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Guest editors Keriann Backus, Zhengying Pan and Lyn Jones introduce the themed collection on Covalent Drug Discovery.

[Image: see text] Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide engage cereblon and mediate a protein interface with neosubstrates such as zinc finger transcription factors promoting their polyubiquitination and degradation. The IMiDs have garnered considerable excitement in drug discovery, leading to exploration of targeted protein degradation strategies. Although the molecular modes-of-action of the IMiDs and related degraders have been the subject of intense research, their pharmacokinetics and disposition have been relatively understudied. Here, we assess the effects of physicochemistry of the IMiDs, the phthalimide EM-12, and the candidate drug CC-220 (iberdomide) on lipophilicity, solubility, metabolism, permeability, intracellular bioavailability, and cell-based potency. The insights yielded in this study will enable the rational property-based design and development of targeted protein degraders in the future.

Covalent drugs and chemical probes often possess pharmacological advantages over reversible binding ligands, such as enhanced potency and pharmacodynamic duration. The highly nucleophilic cysteine thiol is commonly targeted using acrylamide electrophiles, but the amino acid is rarely present in protein binding sites. Sulfonyl exchange chemistry has expanded the covalent drug discovery toolkit by enabling the rational design of irreversible inhibitors targeting tyrosine, lysine, serine and threonine. Probes containing the sulfonyl fluoride warhead have also been shown to serendipitously label histidine residues in proteins. Histidine targeting is an attractive prospect because the residue is frequently proximal to protein small molecule ligands and the imidazole side chain possesses desirable nucleophilicity. We recently reported the design of cereblon molecular glues to site-selectively modify a histidine in the thalidomide binding site using sulfonyl exchange chemistry. We believe that histidine targeting holds great promise for future covalent drug development and this Opinion highlights these opportunities.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional compounds that recruit the E3 ubiquitin ligase machinery to proteins of interest, resulting in their ubiquitination and subsequent proteasomal degradation. Targeted protein degradation has generated considerable interest in drug discovery because inhibition of one particular function of a protein often does not deliver the therapeutic efficacy that results from whole-protein depletion. However, the physicochemistry and intrinsically complex pharmacology of PROTACs present challenges, particularly for the development of orally bioavailable drugs. Here we describe the application of a translational pharmacology framework (called the four pillars) to expedite PROTAC development by informing pharmacokinetic-pharmacodynamic (PKPD) understanding and helping elucidate structure-activity relationships. Experimental methods are reviewed that help illuminate exposure of the drug or probe at the site of action (pillar 1) and engagement of its target(s) (pillar 2) that drive functional pharmacological effects (pillar 3) resulting in modulation of a relevant phenotype (pillar 4). We hope the guidance will be useful to those developing targeted protein degraders and help establish PROTAC molecules as robust target validation chemical probes.

Targeted covalent inhibitors have regained widespread attention in drug discovery and have emerged as powerful tools for basic biomedical research. Fueled by considerable improvements in mass spectrometry sensitivity and sample processing, chemoproteomic strategies have revealed thousands of proteins that can be covalently modified by reactive small molecules. Fragment-based drug discovery, which has traditionally been used in a target-centric fashion, is now being deployed on a proteome-wide scale thereby expanding its utility to both the discovery of novel covalent ligands and their cognate protein targets. This powerful approach is allowing ‘high-throughput’ serendipitous discovery of cryptic pockets leading to the identification of pharmacological modulators of proteins previously viewed as “undruggable”. The reactive fragment toolkit has been enabled by recent advances in the development of new chemistries that target residues other than cysteine including lysine and tyrosine. Here, we review the emerging area of covalent fragment-based ligand discovery, which integrates the benefits of covalent targeting and fragment-based medicinal chemistry. We discuss how the two strategies synergize to facilitate the efficient discovery of new pharmacological modulators of established and new therapeutic target proteins., Covalent fragment-based ligand discovery greatly facilitates the discovery of useful fragments for drug discovery and helps unveil chemical-tractable biological targets in native biological systems.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (β6 Glu → Val) on the β-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of the list of targetable amino acid residues is required to augment the synthetic bology toolkit of site-selective protein modifications. This work describes the first rational targeting of a specific histidine residue in a protein binding site using sulfonyl exchange chemistry. Structure-based drug design was used to incorporate sulfonyl fluoride and triazole reactive groups into the isoindolinone thalidomide congener EM12 to yield potent covalent inhibitors of the cereblon E3 ubiquitin ligase complex through engagement of His353. Conversely, the fluorosulfate derivative EM12-FS labels His353, but degrades a novel neosubstrate, the protein N-terminal glutamine amidohydrolase NTAQ1, which is involved in the N-end rule pathway and DNA damage response. Targeted protein degradation using cereblon ligands has become an important new drug discovery modality and the chemical probes and covalent labeling strategy described here will broadly impact this exciting area of therapeutic research.

