Targeting the CBM complex causes Treg cells to prime tumours for immune checkpoint therapy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

Microglia-derived ASC specks cross-seed amyloid-ß in Alzheimer's disease.

The spreading of pathology within and between brain areas is a hallmark of neurodegenerative disorders. In patients with Alzheimer's disease, deposition of amyloid-ß is accompanied by activation of the innate immune system and involves inflammasome-dependent formation of ASC specks in microglia. ASC specks released by microglia bind rapidly to amyloid-ß and increase the formation of amyloid-ß oligomers and aggregates, acting as an inflammation-driven cross-seed for amyloid-ß pathology. Here we show that intrahippocampal injection of ASC specks resulted in spreading of amyloid-ß pathology in transgenic double-mutant APPSwePSEN1dE9 mice. By contrast, homogenates from brains of APPSwePSEN1dE9 mice failed to induce seeding and spreading of amyloid-ß pathology in ASC-deficient APPSwePSEN1dE9 mice. Moreover, co-application of an anti-ASC antibody blocked the increase in amyloid-ß pathology in APPSwePSEN1dE9 mice. These findings support the concept that inflammasome activation is connected to seeding and spreading of amyloid-ß pathology in patients with Alzheimer's disease.

Discovery and characterization of small-molecule inhibitors of NLRP3 and NLRC4 inflammasomes.

Inflammasomes are macromolecular complexes involved in the host response to external and endogenous danger signals. Inflammasome-mediated sterile inflammation plays a central role in several human conditions such as autoimmune diseases, type-2 diabetes, and neurodegenerative disorders, indicating inflammasomes could be appealing therapeutic targets. Previous work has demonstrated that inhibiting the ATPase activity of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3), disrupts inflammasome assembly and function. However, there is a necessity to find new potent compounds with therapeutic potential. Here we combine computational modeling of the target and virtual screening to discover a group of novel compounds predicted to inhibit NLRP3. We characterized the best compounds and determined their potency, specificity, and ability to inhibit processes downstream from NLRP3 activation. Moreover, we analyzed in mice the competence of a lead candidate to reduce lipopolysaccharide-induced inflammation. We also validated the active pharmacophore shared among all the NLRP3 inhibitors, and through computational docking, we clarify key structural features for compound positioning within the inflammasome ATP-binding site. Our study sets the basis for rational design and optimization of inflammasome-targeting probes and drugs.

Safranal inhibits NLRP3 inflammasome activation by preventing ASC oligomerization.

NLRP3 inflammasome is involved in several chronic inflammatory diseases. The inflammatory effect of the NLRP3 inflammasome is executed through IL-1ß and IL-18. Therefore, IL-1ß is one of the primary targets in chronic inflammatory conditions. However, current treatment regimens are dependent on anti- IL-1ß biologicals. The therapies targeting IL-1ß through inhibition of NLRP3 inflammasome are thus being actively explored. We identified safranal, a small molecule responsible for the essence of saffron as a potential inhibitor of the NLRP3 inflammasome. Safranal significantly suppressed the release of IL-1ß from ATP stimulated J774A.1 and bone marrow-derived macrophages (BMDMs) by regulating CASP1 and CASP8 dependent cleavage of pro-IL-1ß. Safranal markedly suppressed the expression of NLRP3 and its ATPase activity. Safranal treatment enhanced the expression of NRF2, whereas, si-RNA mediated silencing of Nrf2 abrogated the anti-NLRP3 effect of safranal. Furthermore, safranal inhibited ASC oligomerization and formation of ASC specks. Safranal also displayed anti-NLRP3 activity in multiple mice models. Treatment of animals with safranal reduced the production of IL-1ß in ATP elicited peritoneal inflammation, MSU induced air pouch inflammation, and MSU injected foot paw edema in mice. Thus, our data projects safranal as a potential preclinical drug candidate against NLRP3 inflammasome triggered chronic inflammation.

Role of CARD9 in inflammatory signal pathway of peritoneal macrophages in severe acute pancreatitis.

