Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

African swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2',3'-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2',3'-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2',3'-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2',3'-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2',3'-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2',3'-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2',3'-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2',3'-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2',3'-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

2',3'-Cyclic GMP-AMP Dinucleotides for STING-Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis.

The cGAS-STING pathway discovered ten years ago is an important component of the innate immune system. Activation of cGAS-STING triggers downstream signalling, such as TBK1-IRF3, NF-κB and autophagy, which in turn leads to antipathogen responses, durable antitumour immunity or autoimmune diseases. 2',3'-Cyclic GMP-AMP dinucleotides (2',3'-cGAMP), the key second messengers produced by cGAS, play a pivotal role in cGAS-STING signalling by binding and activating STING. Thus, 2',3'-cGAMP has immunotherapeutic potential, which in turn has stimulated research on the design and synthesis of 2',3'-cGAMP analogues for clinical applications over the past ten years. This review presents the discovery, metabolism, and function of 2',3'-cGAMP in the cGAS-STING innate immune signalling axis. The enzymatic and chemical syntheses of 2',3'-cGAMP analogues as STING-targeting therapeutics are also summarized.

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme.

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5' capping of viral RNAs. The formation of the 5' 7-methylguanosine (m7G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5' triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5' end of viral RNA via a 5' to 5' triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.

REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps.

BRCA1/2 mutant tumor cells display an elevated mutation burden, the etiology of which remains unclear. Here, we report that these cells accumulate ssDNA gaps and spontaneous mutations during unperturbed DNA replication due to repriming by the DNA primase-polymerase PRIMPOL. Gap accumulation requires the DNA glycosylase SMUG1 and is exacerbated by depletion of the translesion synthesis (TLS) factor RAD18 or inhibition of the error-prone TLS polymerase complex REV1-Polζ by the small molecule JH-RE-06. JH-RE-06 treatment of BRCA1/2-deficient cells results in reduced mutation rates and PRIMPOL- and SMUG1-dependent loss of viability. Through cellular and animal studies, we demonstrate that JH-RE-06 is preferentially toxic toward HR-deficient cancer cells. Furthermore, JH-RE-06 remains effective toward PARP inhibitor (PARPi)-resistant BRCA1 mutant cells and displays additive toxicity with crosslinking agents or PARPi. Collectively, these studies identify a protective and mutagenic role for REV1-Polζ in BRCA1/2 mutant cells and provide the rationale for using REV1-Polζ inhibitors to treat BRCA1/2 mutant tumors.

A cGAS-dependent response links DNA damage and senescence in alveolar epithelial cells: a potential drug target in IPF.

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5-7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.

Cooperative DNA binding mediated by KicGAS/ORF52 oligomerization allows inhibition of DNA-induced phase separation and activation of cGAS.

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.

Macrodiolide Diversification Reveals Broad Immunosuppressive Activity That Impairs the cGAS-STING Pathway.

The development of new immunomodulatory agents can impact various areas of medicine. In particular, compounds with the ability to modulate innate immunological pathways hold significant unexplored potential. Herein, we report a modular synthetic approach to the macrodiolide natural product (-)-vermiculine, an agent previously shown to possess diverse biological effects, including cytotoxic and immunosuppressive activity. The synthesis allows for a high degree of flexibility in modifying the macrocyclic framework, including the formation of all possible stereoisomers. In total, 18 analogues were prepared. Two analogues with minor structural modifications showed clearly enhanced cancer cell line selectivity and reduced toxicity. Moreover, these compounds possessed broad inhibitory activity against innate immunological pathways in human PBMCs, including the DNA-sensing cGAS-STING pathway. Initial mechanistic characterization suggests a surprising impairment of the STING-TBK1 interaction.

Synthesis and Pharmacological Evaluation of Tetrahydro-γ-carboline Derivatives as Potent Anti-inflammatory Agents Targeting Cyclic GMP-AMP Synthase.

The activation of cyclic GMP-AMP synthase (cGAS) by double-stranded DNA is implicated in the pathogenesis of many hyperinflammatory and autoimmune diseases, and the cGAS-targeting small molecule has emerged as a novel therapeutic strategy for treating these diseases. However, the currently reported cGAS inhibitors are far beyond maturity, barely demonstrating in vivo efficacy. Inspired by the structural novelty of compound 5 (G140), we conducted a structural optimization on both its side chain and the central tricyclic core, leading to several subseries of compounds, including those unexpectedly cyclized complex ones. Compound 25 bearing an N-glycylglycinoyl side chain was identified as the most potent one with cellular IC50 values of 1.38 and 11.4 µM for h- and m-cGAS, respectively. Mechanistic studies confirmed its direct targeting of cGAS. Further, compound 25 showed superior in vivo anti-inflammatory effects in the lipopolysaccharide-induced mouse model. The encouraging result of compound 25 provides solid evidence for further pursuit of cGAS-targeting inhibitors as a new anti-inflammatory treatment.

