N-Containing triterpenoid saponins from Mussaenda densiflora and identification of heinsiagenin A as a potent immunosuppressant.

Eleven triterpenoid saponins, including five new compounds, which were named densiflorasides A - E (1 - 5), were isolated from aerial parts of Mussaenda densiflora (Rubiaceae). Their structures were elucidated based on spectroscopic and single-crystal X-ray diffraction analyses and chemical methods. All the isolated compounds and the aglycone heinsiagenin A were evaluated for their immunosuppressive and antiosteoclastogenic activities in vitro. Compounds 6 - 8 and heinsiagenin A inhibited osteoclastogenesis, with IC50 values ranging from 8.24 to 17.7 µM. Furthermore, compounds 3, 6 - 8, and heinsiagenin A significantly inhibited T-cell proliferation, with IC50 values ranging from 2.56 to 8.60 µM, and compounds 3 - 5 and 11 inhibited the proliferation of B lymphocytes, with IC50 values ranging from 1.29 to 8.49 µM. Further in vivo experiments indicated that heinsiagenin A could significantly attenuate IMQ-induced psoriasis and DSS-induced colitis in mice.

Macrolide sesquiterpene pyridine alkaloids from Celastrus monospermus and evaluation of their immunosuppressive and anti-osteoclastogenesis activities.

Phytochemical investigation of the stems of Celastrus monospermus Roxb enabled isolation and identification of fifteen new macrolide sesquiterpene pyridine alkaloids (1-15) along with five known analogues. Their structures were elucidated by comprehensive spectroscopic analysis (NMR, HRESIMS, IR, UV), chemical hydrolysis, and single crystal X-ray diffraction analysis. Bioassay of the abundant isolates revealed that seven compounds inhibited the proliferation of B lymphocytes with IC50 values ranging between 1.4 and 19.9 µM. Among them, celasmondine C (3) could significantly promote the apoptosis of activated B lymphocyte, especially late-stage apoptosis. Besides, compounds 3, 16, and 20 exhibited potent suppression of osteoclast formation at a concentration of 1.0 µM. This investigation enriched the chemical diversity of macrolide sesquiterpene pyridine alkaloids, and supported evidence for the development of new immunosuppressive and anti-osteoclastogenesis agents.

DZ2002 alleviates corneal angiogenesis and inflammation in rodent models of dry eye disease via regulating STAT3-PI3K-Akt-NF-κB pathway.

Dry eye disease (DED) is a prevalent ocular disorder with a multifactorial etiology. The pre-angiogenic and pre-inflammatory milieu of the ocular surface plays a critical role in its pathogenesis. DZ2002 is a reversible type III S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitor, which has shown excellent anti-inflammatory and immunosuppressive activities in vivo and in vitro. In this study, we evaluated the therapeutic potential of DZ2002 in rodent models of DED. SCOP-induced dry eye models were established in female rats and mice, while BAC-induced dry eye model was established in female rats. DZ2002 was administered as eye drops (0.25%, 1%) four times daily (20 µL per eye) for 7 or 14 consecutive days. We showed that topical application of DZ2002 concentration-dependently reduced corneal neovascularization and corneal opacity, as well as alleviated conjunctival irritation in both DED models. Furthermore, we observed that DZ2002 treatment decreased the expression of genes associated with angiogenesis and the levels of inflammation in the cornea and conjunctiva. Moreover, DZ2002 treatment in the BAC-induced DED model abolished the activation of the STAT3-PI3K-Akt-NF-κB pathways in corneal tissues. We also found that DZ2002 significantly inhibited the proliferation, migration, and tube formation of human umbilical endothelial cells (HUVECs) while downregulating the activation of the STAT3-PI3K-Akt-NF-κB pathway. These results suggest that DZ2002 exerts a therapeutic effect on corneal angiogenesis in DED, potentially by preventing the upregulation of the STAT3-PI3K-Akt-NF-κB pathways. Collectively, DZ2002 is a promising candidate for ophthalmic therapy, particularly in treating DED.

Artemisinin derivative SM934 in the treatment of autoimmune and inflammatory diseases: therapeutic effects and molecular mechanisms.

