Human cGAS Research Articles

In Vitro Cleavage Assay to Characterize DENV NS2B3 Antagonism of cGAS.

cGAS is a key cytosolic dsDNA receptor that senses viral infection and elicits interferon production through the cGAS-cGAMP-STING axis. cGAS is activated by dsDNA from viral and bacterial origins as well as dsDNA leaked from damaged mitochondria and nucleus. Eventually, cGAS activation launches the cell into an antiviral state to restrict the replication of both DNA and RNA viruses. Throughout the long co-evolution, viruses devise many strategies to evade cGAS detection or suppress cGAS activation. We recently reported that the Dengue virus protease NS2B3 proteolytically cleaves human cGAS in its N-terminal region, effectively reducing cGAS binding to DNA and consequent production of the second messenger cGAMP. Several other RNA viruses likely adopt the cleavage strategy. Here, we describe a protocol for the purification of recombinant human cGAS and Dengue NS2B3 protease, as well as the in vitro cleavage assay.

MTORC2-driven chromatin cGAS mediates chemoresistance through epigenetic reprogramming in colorectal cancer

Cyclic GMP–AMP synthase (cGAS), a cytosolic DNA sensor that initiates a STING-dependent innate immune response, binds tightly to chromatin, where its catalytic activity is inhibited; however, mechanisms underlying cGAS recruitment to chromatin and functions of chromatin-bound cGAS (ccGAS) remain unclear. Here we show that mTORC2-mediated phosphorylation of human cGAS serine 37 promotes its chromatin localization in colorectal cancer cells, regulating cell growth and drug resistance independently of STING. We discovered that ccGAS recruits the SWI/SNF complex at specific chromatin regions, modifying expression of genes linked to glutaminolysis and DNA replication. Although ccGAS depletion inhibited cell growth, it induced chemoresistance to fluorouracil treatment in vitro and in vivo. Moreover, blocking kidney-type glutaminase, a downstream ccGAS target, overcame chemoresistance caused by ccGAS loss. Thus, ccGAS coordinates colorectal cancer plasticity and acquired chemoresistance through epigenetic patterning. Targeting both mTORC2–ccGAS and glutaminase provides a promising strategy to eliminate quiescent resistant cancer cells.

The structural basis for 2′−5′/3′−5′-cGAMP synthesis by cGAS

cGAS activates innate immune responses against cytosolic double-stranded DNA. Here, by determining crystal structures of cGAS at various reaction stages, we report a unifying catalytic mechanism. apo-cGAS assumes an array of inactive conformations and binds NTPs nonproductively. Dimerization-coupled double-stranded DNA-binding then affixes the active site into a rigid lock for productive metal•substrate binding. A web-like network of protein•NTP, intra-NTP, and inter-NTP interactions ensures the stepwise synthesis of 2′−5′/3′−5′-linked cGAMP while discriminating against noncognate NTPs and off-pathway intermediates. One divalent metal is sufficient for productive substrate binding, and capturing the second divalent metal is tightly coupled to nucleotide and linkage specificities, a process which manganese is preferred over magnesium by 100-fold. Additionally, we elucidate how mouse cGAS achieves more stringent NTP and linkage specificities than human cGAS. Together, our results reveal that an adaptable, yet precise lock-and-key-like mechanism underpins cGAS catalysis.

Species-specific cleavage of cGAS by picornavirus protease 3C disrupts mitochondria DNA-mediated immune sensing.

RNA viruses cause numerous infectious diseases in humans and animals. The crosstalk between RNA viruses and the innate DNA sensing pathways attracts increasing attention. Recent studies showed that the cGAS-STING pathway plays an important role in restricting RNA viruses via mitochondria DNA (mtDNA) mediated activation. However, the mechanisms of cGAS mediated innate immune evasion by RNA viruses remain unknown. Here, we report that seneca valley virus (SVV) protease 3C disrupts mtDNA mediated innate immune sensing by cleaving porcine cGAS (pcGAS) in a species-specific manner. Mechanistically, a W/Q motif within the N-terminal domain of pcGAS is a unique cleavage site recognized by SVV 3C. Three conserved catalytic residues of SVV 3C cooperatively contribute to the cleavage of pcGAS, but not human cGAS (hcGAS) or mouse cGAS (mcGAS). Additionally, upon SVV infection and poly(dA:dT) transfection, pcGAS and SVV 3C colocalizes in the cells. Furthermore, SVV 3C disrupts pcGAS-mediated DNA binding, cGAMP synthesis and interferon induction by specifically cleaving pcGAS. This work uncovers a novel mechanism by which the viral protease cleaves the DNA sensor cGAS to evade innate immune response, suggesting a new antiviral approach against picornaviruses.