Immune sensing of mouse polyomavirus DNA by p204 and cGAS DNA sensors.

The mechanism by which DNA viruses interact with different DNA sensors and their connection with the activation of interferon (IFN) type I pathway are poorly understood. We investigated the roles of protein 204 (p204) and cyclic guanosine-adenosine synthetase (cGAS) sensors during infection with mouse polyomavirus (MPyV). The phosphorylation of IFN regulatory factor 3 (IRF3) and the stimulator of IFN genes (STING) proteins and the upregulation of IFN beta (IFN-ß) and MX Dynamin Like GTPase 1 (MX-1) genes were detected at the time of replication of MPyV genomes in the nucleus. STING knockout abolished the IFN response. Infection with a mutant virus that exhibits defective nuclear entry via nucleopores and that accumulates in the cytoplasm confirmed that replication of viral genomes in the nucleus is required for IFN induction. The importance of both DNA sensors, p204 and cGAS, in MPyV-induced IFN response was demonstrated by downregulation of the IFN pathway observed in p204-knockdown and cGAS-knockout cells. Confocal microscopy revealed the colocalization of p204 with MPyV genomes in the nucleus. cGAS was found in the cytoplasm, colocalizing with viral DNA leaked from the nucleus and with DNA within micronucleus-like bodies, but also with the MPyV genomes in the nucleus. However, 2'3'-Cyclic guanosine monophosphate-adenosine monophosphate synthesized by cGAS was detected exclusively in the cytoplasm. Biochemical assays revealed no evidence of functional interaction between cGAS and p204 in the nucleus. Our results provide evidence for the complex interactions of MPyV and DNA sensors including the sensing of viral genomes in the nucleus by p204 and of leaked viral DNA and micronucleus-like bodies in the cytoplasm by cGAS.

The cGAS-STING pathway as a therapeutic target in inflammatory diseases.

The cGAS-STING signalling pathway has emerged as a key mediator of inflammation in the settings of infection, cellular stress and tissue damage. Underlying this broad involvement of the cGAS-STING pathway is its capacity to sense and regulate the cellular response towards microbial and host-derived DNAs, which serve as ubiquitous danger-associated molecules. Insights into the structural and molecular biology of the cGAS-STING pathway have enabled the development of selective small-molecule inhibitors with the potential to target the cGAS-STING axis in a number of inflammatory diseases in humans. Here, we outline the principal elements of the cGAS-STING signalling cascade and discuss the general mechanisms underlying the association of cGAS-STING activity with various autoinflammatory, autoimmune and degenerative diseases. Finally, we outline the chemical nature of recently developed cGAS and STING antagonists and summarize their potential clinical applications.

Conformational dynamics is critical for the allosteric inhibition of cGAS upon acetyl-mimic mutations.

Detection of cytosolic dsDNA by cyclic GMP-AMP synthase (cGAS) is critical for the immune system to sense and fight against infection, but chronic activation of cGAS by self-DNA leads to autoimmune diseases without effective treatment yet. It was found that acetylation on either Lys384, Lys394, or Lys414 could inhibit the catalytic production of cGAMP by cGAS, and further suppressed self-DNA-induced autoimmunity. However, the implied mechanism remains unclear. Here, extensive molecular dynamics simulations combined with multiple analytical approaches were employed to uncover the allosteric inhibition mechanisms by using the K-to-Q mutations to mimic acetylation. Results suggested that the exterior loops contributed most to the conformational dynamics of cGAS, and two concerted intrinsic motions were observed: the inward/outward or twisting movement for the outer appendage of lobe 1 and the open/closed swing of the active-site loops. Mutations slightly affected the binding of dsDNA and cGAMP. The shift of the conformational sampling of the active-site loops or residues around cGAMP upon mutation might potentially explain the inhibition of cGAS activity. Moreover, the intra- and inter-molecular coupling was weakened upon mutations more or less but via distinct pathways. Hence, conformational dynamics play a vital role in the allosteric inhibition of cGAS upon the studied acetyl-mimic mutations. As the studied acetyl-mimic mutations are located at either the inter-lobe or inter-molecular interfaces, hence except for acetylation, our findings might help the development of new therapeutics against autoimmune diseases due to abnormal cGAS activation by designing inter-lobe or intermolecular allosteric inhibitors.