Artemisinin and its derivatives are the well-known anti-malarial drugs derived from a traditional Chinese medicine. In addition to antimalarial, artemisinin and its derivatives possess distinguished anti-cancer, anti-oxidant, anti-inflammatory and anti-viral activities, but the poor solubility and low bioavailability hinder their clinical application. In the last decades a series of new water-soluble and oil-soluble derivatives were synthesized. Among them, we have found a water-soluble derivative ß-aminoarteether maleate (SM934) that exhibits outstanding suppression on lymphocytes proliferation in immunosuppressive capacity and cytotoxicity screening assays with 35-fold higher potency than dihydroartemisinin. SM934 displays significant therapeutic effects on various autoimmune and inflammatory diseases, including systemic lupus erythematosus, antiphospholipid syndrome nephropathy, membranous nephropathy, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and dry eye disease. Here, we summarize the immunomodulatory effects, anti-inflammatory, anti-oxidative and anti-fibrosis activities of SM934 in disease-relevant animal models and present the probable pharmacological mechanisms involved in its therapeutic efficacy. This review also delineates a typical example of natural product-based drug discovery, which might further vitalize natural product exploration and development in pharmacotherapy.

Heme protects intestinal mucosal barrier in DSS-induced colitis through regulating macrophage polarization in both HO-1-dependent and HO-1-independent way.

Hemoglobin-derived heme was reported to play protective roles in hemorrhagic diseases by modulating the macrophages toward recovery. Mucosal bleeding is one of the pathological features of inflammatory bowel diseases (IBD). However, whether heme provides anti-inflammatory profiles in macrophages, thus contributing to the intestinal mucosal barrier protection, is unclear. In the current study, we investigated the beneficial effects of heme on DSS-induced colitis mice and explored the underlying mechanisms. In vivo, systemic heme supplementation by hemin injection relieved intestinal inflammation and remedied intestinal mucosal barrier damage by correcting abnormal intestinal macrophage polarization. In vitro, we confirmed the reciprocally regulating effects of hemin on M1/M2 macrophage polarization in BMDM. Intriguingly, with knockdown of HO-1, the inhibiting effects of hemin on M1 polarization were maintained, while the promoting effects on M2 polarization were reversed. Further research proved that hemin repressed the inflammatory profiles in macrophages through inhibiting the translocation of NF-κB p65 by disrupting IRF5-NF-κB p65 complex formation in Spi-C-dependent way. In conclusion, these results showed that the modification of colon tissue microenvironment with heme supplementation plays a protective role in DSS-induced colitis mice through regulating the macrophage polarization in both HO-1-dependent and HO-1-independent way, indicating a new choice to therapeutically modulate the macrophage function and prevent IBD.

Triptolide analog LLDT-8 ameliorates psoriasis-like dermatitis in BALB/c mice via suppressing the IL-36α signaling pathway.

Triptolide has shown a good immunosuppressive effect on autoimmune diseases. However, the toxicity limited its widely clinical practice. In this study, we investigated the effects and underlying mechanisms of (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative, on a murine psoriasis-like dermatitis model and related cell lines. Here, we showed that LLDT-8 significantly attenuated symptoms of psoriasis-like dermatitis induced by imiquimod (IMQ, a TLR7 agonist) by reducing the psoriasis area and severity index (PASI) score and inflammatory parameters. The action of LLDT-8 was involved in down-regulated interleukin (IL)-36α expression and blocked IL-36α pathway by LC-MS-based label-free quantitative (LFQ) proteomic approach and further experiments. Meanwhile, we observed that LLDT-8 significantly inhibited the expression of IL-36α in R837-treated bone marrow-derived dendritic cells (BMDCs). In conclusion, LLDT-8 notably alleviated IMQ-induced psoriasis-like skin inflammation via suppressing the IL-36α signaling pathway, suggesting LLDT-8 might be a potential drug for the treatment of psoriasis.

Rh-Catalyzed C-H alkylation enabling modular synthesis of CF3-substituted benzannulated macrocyclic inhibitors of B cell responses.