Previous studies revealed that caspase recruitment domain protein 9 (CARD9) was involved in severe acute pancreatitis (SAP) inflammation and that interfering with its expression in vivo could inhibit inflammation. However, the specific mechanism is unknown. This study aimed to discover the related signal pathways of CARD9 in macrophages. SiRNA interference technology was used in vivo and in vitro to detect CARD9-related signal pathways in peritoneal macrophages. Furthermore, Toll-like receptor 4 (TLR4) and membrane-associated C-type lectin-1 (Dectin-1) pathways in macrophages were activated specially to looking for the upstream signal path of CARD9. Results showed up-regulation of CARD9 expression in peritoneal macrophages of SAP rats (P < .05). CARD9 siRNA alleviated inflammatory cytokines, and inhibited the phosphorylation of NF-κB and p38MAPK in peritoneal macrophages in vivo or in vitro. Meanwhile, CARD9 siRNA reduced the concentration of CARD9 and Bcl10 in peritoneal macrophages, and TLR4 and Dectin-1 took part in CARD9 signal pathways in macrophages. In conclusion, there is an inflammation signal pathway comprised of TLR4/Dectin-1-CARD9-NF-κB/p38MAPK activated in macrophages in SAP. Blockade of CARD9 expression in macrophages can effectively alleviate SAP inflammation.

IC100: a novel anti-ASC monoclonal antibody improves functional outcomes in an animal model of multiple sclerosis.

BACKGROUND: The inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) is involved in immune signaling by bridging the interactions between inflammasome sensors and caspase-1. Strong experimental evidence has shown that ASC-/- mice are protected from disease progression in animal models of multiple sclerosis (MS), suggesting that targeting inflammasome activation via ASC inhibition may be a promising therapeutic strategy in MS. Thus, the goal of our study is to test the efficacy of IC100, a novel humanized antibody targeting ASC, in preventing and/or suppressing disease in the experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: We employed the EAE model of MS where disease was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55). Mice were treated with vehicle or increasing doses of IC100 (10, 30, and 45 mg/kg) and clinical disease course was evaluated up to 35 days post EAE induction. Immune cell infiltration into the spinal cord and microglia responses were assessed. RESULTS: We show that IC100 treatment reduced the severity of EAE when compared to vehicle-treated controls. At a dose of 30 mg/kg, IC100 significantly reduced the number of CD4+ and CD8+ T cells and CD11b+MHCII+ activated myeloid cells entering the spinal cord from the periphery, and reduced the number of total and activated microglia. CONCLUSIONS: These data indicate that IC100 suppresses the immune-inflammatory response that drives EAE development and progression, thereby identifying ASC as a promising target for the treatment of MS as well as other neurological diseases with a neuroinflammatory component.

CRID3, a blocker of apoptosis associated speck like protein containing a card, ameliorates murine spinal cord injury by improving local immune microenvironment.

BACKGROUND: After spinal cord injury (SCI), destructive immune cell subsets are dominant in the local microenvironment, which are the important mechanism of injury. Studies have shown that inflammasomes play an important role in the inflammation following SCI, and apoptosis-associated speck-like protein containing a card (ASC) is the adaptor protein shared by inflammasomes. Therefore, we speculated that inhibiting ASC may improve the local microenvironment of injured spinal cord. Here, CRID3, a blocker of ASC oligomerization, was used to study its effect on the local microenvironment and the possible role in neuroprotection following SCI. METHODS: Murine SCI model was created using an Infinite Horizon impactor at T9 vertebral level with a force of 50 kdynes and CRID3 (50 mg/kg) was intraperitoneally injected following injury. ASC and its downstream molecules in inflammasome signaling pathway were measured by western blot. The immune cell subsets were detected by immunohistofluorescence (IHF) and flow cytometry (FCM). The spinal cord fibrosis area, neuron survival, myelin preservation, and functional recovery were assessed. RESULTS: Following SCI, CRID3 administration inhibited inflammasome-related ASC and caspase-1, IL-1ß, and IL-18 activation, which consequently suppressed M1 microglia, Th1 and Th1Th17 differentiation, and increased M2 microglia and Th2 differentiation. Accordingly, the improved histology and behavior have also been found. CONCLUSIONS: CRID3 may ameliorate murine SCI by inhibiting inflammasome activation, reducing proinflammatory factor production, restoring immune cell subset balance, and improving local immune microenvironment, and early administration may be a promising therapeutic strategy for SCI.

Host CARD11 Inhibits Newcastle Disease Virus Replication by Suppressing Viral Polymerase Activity in Neurons.