Structure-Based Drug Design of Phenazopyridine Derivatives as Inhibitors of Rev1 Interactions in Translesion Synthesis.

Rev1 is a protein scaffold of the translesion synthesis (TLS) pathway, which employs low-fidelity DNA polymerases for replication of damaged DNA. The TLS pathway helps cancers tolerate DNA damage induced by genotoxic chemotherapy, and increases mutagenesis in tumors, thus accelerating the onset of chemoresistance. TLS inhibitors have emerged as potential adjuvant drugs to enhance the efficacy of first-line chemotherapy, with the majority of reported inhibitors targeting protein-protein interactions (PPIs) of the Rev1 C-terminal domain (Rev1-CT). We previously identified phenazopyridine (PAP) as a scaffold to disrupt Rev1-CT PPIs with Rev1-interacting regions (RIRs) of TLS polymerases. To explore the structure-activity relationships for this scaffold, we developed a protocol for co-crystallization of compounds that target the RIR binding site on Rev1-CT with a triple Rev1-CT/Rev7R124A /Rev3-RBM1 complex, and solved an X-ray crystal structure of Rev1-CT bound to the most potent PAP analogue. The structure revealed an unexpected binding pose of the compound and informed changes to the scaffold to improve its affinity for Rev1-CT. We synthesized eight additional PAP derivatives, with modifications to the scaffold driven by the structure, and evaluated their binding to Rev1-CT by microscale thermophoresis (MST). Several second-generation PAP derivatives showed an affinity for Rev1-CT that was improved by over an order of magnitude, thereby validating the structure-based assumptions that went into the compound design.

Inhibition of d-glycero-ß-d-manno-heptose 1-phosphate adenylyltransferase from Burkholderia pseudomallei by epigallocatechin gallate and myricetin.

Flavonoids play beneficial roles in various human diseases. In this study, a flavonoid library was employed to probe inhibitors of d-glycero-ß-d-manno-heptose-1-phosphate adenylyltransferase from Burkholderia pseudomallei (BpHldC) and two flavonoids, epigallocatechin gallate (EGCG) and myricetin, have been discovered. BpHldC is one of the essential enzymes in the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway constructing lipopolysaccharide of B. pseudomallei. Enzyme kinetics study showed that two flavonoids work through different mechanisms to block the catalytic activity of BpHldC. Among them, a docking study of EGCG was performed and the binding mode could explain its competitive inhibitory mode for both ATP and ßG1P. Analyses with EGCG homologs could reveal the important functional moieties, too. This study is the first example of uncovering the inhibitory activity of flavonoids against the ADP-l-glycero-ß-d-manno-heptose biosynthesis pathway and especially targeting HldC. Since there are no therapeutic agents and vaccines available against melioidosis, EGCG and myricetin can be used as templates to develop antibiotics over B. pseudomallei.

REV1 inhibitor JH-RE-06 enhances tumor cell response to chemotherapy by triggering senescence hallmarks.

REV1/POLζ-dependent mutagenic translesion synthesis (TLS) promotes cell survival after DNA damage but is responsible for most of the resulting mutations. A novel inhibitor of this pathway, JH-RE-06, promotes cisplatin efficacy in cancer cells and mouse xenograft models, but the mechanism underlying this combinatorial effect is not known. We report that, unexpectedly, in two different mouse xenograft models and four human and mouse cell lines we examined in vitro cisplatin/JH-RE-06 treatment does not increase apoptosis. Rather, it increases hallmarks of senescence such as senescence-associated ß-galactosidase, increased p21 expression, micronuclei formation, reduced Lamin B1, and increased expression of the immune regulators IL6 and IL8 followed by cell death. Moreover, although p-γ-H2AX foci formation was elevated and ATR expression was low in single agent cisplatin-treated cells, the opposite was true in cells treated with cisplatin/JH-RE-06. These observations suggest that targeting REV1 with JH-RE-06 profoundly affects the nature of the persistent genomic damage after cisplatin treatment and also the resulting physiological responses. These data highlight the potential of REV1/POLζ inhibitors to alter the biological response to DNA-damaging chemotherapy and enhance the efficacy of chemotherapy.

Structural mechanism of cGAS inhibition by the nucleosome.

The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons 2-7. cGAS is also present inside the cell nucleus, which is replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Small molecule inhibition of human cGAS reduces total cGAMP output and cytokine expression in cells.