Inspired by aspirin and chalcone, herein, we describe a modular biomimetic strategy to achieve a new class of CF3-bearing benzannulated macrolactams. The key to the success of macrolactams was the utilization of a highly chemoselective Rh(iii)-catalyzed native carboxylic acid-directed C-H alkylation. Moreover, the unique CF3-containing benzannulated macrocycles showed decent immunosuppressive effects on B cells in vitro, including proliferation, activation, and antibody production upon specific stimulation implicating TLR and BCR signaling.

Synthesis and pharmacological evaluation of choroquine derivatives bearing long aminated side chains as antivirus and anti-inflammatory agents.

Starting from the antimalarial drugs chloroquine and hydroxychloroquine, we conducted a structural optimization on the side chain of chloroquine by introducing amino substituted longer chains thus leading to a series of novel aminochloroquine derivatives. Anti-infectious effects against SARS-Cov2 spike glycoprotein as well as immunosuppressive and anti-inflammatory activities of the new compounds were evaluated. Distinguished immunosuppressive activities on the responses of T cell, B cell and macrophages upon mitogen and pathogenic signaling were manifested. Compounds 9-11 displayed the most promising inhibitory effects both on cellular proliferation and on the production of multiple pro-inflammatory cytokines, including IL-17, IFN-γ, IL-6, IL-1ß and TNF-α, which might be insightful in the pursuit of treatment for immune disorders and inflammatory diseases.

Minor immunosuppressive spiroorthoester group-containing pregnane glycosides from the root barks of Periploca sepium.

LC-MS guided chemical investigation of the periploside-rich extract of the root barks of Periploca sepium afforded six new minor pregnane glycosides, named periplosides A1-A6 (1-6). Their structures were characterized on the basis of extensive spectroscopic analysis. Compounds 1-6 were evaluated for their inhibitory activities against the proliferation of T and B lymphocytes in vitro, among them, compound 5 exhibited significant inhibitory activities and the most favorite selective index (SI) values against the proliferation of T lymphocyte (IC50 = 0.30 µM, SI = 176) and B lymphocyte (IC50 = 0.55 µM, SI = 97).

NOD-like receptors in autoimmune diseases.

Autoimmune diseases are chronic immune diseases characterized by dysregulation of immune system, which ultimately results in a disruption in self-antigen tolerance. Cumulative data show that nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) play essential roles in various autoimmune diseases, such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), psoriasis, multiple sclerosis (MS), etc. NLR proteins, consisting of a C-terminal leucine-rich repeat (LRR), a central nucleotide-binding domain, and an N-terminal effector domain, form a group of pattern recognition receptors (PRRs) that mediate the immune response by specifically recognizing cellular pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and triggering numerous signaling pathways, including RIP2 kinase, caspase-1, nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and so on. Based on their N-terminal domain, NLRs are divided into five subfamilies: NLRA, NLRB, NLRC, NLRP, and NLRX1. In this review, we briefly describe the structures and signaling pathways of NLRs, summarize the recent progress on NLR signaling in the occurrence and development of autoimmune diseases, as well as highlight numerous natural products and synthetic compounds targeting NLRs for the treatment of autoimmune diseases.

A novel tricyclic BTK inhibitor suppresses B cell responses and osteoclastic bone erosion in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by joint leukocyte infiltration, synovial inflammation and bone damage result from osteoclastogenesis. Bruton's tyrosine kinase (BTK) is a key regulator of B cell receptor (BCR) and Fc gamma receptor (FcγR) signaling involved in the pathobiology of RA and other autoimmune disorders. SOMCL-17-016 is a potent and selective tricyclic BTK inhibitor, structurally distinct from other known BTK inhibitors. In present study we investigated the therapeutic efficacy of SOMCL-17-016 in a mouse collagen-induced arthritis (CIA) model and underlying mechanisms. CIA mice were administered SOMCL-17-016 (6.25, 12.5, 25 mg·kg-1·d-1, ig), or ibrutinib (25 mg·kg-1·d-1, ig) or acalabrutinib (25 mg·kg-1·d-1, ig) for 15 days. We showed that oral administration of SOMCL-17-016 dose-dependently ameliorated arthritis severity and bone damage in CIA mice; it displayed a higher in vivo efficacy than ibrutinib and acalabrutinib at the corresponding dosage. We found that SOMCL-17-016 administration dose-dependently inhibited anti-IgM-induced proliferation and activation of B cells from CIA mice, and significantly decreased anti-IgM/anti-CD40-stimulated RANKL expression in memory B cells from RA patients. In RANKL/M-CSF-stimulated RAW264.7 cells, SOMCL-17-016 prevented osteoclast differentiation and abolished RANK-BTK-PLCγ2-NFATc1 signaling. In summary, this study demonstrates that SOMCL-17-016 presents distinguished therapeutic effects in the CIA model. SOMCL-17-016 exerts a dual inhibition of B cell function and osteoclastogenesis, suggesting that it to be a promising drug candidate for RA treatment.

