumor Activity Without Increasing Lactate Production.

Metformin and other biguanides represent a new class of inhibitors of mitochondrial complex I that show promising antitumor effects. However, stronger inhibition of mitochondrial complex I is generally associated with upregulation of glycolysis and higher risk of lactic acidosis. Herein we report a novel biguanide derivative, N-cystaminylbiguanide (MC001), which was found to inhibit mitochondrial complex I with higher potency while inducing lactate production to a similar degree as metformin.Furthermore, MC001 was found to efficiently inhibit a panel of colorectal cancer (CRC) cells in vitro and to suppress tumor growth in a HCT116 xenograft nude mouse model, while not enhancing lactate production relative to metformin, exhibiting a superior safety profile to other potent biguanides such as phenformin. Mechanistically, MC001 efficiently inhibits mitochondrial complex I, activates AMPK, and represses mTOR, leading to cell-cycle arrest and apoptosis. Notably, MC001 inhibits both oxidative phosphorylation (OXPHOS) and glycolysis. We therefore propose that MC001 warrants further investigation in cancer treatment.

STAT3 inhibition enhances CDN-induced STING signaling and antitumor immunity.

Cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a key regulator in innate immunity and has emerged as a promising drug target in cancer treatment, but the utility of this pathway in therapeutic development is complicated by its dichotomous roles in tumor development and immunity. The activation of the STING pathway and the induced antitumor immunity could be attenuated by the feedback activation of IL-6/STAT3 pathway. Here we reported that STAT3 inhibition significantly enhanced the intensity and duration of STING signaling induced by the STING agonist c-diAM(PS)2. Such sensitization effect of STAT3 inhibition on STING signaling depended on STING rather than cGAS, which was mediated by simultaneously upregulating the positive modulators and downregulating the negative modulators of the STING pathway. Furthermore, the combination treatment with the STAT3 inhibitor and STING agonist markedly regressed tumor growth in syngeneic mice by increasing CD8+ T cells and reducing regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment. Our work provides a rationale for the combination of STAT3 inhibitors and STING agonists in cancer immunotherapy.

Structural basis for dimerization of the death effector domain of the F122A mutant of Caspase-8.

Caspase-8 is an apoptotic protease that is activated by a proximity-induced dimerization mechanism within the death-inducing signaling complex (DISC). The death effector domain (DED) of caspase-8 is involved in protein-protein interactions and is essential for the activation. Here, we report two crystal structures of the dimeric DEDs of the F122A mutant of caspase-8, both of which illustrate a novel domain-swapped dimerization, while differ in the relative orientation of the two subunits and the solvent exposure of the conserved hydrophobic patch Phe122/Leu123. We demonstrate that mutations disrupting the dimerization of the DEDs abrogate the formation of cellular death effector filaments (DEFs) and the induced apoptosis by overexpressed DEDs. Furthermore, such dimerization-disrupting mutations also impair the activation of the full-length caspase-8 and the downstream apoptosis cascade. The structures provide new insights into understanding the mechanism underlying the activation of procaspase-8 within the DISC and DEFs.

Identification of α-Mangostin as an Agonist of Human STING.

The xanthone derivate 5',6'-dimethylxanthenone-4-acetic acid (DMXAA, also known as ASA404 or vadimezan) is a potent agonist of murine STING (stimulator of interferon genes), but cannot activate human STING. Herein we report that α-mangostin, which bears the xanthone skeleton, is an agonist of human STING, but activates murine STING to a lesser extent. Biochemical and cell-based assays indicate that α-mangostin binds to and activates human STING, leading to activation of the downstream interferon regulatory factor (IRF) pathway and production of type I interferons. Furthermore, our studies show that α-mangostin has the potential to repolarize human monocyte-derived M2 macrophages to the M1 phenotype. The agonist effect of α-mangostin in the STING pathway might account for its antitumor and antiviral activities.

Dissociation of haemolytic and oligomer-preventing activities of gramicidin S derivatives targeting the amyloid-ß N-terminus.

The intrinsic haemolysis of an amyloid-ß (Aß) N-terminal targeting gramicidin S derivative was successfully dissociated from its Aß oligomer-preventing activities via Ala-scanning-based regulation of molecular amphiphilicity. The representative analogue DGR-7 shows low toxicity but significant efficiency in preventing Aß oligomers and reducing amyloid plaques in APP/PS1 transgenic AD mice.

Mitochondria-Targeted Approach: Remarkably Enhanced Cellular Bioactivities of TPP2a as Selective Inhibitor and Probe toward TrxR.