The cGAS-STING pathway is a major mechanism that mammalian cells utilize to detect cytoplasmic dsDNA from incoming viruses, bacteria, or self. CYCLIC GMP-AMP SYNTHASE (cGAS) is the sensor protein that directly binds dsDNAs. cGAS synthesizes cyclic GMP-AMP (cGAMP), which binds to the adaptor STIMULATOR OF INTERFERON GENES (STING), activating an INTERFERON REGULATORY FACTOR 3 (IRF3)-mediated immune response. Constitutive activation can result in interferonopathies such as Aicardi-Goutieres Syndrome (AGS) or other lupus-like autoimmune disorders. While inhibitors targeting mouse or human cGAS have been reported, the identification of a small molecule that targets both homologs of cGAS has been challenging. Here, we show that RU.521 is capable of potently and selectively inhibiting mouse and human cGAS in cell lines and human primary cells. This inhibitory activity requires the presence of cGAS, but it cannot suppress an immune response in cells activated by RNA, Toll-like receptor ligands, cGAMP, or recombinant interferon. Importantly, when RU.521 is applied to cells, the production of dsDNA-induced intracellular cGAMP is suppressed in a dose-dependent manner. Our work validates the use of RU.521 for probing DNA-induced innate immune responses and underscores its potential as an ideal scaffold towards pre-clinical development, given its potency against human and mouse cGAS.

Inhibition of double-strand DNA-sensing cGAS ameliorates brain injury after ischemic stroke.

Cytosolic double-stranded DNA (dsDNA) is a danger signal that is tightly monitored and sensed by nucleic acid-sensing pattern recognition receptors. We study the inflammatory cascade on dsDNA recognition and investigate the neuroprotective effect of cyclic GMP-AMP (cGAMP) synthase (cGAS) antagonist A151 and its mechanisms of neuroprotection in a mouse model of experimental stroke. Here, we found that cerebral ischemia promoted the release of dsDNA into the cytosol, where it initiated inflammatory responses by activating the cGAS. A151 effectively reduced the expression of cGAS, absent in melanoma 2 (AIM2) inflammasome, and pyroptosis-related molecules, including caspase-1, gasdermin D, IL-1ß, and IL-18. Furthermore, mice treated with A151 showed a dampened immune response to stroke, with reduced counts of neutrophils, microglia, and microglial production of IL-6 and TNF-α after MCAO. Moreover, A151 administration significantly reduced infarct volume, attenuated neurodeficits, and diminished cell death. Notably, the protective effect of A151 was blocked in a microglia-specific cGAS knockout mouse. These findings offer unique perspectives on stroke pathogenesis and indicate that inhibition of cGAS could attenuate brain inflammatory burden, representing a potential therapeutic opportunity for stroke.

Self DNA perpetuates IPF lung fibroblast senescence in a cGAS-dependent manner.

Senescence and mitochondrial stress are mutually reinforcing age-related processes that contribute to idiopathic pulmonary fibrosis (IPF); a lethal disease that manifests primarily in the elderly. Whilst evidence is accumulating that GMP-AMP synthase (cGAS) is crucial in perpetuating senescence by binding damaged DNA released into the cytosol, its role in IPF is not known. The present study examines the contributions of cGAS and self DNA to the senescence of lung fibroblasts from IPF patients (IPF-LFs) and age-matched controls (Ctrl-LFs). cGAS immunoreactivity was observed in regions of fibrosis associated with fibroblasts in lung tissue of IPF patients. Pharmacological inhibition of cGAS or its knockdown by silencing RNA (siRNA) diminished the escalation of IPF-LF senescence in culture over 7 days as measured by decreased p21 and p16 expression, histone 2AXγ phosphorylation and/or IL-6 production (P < 0.05, n = 5-8). The targeting of cGAS also attenuated etoposide-induced senescence in Ctrl-LFs (P < 0.05, n = 5-8). Levels of mitochondrial DNA (mDNA) detected by qPCR in the cytosol and medium of IPF-LFs or senescence-induced Ctrl-LFs were higher than Ctrl-LFs at baseline (P < 0.05, n = 5-7). The addition of DNAse I (100 U/ml) deaccelerated IPF-LF senescence (P < 0.05, n = 5), whereas ectopic mDNA or the induction of endogenous mDNA release augmented Ctrl-LF senescence in a cGAS-dependent manner (P < 0.05, n = 5). In conclusion, we provide evidence that cGAS reinforces lung fibroblast senescence involving damaged self DNA. The targeting of cGAS to supress senescent-like responses may have potential important therapeutic implications in the treatment of IPF.