The artemisinin analog SM934 alleviates dry eye disease in rodent models by regulating TLR4/NF-κB/NLRP3 signaling.

Dry eye disease (DED) is a multifactorial disorder of the tears and ocular surface characterized by manifestations of dryness and irritation. Although the pathogenesis is not fully illuminated, it is recognized that inflammation has a prominent role in the development and deterioration of DED. ß-aminoarteether maleate (SM934) is a water-soluble artemisinin derivative with anti-inflammatory and immunosuppressive activities. In this study, we established scopolamine hydrobromide (SCOP)-induced rodent model as well as benzalkonium chloride (BAC)-induced rat model to investigate the therapeutic potential of SM934 for DED. We showed that topical application of SM934 (0.1%, 0.5%) significantly increased tear secretion, maintained the number of conjunctival goblet cells, reduced corneal damage, and decreased the levels of inflammatory mediators (TNF-α, IL-6, IL-10, or IL-1ß) in conjunctiva in SCOP-induced and BAC-induced DED models. Moreover, SM934 treatment reduced the accumulation of TLR4-expressing macrophages in conjunctiva, and suppressed the expression of inflammasome components, i.e., myeloid differentiation factor88 (MyD88), Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC), and cleaved caspase 1. In LPS-treated RAW 264.7 cells, we demonstrated that pretreatment with SM934 (10 µM) impeded the upregulation of TLR4 and downstream NF-κB/NLRP3 signaling proteins. Collectively, artemisinin analog SM934 exerts therapeutic benefits on DED by simultaneously reserving the structural integrity of ocular surface and preventing the corneal and conjunctival inflammation, suggested a further application of SM934 in ophthalmic therapy, especially for DED.

Identification of SARS-CoV-2 entry inhibitors among already approved drugs.

To discover effective drugs for COVID-19 treatment amongst already clinically approved drugs, we developed a high throughput screening assay for SARS-CoV-2 virus entry inhibitors using SARS2-S pseudotyped virus. An approved drug library of 1800 small molecular drugs was screened for SARS2 entry inhibitors and 15 active drugs were identified as specific SARS2-S pseudovirus entry inhibitors. Antiviral tests using native SARS-CoV-2 virus in Vero E6 cells confirmed that 7 of these drugs (clemastine, amiodarone, trimeprazine, bosutinib, toremifene, flupenthixol, and azelastine) significantly inhibited SARS2 replication, reducing supernatant viral RNA load with a promising level of activity. Three of the drugs were classified as histamine receptor antagonists with clemastine showing the strongest anti-SARS2 activity (EC50 = 0.95 ± 0.83 µM). Our work suggests that these 7 drugs could enter into further in vivo studies and clinical investigations for COVID-19 treatment.

(-)-Isoscopariusin†A, a Naturally Occurring Immunosuppressive Meroditerpenoid: Structure Elucidation and Scalable Chemical Synthesis.