Host factors play multiple essential roles in the replication and pathogenesis of mammalian neurotropic viruses. However, the cellular proteins of the central nervous system (CNS) involved in avian neurotropic virus infection have not been completely elucidated. Here, we employed a gene microarray to identify caspase recruitment domain-containing protein 11 (CARD11), a lymphoma-associated scaffold protein presenting brain-specific upregulated expression in a virulent neurotropic Newcastle disease virus (NDV)-infected natural host. Chicken primary neuronal cells infected with NDV appeared slightly syncytial and died quickly. CARD11 overexpression inhibited viral replication and delayed cytopathic effects; conversely, depletion of CARD11 enhanced viral replication and cytopathic effects in chicken primary neuronal cells. The inhibition of viral replication by CARD11 could not be blocked with CARD11-Bcl10-MALT1 (CBM) signalosome and NF-κB signaling inhibitors. CARD11 was found to interact directly with the viral phosphoprotein (P) through its CC1 domain and the X domain of P; this X domain also mediated the interaction between P and the viral large polymerase protein (L). The CARD11 CC1 domain and L competitively bound to P via the X domain that hindered the P-L interaction of the viral ribonucleoprotein (RNP) complex, resulting in a reduction of viral polymerase activity in a minigenome assay and inhibition of viral replication. Animal experiments further revealed that CARD11 contributed to viral replication inhibition and neuropathology in infected chicken brains. Taken together, our findings identify CARD11 as a brain-specific antiviral factor of NDV infection in avian species.IMPORTANCE Newcastle disease virus (NDV) substantially impacts the poultry industry worldwide and causes viral encephalitis and neurological disorders leading to brain damage, paralysis, and death. The mechanism of interaction between this neurotropic virus and the avian central nervous system (CNS) is largely unknown. Here, we report that host protein CARD11 presented brain-specific upregulated expression that inhibited NDV replication, which was not due to CARD11-Bcl10-MALT1 (CBM) complex-triggered activation of its downstream signaling pathways. The inhibitory mechanism of viral replication is through the CARD11 CC1 domain, and the viral large polymerase protein (L) competitively interacts with the X domain of the viral phosphoprotein (P), which hampers the P-L interaction, suppressing the viral polymerase activity and viral replication. An in vivo study indicated that CARD11 alleviated neuropathological lesions and reduced viral replication in chicken brains. These results provide insight into the interaction between NDV infection and the host defense in the CNS and a potential antiviral target for viral neural diseases.

Dehydrocostus lactone inhibits NLRP3 inflammasome activation by blocking ASC oligomerization and prevents LPS-mediated inflammation in vivo.

Uncontrolled activation of NLRP3 inflammasome initiates a series of human inflammatory diseases. Targeting NLRP3 inflammasome has attracted considerable attention in developing potential therapeutic interventions. Here, we reported that dehydrocostus lactone (DCL), a main component of Saussurea lappa from the traditional Chinese medicine, inhibited NLRP3 inflammasome-mediated caspase-1 activation and subsequent interleukin (IL)-1ß production in primary mouse macrophages and human peripheral blood mononuclear cells and exerted an inhibitory effect on NLRP3-driven inflammation. Mechanistically, DCL significantly blocked the ASC oligomerization, which is essential for the assembly of activated inflammasome. Importantly, in vivo experiments showed that DCL reduced IL-1ß secretion and peritoneal neutrophils recruitment in LPS-mediated inflammation mouse model, which is demonstrated to be NLRP3 dependent. These results suggest that DCL is a potent pharmacological inhibitor of NLRP3 inflammasome and may be developed as a therapeutic drug for treating NLRP3-associated diseases.

Small-molecule inhibitors directly target CARD9 and mimic its protective variant in inflammatory bowel disease.

Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.

Long Non-Coding RNA LINC01260 Inhibits the Proliferation, Migration and Invasion of Spinal Cord Glioma Cells by Targeting CARD11 Via the NF-κB Signaling Pathway.