Discovery of novel cGAS inhibitors based on natural flavonoids

Cyclic GMP-AMP synthase (cGAS) plays an important role in the inflammatory response. It has been reported that aberrant activation of cGAS is associated with a variety of immune-mediated inflammatory disorders. The development of small molecule inhibitors of cGAS has been considered as a promising therapeutic strategy for the diseases. Flavonoids, a typical class of natural products, are known for their anti-inflammatory activities. Although cGAS is closely associated with inflammation, the potential effects of natural flavonoid compounds on cGAS have been rarely studied. Therefore, we screened an in-house natural flavonoid library by pyrophosphatase (PPiase) coupling assay and identified novel cGAS inhibitors baicalein and baicalin. Subsequently, crystal structures of the two natural flavonoids in complex with human cGAS were determined, which provide mechanistic insight into the anti-inflammatory activities of baicalein and baicalin at the molecular level. After that, a virtual screening based on the crystal structures of baicalein and baicalin in complex with human cGAS was performed. As a result, compound C20 was identified to inhibit both human and mouse cGAS with IC50 values of 2.28 and 1.44 μM, respectively, and its detailed interactions with human cGAS were further revealed by the X-ray crystal structure determination. These results demonstrate the potential of natural products used as hits in drug discovery and provide valuable hints for further development of cGAS inhibitors.

Investigating the Activation Mechanism Differences between Human and Mouse cGAS by Molecular Dynamics Simulations.

Cyclic GMP-AMP synthase (cGAS) has been widely investigated as a drug target for its crucial role in innate immunity. However, the inhibitors designed using mouse model were often shown to be ineffective for humans. This outcome indicates that the activation mechanisms of human and mouse cGAS (mcGAS) are different. The cGAS activation is achieved by dimerization via binding to DNA, the detailed mechanism of which, however, is not entirely clear. To investigate these mechanisms, molecular dynamics (MD) simulations were performed on several states of four types of cGAS, namely, the mcGAS, the wild-type and A- and C-type mutations of human cGAS (hcGAS). We find that sequence differences between hcGAS and mcGAS can directly affect the protein structure stability, especially that of the siteB domain. The sequence and structural differences also contribute to DNA-binding differences. In addition, the conformational fluctuations of cGAS are found to correlate with the regulation of catalytic capacity. More importantly, we illustrate that dimerization enhances the correlation among distant residues and significantly reinforces the allosteric signal transmission among the DNA-binding interfaces and the catalytic pocket, which facilitates rapid immune response to cytosolic DNA. We conclude that siteB domain plays a prominent role in mcGAS activation, while siteA domain is key to hcGAS activation.

T6SS-5 and the cGAS-STING pathway in Burkholderia pseudomallei infection and immunity

Burkholderia pseudomallei is a causative agent of melioidosis that can infect humans and animals in endemic countries, specifically in Southeast Asia and tropical Australia. A fundamental component for the pathogenesis of Burkholderia pseudomallei is the capability of the bacterium to enter, survive, replicate, and cause disease in a host cell by inducing the host cell fusion. Cell fusion results in multinucleated-giant cell formation, thus enabling the dissemination of Burkholderia pseudomallei intracellularly. cGAS reacts to Burkholderia pseudomallei infection by activating the cGAS-STING pathway and subsequently limiting host’s aberrant cell division and cellular replication by inducing autophagic cell death. In this review, we discuss the host-pathogen interactions between the type VI secretion system 5 (T6SS-5) of Burkholderia pseudomallei and human cGAS pathway in melioidosis infections. Since T6SS-5 is a main virulent factor in Burkholderia pseudomallei and the cGAS pathway is vital for host immune response, elucidating their functions is important for better understanding the pathogenesis of Burkholderia pseudomallei.

Species-specific self-DNA detection mechanisms by mammalian cyclic GMP-AMP synthases.

