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.

Cyclopeptide Inhibitors Target the N-Terminal Tail of STING and Alleviate Autoinflammation.

Cyclic GMP-AMP synthase (cGAS)- stimulator of interferon genes (STING) signaling pathway is a crucial component of innate immunity that plays a vital role in protecting against pathogen infections and cellular stress. However, aberrant activation of cGAS-STING pathway is associated with inflammatory and autoimmune diseases. Here, we developed cyclopeptide STING inhibitors by cyclizing the N-terminal tail (NTT) of STING. These cyclopeptides selectively inhibited the activation of STING pathway in human or murine cell lines. Mechanistically, the inhibitors directly bound to STING, and subsequently blocked the aggregation and activation of STING. In addition, the optimal inhibitor STi-2 significantly suppressed the elevated levels of type I interferon and proinflammatory cytokines in primary macrophages derived from Trex1-/- mice and systemic inflammation in Trex1-/- mice. Overall, our work facilitates the development of specific inhibitors of STING as potential therapies in the treatment of cGAS-STING associated autoinflammatory diseases.

Development of a novel anti-inflammatory recombinant uricase with extended half-life for gout therapy.

Gout is a form of inflammatory arthritis that results from elevated serum uric acid levels and the deposition of urate crystals in multiple joints. The inflammatory response during an acute gout attack is mediated by the activation of the NLRP3 inflammasome, leading to the release of IL-1ß and inducing a localized tissue inflammatory response. Urate lowering therapies such as Pegloticase effectively reduce serum uric acid levels but are generally associated with an increase in acute gout flares. In this study, we developed a long-acting anti-inflammatory recombinant uricase by sequential fusing interleukin-1 receptor antagonist (IL-1Ra) and albumin-binding domain (ABD) with the N-terminal end of Arthrobacter globiformis uricase (AgUox). The recombinant uricase has longer in vivo half-life, and significantly alleviates monosodium urate (MSU) crystals induced inflammation in mouse model compared with the wild-type AgUox. This long-acting anti-inflammatory recombinant uricase has the potential to be developed as an effective urate lowering therapy with better safety profiles.

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.

PFKFB3-mediated endothelial glycolysis promotes pulmonary hypertension.

Increased glycolysis in the lung vasculature has been connected to the development of pulmonary hypertension (PH). We therefore investigated whether glycolytic regulator 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3)-mediated endothelial glycolysis plays a critical role in the development of PH. Heterozygous global deficiency of Pfkfb3 protected mice from developing hypoxia-induced PH, and administration of the PFKFB3 inhibitor 3PO almost completely prevented PH in rats treated with Sugen 5416/hypoxia, indicating a causative role of PFKFB3 in the development of PH. Immunostaining of lung sections and Western blot with isolated lung endothelial cells showed a dramatic increase in PFKFB3 expression and activity in pulmonary endothelial cells of rodents and humans with PH. We generated mice that were constitutively or inducibly deficient in endothelial Pfkfb3 and found that these mice were incapable of developing PH or showed slowed PH progression. Compared with control mice, endothelial Pfkfb3-knockout mice exhibited less severity of vascular smooth muscle cell proliferation, endothelial inflammation, and leukocyte recruitment in the lungs. In the absence of PFKFB3, lung endothelial cells from rodents and humans with PH produced lower levels of growth factors (such as PDGFB and FGF2) and proinflammatory factors (such as CXCL12 and IL1ß). This is mechanistically linked to decreased levels of HIF2A in lung ECs following PFKFB3 knockdown. Taken together, these results suggest that targeting PFKFB3 is a promising strategy for the treatment of PH.

Comparative pharmacokinetic profile of cyclosporine (CsA) with a decapeptide and a linear analogue.

The synthesis and in vivo pharmacokinetic profile of an analogue of cyclosporine is disclosed. An acyclic congener was also profiled in in vitro assays to compare cell permeability. The compounds possess similar calculated and measured molecular descriptors however have different behaviors in an RRCK assay to assess cell permeability.

Inhibitory effect of flavonoids on human glutaminyl cyclase.

Glutaminyl cyclase (QC) plays an important role in the pathogenesis of Alzheimer's disease (AD) and can be a potential target for the development of novel anti-AD agents. However, the study of QC inhibitors are still less. Here, phenol-4' (R1-), C5-OH (R2-) and C7-OH (R3-) modified apigenin derivatives were synthesized as a new class of human QC (hQC) inhibitors. The efficacy investigation of these compounds was performed by spectrophotometric assessment and the structure-activity relationship (SAR) was evaluated. Molecular docking was also carried out to analyze the binding mode of the synthesized flavonoid to the active site of hQC.