BACKGROUND/AIMS: Spinal cord glioma is a highly aggressive malignancy that commonly results in high mortality due to metastasis, high recurrence and limited treatment regimens. This study aims to elucidate the effects of long non-coding RNA LINC01260 (LINC01260) on the proliferation, migration and invasion of spinal cord glioma cells by targeting Caspase recruitment domain family, member 11 (CARD11) via nuclear factor kappa B (NF-κB) signaling. METHODS: The Multi Experiment Matrix (MEM) website was used for target gene prediction, and the DAVID database was used for analysis of the relationship between CARD11 and the NF-κB pathway. In total, 60 cases of glioma tissues and adjacent normal tissues were collected. Human U251 glioma cells were grouped into blank, negative control (NC), LINC01260 vector, CARD11 vector, siRNA-LINC01260, siRNA-CARD11, LINC01260 vector + CARD11 vector and LINC01260 + siRNA-CARD11 groups. A dual-luciferase reporter assay was conducted to verify the target relationship between LINC01260 and CARD11. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were employed to assess expression of LINC01260, E-cadherin, p53, CARD11, Ki67, N-cadherin, matrix metalloproteinase (MMP)-9, NF-κBp65 and NF-κBp50. MTT, flow cytometry, wound-healing and Transwell assays were performed to examine cell viability, the cell cycle, apoptosis, invasion and migration. Tumor growth was assessed through xenografts in nude mice. RESULTS: CARD11 was confirmed to be a target gene of LINC01260 and was found to be involved in regulating the NF-κB pathway. Compared with adjacent normal tissues, glioma tissues showed reduced expression of LINC01260 and elevated expression of CARD11 and genes related to apoptosis, invasion and migration; activation of NF-κB signaling was also observed. In contrast to the blank and NC groups, an elevated number of cells arrested in G1 phase, increased apoptosis and reduced cell proliferation, invasion and number of cells arrested in S and G2 phases, as well as tumor growth were found for the LINC01260 vector and siRNA-CARD11 groups. CONCLUSIONS: Our findings demonstrate that overexpression of LINC01260 inhibits spinal cord glioma cell proliferation, migration and invasion by targeting CARD11 via NF-κB signaling suppression.

CARD9 gene silencing with siRNA protects rats against severe acute pancreatitis: CARD9-dependent NF-κB and P38MAPKs pathway.

We previously reported the up-regulation of caspase recruitment domain 9 (CARD9) expressions in severe acute pancreatitis (SAP) patients, but little is known about its regulation. In this study, small interfering RNA (siRNA) was used to reduce the levels of CARD9 expression in sodium taurocholate-stimulated SAP rats. CARD9 was overexpressed in SAP rats, which correlated with the severity of pancreatitis. When compared to the untreated group, the cohort that received the siRNA treatment demonstrated a significant reduction in pancreatic injury, neutrophil infiltration, myeloperoxidase activity and pro-inflammatory cytokines. Furthermore, siRNAs showed that the reduction of CARD9 in SAP rats down-regulated the expression of NF-κBp65 and P38MAPK which are involved in the transcription and release of a wide variety of inflammatory cytokines. These findings provide evidence that CARD9 is up-regulated in SAP rats and acts as a potential therapeutic target for the treatment thereof. Blocking the activation of NF-κB and P38MAPK via siRNA-mediated gene knock-down of CARD9 appears to reduce the inflammatory response in pancreatic tissue.

SGLT-2 Inhibition with Dapagliflozin Reduces the Activation of the Nlrp3/ASC Inflammasome and Attenuates the Development of Diabetic Cardiomyopathy in Mice with Type 2 Diabetes. Further Augmentation of the Effects with Saxagliptin, a DPP4 Inhibitor.

PURPOSE: We assessed whether (1) dapagliflozin (Dapa, an SGLT2-inhibitor) attenuates the deterioration of heart function Nlrp3 and inflammasome activation in diabetic mice. (2) The effects can be augmented with saxagliptin (Saxa), a DDP4-inhibitor. (3) Dapa effect is possibly SGLT2-independent on cardiofibroblasts in vitro. METHODS: Type 2 diabetic (BTBR ob/ob) and wild-type (WT) mice received vehicle, Dapa, or Dapa+Saxa for 8 weeks. Glucose tolerance test and echocardiogram were performed. Cardiofibroblasts from WT and BTBR hearts were incubated with Dapa and exposed to LPS. RESULTS: Left ventricular ejection fraction (LVEF) was 81 ± 1% in the WT and 53 ± 1% in the T2D-cont mice. Dapa and Dapa+Saxa improved LVEF to 68 ± 1 and 74.6 ± 1% in the BTBR mice (p < 0.001). The mRNA levels of NALP3, ASC, IL-1ß, IL-6, caspase-1, and TNFα were significantly higher in the BTBR compared to the WT hearts; and Dapa and Dapa+Saxa significantly attenuated these levels. Likewise, protein levels of NLRP3, TNFα, and caspase-1 were higher in the BTBR compared to the WT hearts and Dapa, and to a greater extent Dapa+Saxa, attenuated the increase in the BTBR mice. Collagen-1 and collagen-3 mRNA levels significantly increased in the BTBR mice and these increases were attenuated by Dapa and Dapa+Saxa. P-AMPK/total-AMPK ratio was significantly lower in the BTBR mice than in the WT mice. Dapa and Dapa+Saxa equally increased the ratio in the BTBR mice. This in vitro study showed that NALP3, ASC, IL-1ß, and caspase-1 mRNA levels were higher in the BTBR cardiofibroblasts and attenuated with Dapa. The effect was AMPK-dependent and SGLT1-independent. CONCLUSIONS: Dapa attenuated the activation of the inflammasome, fibrosis, and deterioration of LVEF in BTBR mice. The anti-inflammatory, anti-fibrotic effects are likely SGLT2- and glucose-lowering-independent, as they were replicated in the in vitro model. The effects on remodeling were augmented when Saxa was added to Dapa. Yet, adding Saxa to Dapa did not result in a greater effect on myocardial fibrosis and collagen levels.