The mechanisms by which innate immune receptors mediate self-nonself discrimination are unclear. In this study, we found species-specific molecular determinants of self-DNA reactivity by cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase (cGAS). Human cGAS contained a catalytic domain that was intrinsically self-DNA reactive and stimulated interferon responses in diverse cell types. This reactivity was prevented by an upstream amino (N)-terminal domain. The cGAS proteins from several nonhuman primate species exhibited a similar pattern of self-DNA reactivity in cells, but chimpanzee cGAS was inactive even when its amino-terminal domain was deleted. In contrast, the N terminus of mouse cGAS promoted self-DNA reactivity. When expressed within tumors, only self-DNA-reactive cGAS proteins protected mice from tumor-induced lethality. In vitro studies of DNA- or chromatin-induced cGAS activation did not reveal species-specific activities that correlate with self-DNA reactivity observed in macrophages. Cell biological analysis revealed that self-DNA reactivity by human cGAS, but not mouse cGAS, correlated with localization to mitochondria. We found that epitope tag positions affected self-DNA reactivity in cells and that DNA present in cell lysates undermines the reliability of cGAS biochemical fractionations. These studies reveal species-specific diversity of cGAS functions, even within the primate lineage, and highlight experimental considerations for the study of this innate immune receptor.

Cytosolic d-type CpG-oligonucleotides induce a type I interferon response by activating the cGAS-STING signaling pathway.

Cytosolic DNA receptor cyclic GMP-AMP (cGAMP) synthase (cGAS) has been shown to be critically involved in the detection of cytosolic, self- and non-self-DNA, initiating a type I IFN response through the adaptor protein Stimulator of Interferon Genes (STING) and interferon regulatory factor 3 (IRF3). Current studies propose that canonical binding of dsDNA by cGAS depends on DNA length, but not on base sequence. In contrast, activation of TLR9 is sequence dependent. It requires unmethylated CpG dinucleotides in microbial DNA, which is mimicked by synthetic oligodeoxynucleotides (ODN). Here, we provide evidence that d-type ODN (D-ODN), but not K-type ODN (K-ODN), bind to human cGAS and activate downstream signaling. Transfection of D-ODN into a TLR9-deficient, human monocytic cell line (THP-1) induced phosphorylation of IRF3 and secretion of IFN. This response was absent in cells with CRISPR/Cas9-mediated cGAS- or STING-deficiency. Utilizing a protein pulldown approach, we further demonstrate direct binding of D-ODN to cGAS. Induction of a type I IFN response by D-ODN was confirmed in human primary monocytes and monocyte-derived macrophages. These results are relevant to our understanding of self-nonself-discrimination by cGAS and to the pharmacologic effects of ODN, which currently are investigated in clinical studies.

RNF111-facilitated neddylation potentiates cGAS-mediated antiviral innate immune response.

The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase (cGAS) has emerged as a fundamental component fueling the anti-pathogen immunity. Because of its pivotal role in initiating innate immune response, the activity of cGAS must be tightly fine-tuned to maintain immune homeostasis in antiviral response. Here, we reported that neddylation modification was indispensable for appropriate cGAS-STING signaling activation. Blocking neddylation pathway using neddylation inhibitor MLN4924 substantially impaired the induction of type I interferon and proinflammatory cytokines, which was selectively dependent on Nedd8 E2 enzyme Ube2m. We further found that deficiency of the Nedd8 E3 ligase Rnf111 greatly attenuated DNA-triggered cGAS activation while not affecting cGAMP induced activation of STING, demonstrating that Rnf111 was the Nedd8 E3 ligase of cGAS. By performing mass spectrometry, we identified Lys231 and Lys421 as essential neddylation sites in human cGAS. Mechanistically, Rnf111 interacted with and polyneddylated cGAS, which in turn promoted its dimerization and enhanced the DNA-binding ability, leading to proper cGAS-STING pathway activation. In the same line, the Ube2m or Rnf111 deficiency mice exhibited severe defects in innate immune response and were susceptible to HSV-1 infection. Collectively, our study uncovered a vital role of the Ube2m-Rnf111 neddylation axis in promoting the activity of the cGAS-STING pathway and highlighted the importance of neddylation modification in antiviral defense.

Comparative Study of Interactions between Human cGAS and Inhibitors: Insights from Molecular Dynamics and MM/PBSA Studies.