(-)-Isoscopariusin A was isolated from the aerial parts of Isodon scoparius. Chemical synthesis and spectroscopic analysis established its structure as an unsymmetrical meroditerpenoid bearing a sterically congested 6/6/4 tricyclic carbon skeleton with seven continuous stereocenters. A gram-scale synthesis was achieved in 12 steps from commercially available (+)-sclareolide. A cobalt catalyzed, hydrogen atom transfer-based olefin isomerization was used to prepare a trisubstituted alkene, which underwent stereoselective [2+2] cycloaddition with a substituted keteniminium ion generated in situ from the corresponding amide. The cyclobutanone product was further elaborated into the fully substituted cyclobutane core through face-selective homologation, and the two side chains were installed by using nickel-catalyzed cross-electrophile coupling and carbodiimide-mediated esterification, respectively. (-)-Isoscopariusin A displayed selective inhibition of T-cell proliferation.

Overview of therapeutic drug research for COVID-19 in China.

Since the outbreak of novel coronavirus pneumonia (COVID-19) in December 2019, more than 2,500,000 people worldwide have been diagnosed with SARS-CoV-2 as of April 22. In response to this epidemic, China has issued seven trial versions of diagnosis and treatment protocol for COVID-19. According to the information that we have collected so far, this article provides an overview of potential therapeutic drugs and compounds with much attention, including favipiravir and hydroxychloroquine, as well as traditional Chinese medicine, which have been reported with good clinical treatment effects. Moreover, with further understanding of SARS-CoV-2 virus, new drugs targeting specific SARS-CoV-2 viral components arise and investigations on these novel anti-SARS-CoV-2 agents are also reviewed.

Anti-SARS-CoV-2 activities in vitro of Shuanghuanglian preparations and bioactive ingredients.

Human infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) and there is no cure currently. The 3CL protease (3CLpro) is a highly conserved protease which is indispensable for CoVs replication, and is a promising target for development of broad-spectrum antiviral drugs. In this study we investigated the anti-SARS-CoV-2 potential of Shuanghuanglian preparation, a Chinese traditional patent medicine with a long history for treating respiratory tract infection in China. We showed that either the oral liquid of Shuanghuanglian, the lyophilized powder of Shuanghuanglian for injection or their bioactive components dose-dependently inhibited SARS-CoV-2 3CLpro as well as the replication of SARS-CoV-2 in Vero E6 cells. Baicalin and baicalein, two ingredients of Shuanghuanglian, were characterized as the first noncovalent, nonpeptidomimetic inhibitors of SARS-CoV-2 3CLpro and exhibited potent antiviral activities in a cell-based system. Remarkably, the binding mode of baicalein with SARS-CoV-2 3CLpro determined by X-ray protein crystallography was distinctly different from those of known 3CLpro inhibitors. Baicalein was productively ensconced in the core of the substrate-binding pocket by interacting with two catalytic residues, the crucial S1/S2 subsites and the oxyanion loop, acting as a "shield" in front of the catalytic dyad to effectively prevent substrate access to the catalytic dyad within the active site. Overall, this study provides an example for exploring the in vitro potency of Chinese traditional patent medicines and effectively identifying bioactive ingredients toward a specific target, and gains evidence supporting the in vivo studies of Shuanghuanglian oral liquid as well as two natural products for COVID-19 treatment.

CNOT2 facilitates dengue virus infection via negatively modulating IFN-Independent Non-Canonical JAK/STAT pathway.

Dengue virus (DENV) infection is a public health problem worldwide. To establish infection in host cells, DENV require host cellular mechanism to suppress and evade innate immunity for their replication. In this study, Ccr4-Not complex genes were screened by using RNAi approach in DENV-infected A549 and Huh7 cells. We found that CNOT2 plays a proviral role in DENV infection. The expression level of CNOT2 was up-regulated in DENV-infected cells. Down-regulation of CNOT2 significantly reduced DENV RNA replication and protein synthesis. Mechanism study showed that CNOT2 knockdown enhanced JAK-STAT antiviral signaling during DENV infection. Further analysis revealed that CNOT2 negatively modulated IFN-Independent Non-Canonical JAK/STAT pathway by accelerating the mRNA decay of JAK1 and STAT1 via its interaction with CNOT6/6L and CNOT7/8 deadenylases. Overall, these results demonstrate that CNOT2 is a novel negative regulator of the JAK-STAT pathway and supports DENV infection.

Recyclable and superior selective CO2 adsorption of C4B32 and Ca@C4B32: a new category of perfect cubic heteroborospherenes.