Crystal structure of the polo-box domain of polo-like kinase 2.

Polo-like kinase 2 (PLK2) is a crucial regulator in cell cycle progression, DNA damage response, and neuronal activity. PLK2 is characterized by the conserved N-terminal kinase domain and the unique C-terminal polo-box domain (PBD). The PBD mediates diverse functions of PLK2 by binding phosphorylated Ser-pSer/pThr motifs of its substrates. Here, we report the first crystal structure of the PBD of PLK2. The overall structure of the PLK2 PBD is similar to that of the PLK1 PBD, which is composed by two polo boxes each contain ß6α structures that form a 12-stranded ß sandwich domain. The edge of the interface between the two polo boxes forms the phosphorylated Ser-pSer/pThr motifs binding cleft. On the hand, the peripheral regions around the core binding cleft of the PLK2 PBD is distinct from that of the PLK1 PBD, which might confer the substrate specificity of the PBDs of the polo-like kinase family.

Crystal structure of the death effector domains of caspase-8.

Caspase-8 is a key mediator in various biological processes such as apoptosis, necroptosis, inflammation, T/B cells activation, and cell motility. Caspase-8 is characterized by the N-terminal tandem death effector domains (DEDs) and the C-terminal catalytic protease domain. The DEDs mediate diverse functions of caspase-8 through homotypic interactions of the DEDs between caspase-8 and its partner proteins. Here, we report the first crystal structure of the DEDs of caspase-8. The overall structure of the DEDs of caspase-8 is similar to that of the DEDs of vFLIP MC159, which is composed of two tandem death effector domains that closely associate with each other in a head-to-tail manner. Structural analysis reveals distinct differences in the region connecting helices α2b and α4b in the second DED of the DEDs between caspase-8 and MC159, in which the helix α3b in MC159 is replaced by a loop in caspase-8. Moreover, the different amino acids in this region might confer the distinct features of solubility and aggregation for the DEDs of caspase-8 and MC159.

Structure-based design of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3ß inhibitors to activate Wnt/ß-catenin pathway.

Deregulation of Wnt/ß-catenin pathway is closely related to the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), and glycogen synthase kinase 3ß (GSK-3ß), the central negative regulator of Wnt pathway, is regarded as an important target for these diseases. Here, we report a series of benzo[e]isoindole-1,3-dione derivatives as selective GSK-3ß inhibitors by rational-design and synthesis, which show high selectivity against GSK-3ß over Cyclin-dependent kinase 2 (CDK2), and significantly activate the cellular Wnt/ß-catenin pathway. The structure-activity relationship of these GSK-3ß inhibitors was also explored by in silico molecular docking.

Development of cyclopeptide inhibitors of cGAS targeting protein-DNA interaction and phase separation.

Cyclic GMP-AMP synthase (cGAS) is an essential sensor of aberrant cytosolic DNA for initiating innate immunity upon invading pathogens and cellular stress, which is considered as a potential drug target for autoimmune and autoinflammatory diseases. Here, we report the discovery of a class of cyclopeptide inhibitors of cGAS identified by an in vitro screening assay from a focused library of cyclic peptides. These cyclopeptides specifically bind to the DNA binding site of cGAS and block the binding of dsDNA with cGAS, subsequently inhibit dsDNA-induced liquid phase condensation and activation of cGAS. The specificity and potency of one optimal lead XQ2B were characterized in cellular assays. Concordantly, XQ2B inhibited herpes simplex virus-1 (HSV-1)-induced antiviral immune responses and enhanced HSV-1 infection in vitro and in vivo. Furthermore, XQ2B significantly suppressed the elevated levels of type I interferon and proinflammatory cytokines in primary macrophages from Trex1-/- mice and systemic inflammation in Trex1-/- mice. XQ2B represents the specific cGAS inhibitor targeting protein-DNA interaction and phase separation and serves as a scaffold for the development of therapies in the treatment of cGAS-dependent inflammatory diseases.

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.

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.

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.

Identification and semisynthesis of (-)-anisomelic acid as oral agent against SARS-CoV-2 in mice.

(-)-Anisomelic acid, isolated from Anisomeles indica (L.) Kuntze (Labiatae) leaves, is a macrocyclic cembranolide with a trans-fused α-methylene-γ-lactone motif. Anisomelic acid effectively inhibits SARS-CoV-2 replication and viral-induced cytopathic effects with an EC50 of 1.1 and 4.3 µM, respectively. Challenge studies of SARS-CoV-2-infected K18-hACE2 mice showed that oral administration of anisomelic acid and subcutaneous dosing of remdesivir can both reduce the viral titers in the lung tissue at the same level. To facilitate drug discovery, we used a semisynthetic approach to shorten the project timelines. The enantioselective semisynthesis of anisomelic acid from the naturally enriched and commercially available starting material (+)-costunolide was achieved in five steps with a 27% overall yield. The developed chemistry provides opportunities for developing anisomelic-acid-based novel ligands for selectively targeting proteins involved in viral infections.