Recent studies have identified cyclic GMP-AMP synthase (cGAS) as an important target for treating autoimmune diseases, and several inhibitors of human cGAS (hcGAS) and their structures in complexation with hcGAS have been reported. However, the mechanisms via which these inhibitors interact with hcGAS are not completely understood. Here, we aimed to assess the performance of molecular mechanics/Poisson–Boltzmann solvent-accessible surface area (MM/PBSA) in evaluating the binding affinity of various hcGAS inhibitors and to elucidate their detailed interactions with hcGAS from an energetic viewpoint. Using molecular dynamics (MD) simulation and MM/PBSA approaches, the estimated free energies were in good agreement with the experimental ones, with a Pearson’s correlation coefficient and Spearman’s rank coefficient of 0.67 and 0.46, respectively. In per-residue energy decomposition analysis, four residues, K362, R376, Y436, and K439 in hcGAS were found to contribute significantly to the binding with inhibitors via hydrogen bonding, salt bridges, and various π interactions, such as π· · ·π stacking, cation· · ·π, hydroxyl· · ·π, and alkyl· · ·π interactions. In addition, we discussed other key interactions between specific residues and ligands, in particular, between H363 and JUJ, F379 and 9BY, and H437 and 8ZM. The sandwiched structures of the inhibitor bound to the guanidinium group of R376 and the phenyl ring of Y436 were also consistent with the experimental data. The results indicated that MM/PBSA in combination with other virtual screening methods, could be a reliable approach to discover new hcGAS inhibitors and thus is valuable for potential treatments of cGAS-dependent inflammatory diseases.

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 triggersthe induction of inflammatory cytokines and type I interferons2-7. 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 forinhibition 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 ofcGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction betweencGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.

Catalytic Promiscuity of cGAS: A Facile Enzymatic Synthesis of 2'-3'-Linked Cyclic Dinucleotides.

Cyclic GMP‐AMP synthase (cGAS) is a cytosolic DNA sensor that catalyzes the synthesis of the cyclic GMP‐AMP dinucleotide 2′3′‐cGAMP. 2′3′‐cGAMP functions as inducer for the production of type I interferons. Derivatives of this important second messenger are highly valuable for pharmaceutical applications. However, the production of these analogues requires complex, multistep syntheses. Herein, human cGAS is shown to react with a series of unnatural nucleotides, thus leading to novel cyclic dinucleotides. Most substrate derivatives with modifications at the nucleobase, ribose, and the α‐thio phosphate were accepted. These results demonstrate the catalytic promiscuity of human cGAS and its utility for the biocatalytic synthesis of cyclic dinucleotide derivatives.

Dephosphorylation of cGAS by PPP6C impairs its substrate binding activity and innate antiviral response

The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosolic DNA derived from microbial pathogens or mis-located cellular DNA. Upon DNA binding, cGAS utilizes GTP and ATP as substrates to synthesize cGAMP, leading to MITA-mediated innate immune response. In this study, we identified the phosphatase PPP6C as a negative regulator of cGAS-mediated innate immune response. PPP6C is constitutively associated with cGAS in un-stimulated cells. DNA virus infection causes rapid disassociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket. Mutation of this serine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection. In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity. PPP6C-deficiency promotes innate immune response to DNA virus in various cells. Our findings suggest that PPP6C-mediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response, which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.

In Silico Screening-Based Discovery of Novel Inhibitors of Human Cyclic GMP-AMP Synthase: A Cross-Validation Study of Molecular Docking and Experimental Testing.

Cyclic GMP-AMP synthase (cGAS) has been recently uncovered to be a promising therapeutic target for immune-associated diseases. Until now, only a few inhibitors have been identified through high-throughput screening campaigns. Here, we reported the discovery of novel inhibitors for the catalytic domain of human cGAS (h-cGASCD) by virtual screening for the first time. To generate a reliable docking mode, we first obtained a high-resolution crystal structure of h-cGASCD in complex with PF-06928215, a known inhibitor of h-cGAS, followed by molecular dynamics simulations on this complex structure. Four fragment hits were identified by the virtual screening together with a thermal shift assay. The crystal structures of these four compounds in complex with h-cGASCD were subsequently determined, and the binding modes of the compounds were similar to those predicted by molecular docking, supporting the reliability of the docking model. In addition, an enzyme activity assay identified compound 18 (IC50 = 29.88 ± 3.20 μM) from the compounds predicted by the virtual screening. A similarity search of compound 18 followed by a second virtual screening led to the discovery of compounds S2 (IC50 = 13.1 ± 0.09 μM) and S3 (IC50 = 4.9 ± 0.26 μM) as h-cGAS inhibitors with improved potency. Therefore, the present study not only provides the validated hit compounds for further development of h-cGAS inhibitors but also demonstrates a cross-validation study of virtual screening, in vitro experimental assays, and crystal structure determination.