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

Covalent protein inhibitors have found considerable utility in medicinal chemistry and chemical biology. Chemical probes and drugs have been rationally designed and fine-tuned to engage cysteine residues predominantly using electrophilic acrylamide warheads that deliver inhibitors with improved pharmacological efficacy, duration and selectivity over reversible binders. However, cysteine is rarely present in protein binding sites and so there is a need to advance inhibitors that engage alternative amino acid residues. This review describes recent progress in the development of covalent small molecules that target residues beyond cysteine. Numerous electrophiles have been incorporated into inhibitors that selectively label various reactive amino acid side chains including lysine, tyrosine, histidine, serine, threonine, glutamate, aspartate and methionine. Several synthetic strategies have facilitated these advances, particularly in the area of sulfonyl-exchange chemistry, though other emerging approaches are presented. We hope the review will inspire future research into next-generation covalent protein inhibitors that positively effect therapeutic discovery and drive fundamental biological insights through novel chemical probe development.

Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such "elimination damage" generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically.

The reaction of small-molecule chemical probes with proteins has been harnessed to develop covalent inhibitor drugs and protein-profiling technologies. This Essay discusses some of the recent enhancements to the chemical biology toolkit that are enabling the study of previously unchartered areas of chemoproteomic space. An analysis of the kinome is used to illustrate the potential for these approaches enable the pursuit of new targets using reactive chemical probes.

New opportunities to advance small-molecule kinase ligands that downregulate their cognate target binding proteins are discussed. Rationally designed heterobifunctional kinase degraders are compared with ATP site ligands that were serendipitously found to cause kinase downregulation. These approaches could be particularly useful in the treatment of cancers since many kinases are known to remodel pro-oncogenic protein-protein interactions, which could be destroyed by small-molecule-mediated kinase depletion.

Correction for ‘Fragment-based covalent ligand discovery’ by Wenchao Lu et al., RSC Chem. Biol., 2021, DOI: 10.1039/d0cb00222d.

Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.

The discovery and selection of a highly potent and selective NaV1.7 inhibitor PF-06456384, designed specifically for intravenous infusion, is disclosed. Extensive in vitro pharmacology and ADME profiling followed by in vivo preclinical PK and efficacy model data are discussed. A proposed protein-ligand binding mode for this compound is also provided to rationalise the high levels of potency and selectivity over inhibition of related sodium channels. To further support the proposed binding mode, potent conjugates are described which illustrate the potential for development of chemical probes to enable further target evaluation.

Irreversible enzyme inhibitors and covalent chemical biology probes often utilize the reaction of a protein cysteine residue with an appropriately positioned electrophile (e.g., acrylamide) on the ligand template. However, cysteine residues are not always available for site-specific protein labeling, and therefore new approaches are needed to expand the toolkit of appropriate electrophiles ("warheads") that target alternative amino acids. We previously described the rational targeting of tyrosine residues in the active site of a protein (the mRNA decapping scavenger enzyme, DcpS) using inhibitors armed with a sulfonyl fluoride electrophile. These inhibitors subsequently enabled the development of clickable probe technology to measure drug-target occupancy in live cells. Here we describe a fluorosulfate-containing inhibitor (aryl fluorosulfate probe (FS-p1)) with excellent chemical and metabolic stability that reacts selectively with a noncatalytic serine residue in the same active site of DcpS as confirmed by peptide mapping experiments. Our results suggest that noncatalytic serine targeting using fluorosulfate electrophilic warheads could be a suitable strategy for the development of covalent inhibitor drugs and chemical probes.