Ellagic Acid-Changed Epigenome of Ribosomal Genes and Condensed RPA194-Positive Regions of Nucleoli in Tumour Cells.

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

Overexpression of CARMA3 is associated with advanced tumor stage, cell cycle progression, and cisplatin resistance in human epithelial ovarian cancer.

CARD recruited membrane associated protein 3 (CARMA3) overexpression has been found in several human cancers. However, its expression pattern and biological roles in human ovarian cancers are not clear. In this study, we examined the expression pattern of CARMA3 in 101 ovarian cancer specimens. We found that 52 (51.5 %) showed CARMA3 overexpression. CARMA3 overexpression positively correlated with tumor histology and advanced FIGO stage. CARMA3 depletion in ovarian cancer cell lines A2780 and HO8910 inhibited ovarian cancer cell proliferation and blocked cell cycle progression. CARMA3 depletion also sensitized ovarian cancer cells to cisplatin-induced cytotoxicity. In addition, Western blot showed that CARMA3 depletion downregulated cyclin D1, cyclin E, and Bcl-2 levels. In conclusion, our data provides evidence that CARMA3 is overexpressed in ovarian cancers and associated with advanced stage. CARMA3 regulates the ovarian cancer cell proliferation, cell cycle progression, and chemoresistance.

Whole-exome sequencing links caspase recruitment domain 11 (CARD11) inactivation to severe combined immunodeficiency.

BACKGROUND: Primary immunodeficiencies represent model diseases for the mechanistic understanding of the human innate and adaptive immune response. They are clinically highly relevant per se because in patients with severe combined immunodeficiency (SCID), infections caused by opportunistic pathogens are typically life-threatening early in life. OBJECTIVES: We aimed at defining and functionally characterizing a novel form of SCID in an infant of consanguineous parents who presented with life-threatening Pneumocystis jirovecii pneumonia using a comprehensive immunologic and whole-exome genetic diagnostic strategy. METHODS: Analysis of leukocyte subpopulations was performed by using multicolor flow cytometry and was combined with stimulation tests for T-cell function. The search for a disease-causing mutation was performed with diagnostic whole-exome sequencing and systematic variant categorization. Reconstitution assays were used for validating the loss-of-function mutation. RESULTS: The novel entity of SCID was characterized by agammaglobulinemia and profoundly deficient T-cell function despite quantitatively normal T and B lymphocytes. Genetic analysis revealed a single pathogenic homozygous nonsense mutation of the caspase recruitment domain 11 (CARD11) gene. In reconstitution assays we demonstrated that the patient-derived truncated CARD11 protein is defective in antigen receptor signaling and nuclear factor κB activation. CONCLUSION: We show that an inactivating CARD11 mutation links defective nuclear factor κB signaling to a novel cause of autosomal recessive SCID.

Identification of an ASC oligomerization inhibitor for the treatment of inflammatory diseases.

The ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) protein is an scaffold component of different inflammasomes, intracellular multiprotein platforms of the innate immune system that are activated in response to pathogens or intracellular damage. The formation of ASC specks, initiated by different inflammasome receptors, promotes the recruitment and activation of procaspase-1, thereby triggering pyroptotic inflammatory cell death and pro-inflammatory cytokine release. Here we describe MM01 as the first-in-class small-molecule inhibitor of ASC that interferes with ASC speck formation. MM01 inhibition of ASC oligomerization prevents activation of procaspase-1 in vitro and inhibits the activation of different ASC-dependent inflammasomes in cell lines and primary cultures. Furthermore, MM01 inhibits inflammation in vivo in a mouse model of inflammasome-induced peritonitis. Overall, we highlight MM01 as a novel broad-spectrum inflammasome inhibitor for the potential treatment of multifactorial diseases involving the dysregulation of multiple inflammasomes.

Structure-Based Discovery of Potent CARM1 Inhibitors for Solid Tumor and Cancer Immunology Therapy.

CARM1 is a protein arginine methyltransferase and acts as a transcriptional coactivator regulating multiple biological processes. Aberrant expression of CARM1 has been related to the progression of multiple types of cancers, and therefore CARM1 was considered as a promising drug target. In the present work, we report the structure-based discovery of a series of N1-(3-(pyrimidin-2-yl)benzyl)ethane-1,2-diamines as potent CARM1 inhibitors, in which compound 43 displays high potency and selectivity. With the advantage of excellent tissue distribution, compound 43 demonstrated good in vivo efficacy for solid tumors. Furthermore, from the detailed immuno-oncology study with MC38 C57BL/6J xenograft model, we confirmed that this chemical probe 43 has profound effects in tumor immunity, which paves the way for future studies on the modulation of arginine post-translational modification that could be utilized in solid tumor treatment and cancer immunotherapy.

The inhibitor effect of RKIP on inflammasome activation and inflammasome-dependent diseases.

Aberrant inflammasome activation contributes to the pathogenesis of various human diseases, including atherosclerosis, gout, and metabolic disorders. Elucidation of the underlying mechanism involved in the negative regulation of the inflammasome is important for developing new therapeutic targets for these diseases. Here, we showed that Raf kinase inhibitor protein (RKIP) negatively regulates the activation of the NLRP1, NLRP3, and NLRC4 inflammasomes. RKIP deficiency enhanced caspase-1 activation and IL-1ß secretion via NLRP1, NLRP3, and NLRC4 inflammasome activation in primary macrophages. The overexpression of RKIP in THP-1 cells inhibited NLRP1, NLRP3, and NLRC4 inflammasome activation. RKIP-deficient mice showed increased sensitivity to Alum-induced peritonitis and Salmonella typhimurium-induced inflammation, indicating that RKIP inhibits NLRP3 and NLRC4 inflammasome activation in vivo. Mechanistically, RKIP directly binds to apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and competes with NLRP1, NLRP3, or NLRC4 to interact with ASC, thus interrupting inflammasome assembly and activation. The depletion of RKIP aggravated inflammasome-related diseases such as monosodium urate (MSU)-induced gouty arthritis and high-fat diet (HFD)-induced metabolic disorders. Furthermore, the expression of RKIP was substantially downregulated in patients with gouty arthritis or type 2 diabetes (T2D) compared to healthy controls. Collectively, our findings suggest that RKIP negatively regulates NLRP1, NLRP3, and NLRC4 inflammasome activation and is a potential therapeutic target for the treatment of inflammasome-related diseases.

Inhibition of ASC enhances the protective role of salvianolic acid A in traumatic brain injury via inhibition of inflammation and recovery of mitochondrial function.

More than 50 million people are affected by traumatic brain injury (TBI) each year around the world, and nearly half of the population worldwide will have one or more TBI(s) in their lifetime. And in 2017, more than 1.39 billion people in China suffered from TBI, representing nearly 18% of the world population; these were mainly caused by road traffic incidents. Salvianolic acid A is a compound obtained from Salvia miltiorrhiza Bunge, which is one of the active components of many traditional Chinese medicines for the treatment of cardiovascular and cerebrovascular disease, with the effect of inhibition of inflammatory response. ASC is a critical factor in the activation of inflammation response process via promoting the maturation of caspase-1, and activation of NLPR3 under bacterial infection promotes the necrosis of cells in an ASC-dependent manner. However, few studies focus on the effect of ASC in a TBI model. In this study, we found that inhibition of ASC reduced the expression of inflammatory cytokines, and the concentration of calcium and ROS, while it increased the expression of mitochondrial function-related proteins. We further noticed that these effects were regulated by DLK2/MLK3/JNK signalling pathway and might contribute to the treatment of TBI.