The capture and separation of CO2 have attracted significant interest as a strategy to control the global emission of greenhouse gases. From the perspective of environmental protection, it is crucial to explore high-performance adsorbents that can efficiently capture CO2. Herein, we report a density functional theory study on the viability of the heteroborospherene C4B32 for the first time. C2v C4B32 was revealed to be a perfect cubic heteroborospherene with the HOMO-LUMO gap of 3.47 eV at the PBE0 level. Then, we evaluated the potential application of C4B32 in the capture and separation of CO2. Our results indicate that the cubic-like C4B32 can efficiently capture CO2 with a -1.34 eV adsorption energy via chemisorption at the most acidic and basic sites of the cage. The strong interaction between CO2 and C4B32 could be supported by an effective charge transfer and orbital overlap. C4B32 also displayed high selectivity for the separation of CO2 from NH3, N2, CH4, CO, and H2 mixtures. Furthermore, it was feasible to tune the CO2-capture ability of C4B32 by metal-doping, which regulated the Lewis acidity/basicity of the C4B32 surface. In particular, Ca-doping could significantly enhance the CO2-capture ability of C4B32. Our results show that as a highly symmetrical and stable heteroborospherene, C4B32 can be used as a building block for the design and synthesis of novel nanomaterials for the capture and separation of CO2.

[Application of active components from traditional Chinese medicine in treatment of inflammatory bowel disease].

Inflammatory bowel disease(IBD) is a non-specific and chronic recurrent autoimmune disease that involves the gastrointestinal tract. Clinical symptoms of intestinal bleeding, diarrhea, and weight loss threat to human health and induce colorectal cancer. The pathogenesis included living environment, genetic factors, immune cell infiltration and immune stress, weakened mucosal barrier defense and intestinal flora imbalance. At present, clinical treatment drugs mainly include aminosalicylic acid, corticosteroids, immunosuppressants, biological agents, etc., in view of the disadvantages of poor therapeutic effect and expensive price. The active ingredients of traditional Chinese medicine(TCM) in the treatment IBD have various biological activities and multiple targets such as anti-inflammatory, antibacterial, anti-tumor and immune regulation. This article summarized the application and the research progress in protecting intestinal epithelial barrier, maintaining intestinal microbial homeostasis, inhibiting causative factors, and regulating Th1/Th17/Treg balance about TCM in the treatment of IBD. The review provided new ideas for further development of the new drugs on the mechanism based on active ingredients of TCM in IBD treatment.

Topical administration of reversible SAHH inhibitor ameliorates imiquimod-induced psoriasis-like skin lesions in mice via suppression of TNF-α/IFN-γ-induced inflammatory response in keratinocytes and T cell-derived IL-17.

DZ2002, a reversible S-adenosyl-l-homocysteine hydrolase (SAHH) inhibitor with immunosuppressive properties and potent therapeutic activity against various autoimmune diseases in mice. The present study was designed to characterize the potential therapeutic effects of DZ2002 on murine model of psoriasis and reveal the correlated mechanisms. In this report, we demonstrated that in vitro, DZ2002 significantly decreased the expression of pro-inflammatory cytokines and adhesion molecule including IL-1α, IL-1ß, IL-6, IL-8, TNF-α and ICAM-1 by inhibiting the phosphorylation of p38 MAPK, ERK and JNK in TNF-α/IFN-γ-stimulated HaCaT human keratinocytes. Topical administration of DZ2002 alleviated the imiquimod (IMQ)-induced psoriasis-like skin lesions and inflammation in mice, the therapeutic effect was comparable with the Calcipotriol. Moreover, the inflammatory skin disorder was restored by DZ2002 treatment characterized by reducing both of the CD3+ T cell accumulation and the psoriasis-specific cytokines expression. Further, we found that DZ2002 improved IMQ-induced splenomegaly and decreased the frequency of splenic IL-17-producing T cells. Our finding offered the convincing evidence that SAHH inhibitor DZ2002 might attenuate psoriasis by simultaneously interfering the abnormal activation and differentiation of keratinocytes and accumulation of IL-17-producing T cells in skin lesions.