The allure of phenotypic screening, combined with the industry preference for target-based approaches, has prompted the development of innovative chemical biology technologies that facilitate the identification of new therapeutic targets for accelerated drug discovery. A chemogenomic library is a collection of selective small-molecule pharmacological agents, and a hit from such a set in a phenotypic screen suggests that the annotated target or targets of that pharmacological agent may be involved in perturbing the observable phenotype. In this Review, we describe opportunities for chemogenomic screening to considerably expedite the conversion of phenotypic screening projects into target-based drug discovery approaches. Other applications are explored, including drug repositioning, predictive toxicology and the discovery of novel pharmacological modalities.

Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.

Histone deacetylases (HDACs) regulate diverse cellular processes, and are promising targets for a number of diseases. Here we describe the design and utilization of a largazole-based chemical probe to quantitatively measure the intracellular occupancy of HDAC1 and HDAC2 by dacinostat. Surprisingly, the probe was unable to enrich HDAC3 despite its nanomolar potency in a biochemical assay, further proving the necessity of cell-based target occupancy assays to understand compound potency in physiologically-relevant settings. This occupancy assay has the potential to aid the development of novel HDAC1/2 inhibitors in drug discovery.

Cyclin-dependent kinase 7 (CDK7) regulates both cell cycle and transcription, but its precise role remains elusive. We previously described THZ1, a CDK7 inhibitor, which dramatically inhibits super-enhancer associated gene expression. However, potent CDK12/13 off-target activity obscured CDK7s contribution to this phenotype. Here, we describe the discovery of a highly selective, covalent CDK7 inhibitor. YKL-5-124 causes arrest at the G1/S transition and inhibition of E2F-driven gene expression; these effects are rescued by a CDK7 mutant unable to covalently engage YKL-5-124, demonstrating on-target specificity. Unlike THZ1, treatment with YKL-5-124 resulted in no change to Pol II CTD phosphorylation; however, inhibition could be reconstituted by combining YKL-5-124 and THZ531, a selective CDK12/13 inhibitor, revealing potential redundancies in CDK control of gene transcription. These findings highlight the importance of CDK7/12/13 polypharmacology for anti-cancer activity of THZ1 and posit that selective inhibition of CDK7 may be useful for treatment of cancers marked by E2F misregulation.

Phenotypic screening in disease-relevant models identifies small molecule hits with desirable efficacy but often with unknown modes of action. Target identification and validation are integral to successful biomedical research. Technologies are required to validate the biological target (or targets) through which a pharmacological agent is proposed to exert its effects. This work details the rational structure-based design, synthetic preparation and cell-based application of a clickable sulfonyl fluoride chemical probe to directly report on the mechanism of a series of compounds previously discovered in a reporter gene assay. Quantification of drug-target occupancy in living human primary cells enabled a deeper understanding of the molecular pharmacology of the chemotype. The technology described herein should be of broad interest to those involved in chemical biology research and the drug discovery endeavor.

Chemogenomics expedites the discovery of therapeutically-relevant targets from phenotypic screens. However, the vast majority of proteins in the proteome lack selective pharmacological modulators, necessitating the development of new technologies that significantly expand chemogenomic space. Chemoproteomics has emerged as a robust platform to map small molecule-protein interactions in cells using functionalized chemical probes in conjunction with mass spectrometry analysis. Exploration of the ligandable proteome in this manner has led to the development of new pharmacological modulators of diverse proteins. Opportunities to further enhance the impact of chemoproteomics using medicinal chemical biology are described.

Phenotypic screening in disease-relevant models identifies small molecule hits with desirable efficacy but often with unknown modes of action. Target identification and validation are integral to successful biomedical research. Technologies are required to validate the biological target (or targets) through which a pharmacological agent is proposed to exert its effects. This work details the rational structure-based design, synthetic preparation and cell-based application of a clickable sulfonyl fluoride chemical probe to directly report on the mechanism of a series of compounds previously discovered in a reporter gene assay. Quantification of drug-target occupancy in living human primary cells enabled a deeper understanding of the molecular pharmacology of the chemotype. The technology described herein should be of broad interest to those involved in chemical biology research and the drug discovery endeavor.