A mitochondria-targeted approach was developed to increase the cellular bioactivities of thioredoxin reductase (TrxR) inhibitors. By being conjugated with a triphenylphosphine (TPP) motif to a previously found TrxR inhibitor 2a, the resulted compound TPP2a can target subcellular mitochondria and efficiently inhibit cellular TrxR, leading to remarkably increased cellular ROS level and mitochondrial apoptosis of HeLa cancer cells. The cellular bioactivities of TPP2a, including its cytotoxicity against a panel of cancer cell lines, dramatically elevated compared with its parental compound 2a. The selectively and covalently interaction of TPP2a with subcellular mitochondrial TrxR was validated by fluorescent microscopy. Moreover, a nonspecific signal quenching coupled strategy was proposed based on the environmentally sensitive fluorescence of TPP2a, which makes it possible to label TrxR by removing the nonspecific backgrounds caused by TPP2a under complex biosettings such as cellular lysates and living cells, implicating a potential of TPP2a for TrxR-specific labeling.

Distinct tubulin dynamics in cancer cells explored using a highly tubulin-specific fluorescent probe.

A highly specific fluorescent probe (OC9) was discovered exhibiting tubulin-specific affinity fluorescence, which allowed selective labeling of cellular tubulin in microtubules. Moreover, distinct tubulin dynamics in various cellular bio-settings such as drug resistant or epithelial-mesenchymal transition (EMT) cancer cells were directly observed for the first time via OC9 staining.

Identification of green tea catechins as potent inhibitors of the polo-box domain of polo-like kinase 1.

Polo-like kinase 1 (PLK1) plays crucial functions in multiple stages of mitosis and is considered to be a potential drug target for cancer therapy. The functions of PLK1 are mediated by its N-terminal kinase domain and C-terminal polo-box domain (PBD). Most inhibitors targeting the kinase domain of PLK1 have a selectivity issue because of a high degree of structural conservation within kinase domains of all protein kinases. Here, we combined virtual and experimental screenings to identify green tea catechins as potent inhibitors of the PLK1 PBD. Initially, (-)-epigallocatechin, one of the main components of green tea polyphenols, was found to significantly block the binding of fluorescein-labeled phosphopeptide to the PBD at a concentration of 10 µm. Next, additional catechins were evaluated for their dose-dependent inhibition of the PBD and preliminary structure-activity relationships were derived. Cellular analysis further showed that catechins interfere with the proper subcellular localization of PLK1, lead to cell-cycle arrest in the S and G2M phases, and induce growth inhibition of several human cancer cell types, such as breast adenocarcinoma (MCF7), lung adenocarcinoma (A549), and cervical adenocarcinoma (HeLa). Our data provides new insight into understanding the anticancer activities of green tea catechins.

Structural basis for the inhibition of Polo-like kinase 1.

Polo-like kinase 1 (PLK1) is a master regulator of mitosis and is considered a potential drug target for cancer therapy. PLK1 is characterized by an N-terminal kinase domain (KD) and a C-terminal Polo-box domain (PBD). The KD and PBD are mutually inhibited, but the molecular mechanisms of the autoinhibition remain unclear. Here we report the 2.3-Å crystal structure of the complex of the Danio rerio KD and PBD together with a PBD-binding motif of Drosophila melanogaster microtubule-associated protein 205 (Map205(PBM)). The structure reveals that the PBD binds and rigidifies the hinge region of the KD in a distinct conformation from that of the phosphopeptide-bound PBD. This structure provides a framework for understanding the autoinhibitory mechanisms of PLK1 and also sheds light on the activation mechanisms of PLK1 by phosphorylation or phosphopeptide binding.

A novel cyclinE/cyclinA-CDK inhibitor targets p27(Kip1) degradation, cell cycle progression and cell survival: implications in cancer therapy.

p27(Kip1) (p27) binds and inhibits the cyclin E- or cyclin A-associated cyclin-dependent kinases (CDKs)2 and other CDKs, and negatively regulates G1-G2 cell cycle progression. To develop specific CDK inhibitors, we have modeled the interaction between p27 and cyclin A-CDK2, and designed a novel compound that mimics p27 binding to cyclin A-CDK2. The chemically synthesized inhibitor exhibited high potency and selective inhibition towards cyclin E/cyclin A-CDK2 kinase in vitro but not other kinases. To facilitate permeability of the inhibitor, a cell penetrating peptide (CPP) was conjugated to the inhibitor to examine its effect in several cancer cell lines. The CPP-conjugated inhibitor significantly inhibited the proliferation of cancer cells. The treatment of the inhibitor resulted in the increased accumulation of p27 and p21(Cip1/Waf1) (p21) and hypo-phosphorylation of retinoblastoma protein (Rb). The degradation of p27, mediated through the phosphorylation of threonine-187 in p27, was also inhibited. Consequently, exposure of cells to the inhibitor caused cell cycle arrest and apoptosis. We conclude that specific cyclinE/cyclin A-CDK2 inhibitors can be developed based on the interaction between p27 and cyclin/CDK to block cell cycle progression to prevent tumor growth and survival.