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.

Innate immunity and bacterial conflict: the deep origins of cGAS-STING signaling and discovery of uncharacterized animal immunity pathways

Abstract Just as animal innate immunity pits endogenous response pathways against invading extragenic threats, microbiomes are governed by myriad pathways mediating inter-genomic resource procurement, collectively known as ‘conflict systems.’ Leveraging the continually-increasing amount of genome data, we have probed such systems and in the process uncovered the evolutionary origins of core components of animal immunity. This includes the discovery that cyclic-nucleotide synthesizing enzymes like human cGAS and OAS1 and their receptors like STING were acquired from comparable conflict-triggered nucleotide signaling systems in bacteria. While our analyses point to previously-unknown diversity animal innate immunity, particularly in marine invertebrates, this diversity pales in contrast to bacterial pathways which contain a wealth of completely uncharacterized nucleotide synthetase enzymes, nucleotide receptors, and response effector domains. Further, studies from our group point to entirely-uncharacterized components of the animal innate immunity response that have also been acquired from bacterial conflict systems. These include the viral RNA-acting human Leng9 phosphoesterase and the human N4BP2L1 nucleotide kinase. These studies also uncover a connection between immunity-driven second messenger nucleotide generation and a previously-unrecognized receptor domain in the human apoptotic factor MAP3K5.

Phosphorylation of cGAS by CDK1 impairs self-DNA sensing in mitosis

The cyclic GMP-AMP synthase (cGAS) is a widely used DNA sensor, which detects cytosolic DNA species without a preference of self or non-self microbial DNA in interphase to initiate innate immune response. How cGAS is regulated to avoid self-DNA sensing upon nuclear envelope breakdown (NEBD) during mitosis remains enigmatic. Here we show that cGAS is mostly localized in the cytoplasm in interphase and rapidly translocated to chromosomes upon NEBD in mitosis. The major mitotic kinase CDK1-cyclin B complex phosphorylates human cGAS at S305 or mouse cGAS at S291, which inhibits its ability to synthesize cGAMP upon mitotic entry. The type 1 phosphatase PP1 dephosphorylates cGAS upon mitotic exit to enable its DNA sensing ability. Our findings reveal a mechanism on how the DNA sensor cGAS is post-translationally regulated by cell cycle-dependent enzymes to ensure its proper activation for host defense of cytosolic DNA in interphase and inert to self-DNA in mitosis.

Discovery of Small-Molecule Cyclic GMP-AMP Synthase Inhibitors.

Cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) (cGAS), a cytosolic DNA sensor, plays an important role in the type I interferon response. DNA from either invading microbes or self-origin triggers the enzymatic activity of cGAS. Aberrant activation of cGAS is associated with various autoimmune disorders. Only one selective probe exists for inhibiting cGAS in cells, while others are limited by their poor cellular activity or specificity, which underscores the urgency for discovering new cGAS inhibitors. Here, we describe the development of new small-molecule human cGAS (hcGAS) inhibitors (80 compounds synthesized) with high binding affinity in vitro and cellular activity. Our studies show CU-32 and CU-76 selectively inhibit the DNA pathway in human cells but have no effect on the RIG-I-MAVS or Toll-like receptor pathways. CU-32 and CU-76 represent a new class of hcGAS inhibitors with activity in cells and provide a new chemical scaffold for designing probes to study cGAS function and development of autoimmune therapeutics.

Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid-phase condensation.

The cyclic GMP-AMP synthase (cGAS)-cGAMP-STING pathway plays a key role in innate immunity, with cGAS sensing both pathogenic and mislocalized DNA in the cytoplasm. Human cGAS (h-cGAS) constitutes an important drug target for control of antiinflammatory responses that can contribute to the onset of autoimmune diseases. Recent studies have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGASCD-DNA interface (labeled site-C; CD, catalytic domain) in the crystal structure of a human SRY.cGASCD-DNA complex, with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation, as monitored by cGAMP formation, gel shift, spin-down, and turbidity assays, as well as time-lapse imaging of liquid droplet formation. We expand on an earlier ladder model of cGAS dimers bound to a pair of parallel-aligned DNAs to propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGAS-DNA interface. We also report the crystal structure of the h-cGASCD-DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development at the DNA-bound h-cGAS level. Finally, we solved the structure of RU.521 bound in two alternate alignments to apo h-cGASCD, thereby occupying more of the catalytic pocket and providing insights into further optimization of active-site-binding inhibitors.