Understanding the binding mode and function of BMS-488043 against HIV-1 viral entry.

A recently discovered small-molecule inhibitor, BMS-488043 (BMS-488), for the invasion of Human immunodeficiency virus Type 1 (HIV-1), shows a high activity against the entry of diversified HIV-1. Docking and molecular dynamic studies have been carried out to understand the binding mode of BMS-488 to gp120 as well as the effect of the small molecule on the conformational change of gp120 induced by CD4 binding. The results indicate that BMS-488 can accommodate in the CD4 binding pocket and interfere the CD4 binding in a noncompetitive mode. The piperazine group of BMS-488 prevents the bridging sheet formation of gp120 induced by the CD4 binding mainly through blocking the rotation of the Trp112 located on the α1 helix of gp120. The bridging sheet formation cannot be blocked for the W112A mutant of gp120 due to the reduced steric hindrance, in agreement with its significant resistance to the BMS inhibitor. The aza-indole ring is likely to interfere the exposure of gp41 by stacking within the ß3-ß5 and LB loops to disrupt the close packing of Pro212-His66-Phe210. The mode of action of BMS-488 also accommodates many mutagenesis results related to BMS-488 activity.

Intermediate-assisted multifunctional catalysis in the conversion of flavin to 5,6-dimethylbenzimidazole by BluB: a density functional theory study.

BluB is a distinct flavin destructase that catalyzes a complex oxygen-dependent conversion of reduced flavin mononucleotide (FMNH(2)) to form 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B(12). The catalyzed mechanism remains a challenge due to the discrepancy between the complexity of the conversion and the relative simplicity of the active site of BluB. In this study, we have explored the detailed conversion mechanism by using the hybrid density functional method B3LYP on an active site model of BluB consisting of 144 atoms. The results indicate that the conversion involves more than 14 sequential steps in two distinct stages. In the first stage, BluB catalyzes the incorporation of dioxygen, and the fragmentation of the isoalloxazine ring of FMNH(2) to form alloxan and the ribityl dimethylphenylenediimine (DMPDI); in the second stage, BluB exploits alloxan as a multifunctional cofactor, such as a proton donor, a proton acceptor, and a hydride acceptor, to catalyze the remaining no fewer than 10 steps of the reaction. The retro-aldol cleavage of the C1'-C2' bond of DMPDI is the rate-determining step with a barrier of about 21.6 kcal/mol, which produces D-erythrose 4-phosphate (E4P) and the ring-closing precursor of DMB. The highly conserved residue Asp32 plays critical roles in multiple steps of the conversion by serving as a proton acceptor or a proton shuttle, and another conserved residue Ser167 plays its catalytic role mainly in the rate-determining step by stabilizing the protonated retro-aldol precursor. These results are consistent with the available experimental observations. More significantly, the novel intermediate-assisted mechanism not only provides significant insights into understanding the mechanism underlying the power of the simple BluB catalyzing the complex conversion of FMNH(2) to DMB, but also represents a new type of intermediate-assisted multifunctional catalysis in an enzymatic reaction.

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.

Theoretical studies on the mechanism and stereoselectivity of Rh(Phebox)-catalyzed asymmetric reductive aldol reaction.

Density functional theory calculations (B3LYP) have been carried out to understand the mechanism and stereochemistry of an asymmetric reductive aldol reaction of benzaldehyde and tert-butyl acrylate with hydrosilanes catalyzed by Rh(Phebox-ip)(OAc)(2)(OH(2)). According to the calculations, the reaction proceeds via five steps: (1) oxidative addition of hydrosilane, (2) hydride migration to carbon-carbon double bond of tert-butyl acrylate, which determines the chirality at C2, (3) tautomerization from rhodium bound C-enolate to rhodium bound O-enolate, (4) intramolecular aldol reaction, which determines the chirality at C3 and consequently the anti/syn-selectivity, and (5) reductive elimination to release aldol product. The hydride migration is the rate-determining step with a calculated activation energy of 23.3 kcal mol(-1). In good agreement with experimental results, the formation of anti-aldolates is found to be the most favorable pathway. The observed Si-facial selectivity in both hydride migration and aldol reaction are well-rationalized by analyzing crucial transition structures. The Re-facial attack transition state is disfavored because of steric hindrance between the isopropyl group of the catalyst and the tert-butyl acrylate.