Novel histone demethylase LSD1 inhibitors selectively target cancer cells with pluripotent stem cell properties.

Histone modification determines epigenetic patterns of gene expression with methylation of histone H3 at lysine 4 (H3K4) often associated with active promoters. LSD1/KDM1 is a histone demethylase that suppresses gene expression by converting dimethylated H3K4 to mono- and unmethylated H3K4. LSD1 is essential for metazoan development, but its pathophysiologic functions in cancer remain mainly uncharacterized. In this study, we developed specific bioactive small inhibitors of LSD1 that enhance H3K4 methylation and derepress epigenetically suppressed genes in vivo. Strikingly, these compounds inhibited the proliferation of pluripotent cancer cells including teratocarcinoma, embryonic carcinoma, and seminoma or embryonic stem cells that express the stem cell markers Oct4 and Sox2 while displaying minimum growth-inhibitory effects on non-pluripotent cancer or normal somatic cells. RNA interference-mediated knockdown of LSD1 expression phenocopied these effects, confirming the specificity of small molecules and further establishing the high degree of sensitivity and selectivity of pluripotent cancer cells to LSD1 ablation. In support of these results, we found that LSD1 protein level is highly elevated in pluripotent cancer cells and in human testicular seminoma tissues that express Oct4. Using these novel chemical inhibitors as probes, our findings establish LSD1 and histone H3K4 methylation as essential cancer-selective epigenetic targets in cancer cells that have pluripotent stem cell properties.

Formal synthesis of cortistatins.

A unified strategy toward the asymmetric facile construction of the [6.7.6.5]oxapentacyclic skeleton of cortistatins is reported, featuring intramolecular Diels-Alder (IMDA), oxidative dearomatization, and an oxy-Michael addition reaction.

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase.

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two-layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn-bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate-determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn-bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate-determining step is the proton transfer from Zn-OH to 6-NH(2) of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate.

Characterization and development of novel small-molecules inhibiting GSK3 and activating Wnt signaling.

Glycogen synthase kinase 3 (GSK3) is an essential component of the Wnt signaling pathway and plays important roles in regulating cell proliferation, differentiation, and apoptosis. As GSK3 is abnormally upregulated in several diseases including type II diabetes, Alzheimer's disease and cancer, it has been regarded as a potential drug target. During zebrafish development, inhibition of GSK3 leads to ectopic activation of the Wnt pathway, resulting in a headless embryo. Using this phenotype as an assay we screened a chemical library of 4000 compounds and identified one novel compound, 3F8, which specifically inhibits eye and forebrain formation in zebrafish embryos, resembling a typical Wnt overexpression phenotype. Cell reporter assays, chemical informatics analysis and in vitro kinase experiments revealed that 3F8 is a selective GSK3 inhibitor, which is more potent than SB216763, a commonly used GSK3 inhibitor. Based on the structure of 3F8, a new generation of compounds inhibiting GSK3 was synthesized and validated by biological assays. Together, 3F8 and its derivatives could be useful as new reagents and potential therapeutic candidates for GSK3 related diseases.

Identifying tumor cell growth inhibitors by combinatorial chemistry and zebrafish assays.

Cyclin-dependent kinases (CDKs) play important roles in regulating cell cycle progression, and altered cell cycles resulting from over-expression or abnormal activation of CDKs observed in many human cancers. As a result, CDKs have become extensive studied targets for developing chemical inhibitors for cancer therapies; however, protein kinases share a highly conserved ATP binding pocket at which most chemical inhibitors bind, therefore, a major challenge in developing kinase inhibitors is achieving target selectivity. To identify cell growth inhibitors with potential applications in cancer therapy, we used an integrated approach that combines one-pot chemical synthesis in a combinatorial manner to generate diversified small molecules with new chemical scaffolds coupled with growth inhibition assay using developing zebrafish embryos. We report the successful identification of a novel lead compound that displays selective inhibitory effects on CDK2 activity, cancer cell proliferation, and tumor progression in vivo. Our approaches should have general applications in developing cell proliferation inhibitors using an efficient combinatorial chemical genetic method and integrated biological assays. The novel cell growth inhibitor we identified should have potential as a cancer therapeutic agent.