SUMMARY Drug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders., Graphical abstract, In brief Motor neuron hyperexcitability is observed in both ALS patients and their iPSC-derived neurons. Combining a high-content live imaging excitability phenotypic assay, high-throughput screening against a cross-annotated chemogenomic library, and bioinformatic enrichment analysis, Huang et al. identify targets modulating the hyperexcitability of ALS patient-derived motor neurons in an unbiased manner.

Histone deacetylases (HDACs) contribute to regulation of gene expression by mediating higher-order chromatin structures. They assemble into large multiprotein complexes that regulate activity and specificity. We report the development of small molecule probes with class IIa and pan-HDAC activity that contain photoreactive crosslinking groups and either a biotin reporter, or a terminal alkyne handle for subsequent bioorthogonal ligation. The probes retained inhibitory activity against recombinant HDAC proteins and caused an accumulation of acetylated histone and tubulin following cell treatment. The versatility of the probes has been demonstrated by their ability to photoaffinity modify HDAC targets in vitro. An affinity enrichment probe was used in conjunction with mass spectrometry proteomics to isolate HDACs and their interacting proteins in a native proteome. The performance of the probes in recombinant versus cell-based systems highlights issues for the development of chemoproteomic technologies targeting class IIa HDACs in particular.

Phenotypic screening is a powerful strategy for identifying active molecules with particular biological effects in cellular or animal disease models. Functionalized chemical probes have been instrumental in revealing new targets and confirming target engagement. However, substantial effort and resources are required to design and synthesize these bioactive probes. In contrast, label-free technologies have the advantage of bypassing the need for chemical probes. Here we highlight the recent developments in label-free methods and discuss the strengths and limitations of each approach.

[Image: see text] Aryl fluorosulfates are finding widespread utility in chemical biology and medicinal chemistry. The context-dependent engagement of tyrosine, lysine, serine, and histidine amino acid residues in functional protein sites has enabled chemogenomic and chemoproteomic techniques that demonstrate considerable promise for drug discovery and biomedical research.

A 1,2,4-triazole motif was employed as a bioisostere for the ester commonly used in muscarinic antagonists, and subsequent integrative conjugation to a β2 agonist quinolinone furnished a new class of bifunctional MABAs for the treatment of COPD. Medicinal chemistry optimization using the principles of 'inhalation by design' furnished a clinical candidate with desirable pharmacological, pharmacokinetic and biopharmaceutical properties.

First introduced by K Barry Sharpless in 2001, the term ‘click chemistry’ soon became a widely used description of chemical reactions that proceed rapidly, cleanly and in a manner that is often compatible with aqueous solutions. Click chemistry is frequently employed throughout the process of drug discovery, and greatly helps advance research programs in the pharmaceutical industry. It facilitates library synthesis to support medicinal chemistry optimization, helps identify the targets and off-targets of drug candidates, and can facilitate the determination of drug efficacy in clinical trials. In the last decade, a large number of patent applications covering the various types and utilities of click chemistry have been filed. In this review, we provide the first analysis of click chemistry applications.

This work describes the first rational targeting of tyrosine residues in a protein binding site by small-molecule covalent probes. Specific tyrosine residues in the active site of the mRNA-decapping scavenger enzyme DcpS were modified using reactive sulfonyl fluoride covalent inhibitors. Structure-based molecular design was used to create an alkyne-tagged probe bearing the sulfonyl fluoride warhead, thus enabling the efficient capture of the protein from a complex proteome. Use of the probe in competition experiments with a diaminoquinazoline DcpS inhibitor permitted the quantification of intracellular target occupancy. As a result, diaminoquinazoline upregulators of survival motor neuron protein that are used for the treatment of spinal muscular atrophy were confirmed as inhibitors of DcpS in human primary cells. This work illustrates the utility of sulfonyl fluoride probes designed to react with specific tyrosine residues of a protein and augments the chemical biology toolkit by these probes uses in target validation and molecular pharmacology.

The mitogen activated protein kinase kinase kinase transforming growth factor-β-activated kinase 1 (TAK1) has emerged as an interesting therapeutic target for inflammatory diseases and cancer. TAK1 is a tightly regulated kinase that represents a key signaling node in cellular responses to inflammatory stimuli, modulating both expression of inflammatory mediators and cell death. The first inhibitors described for TAK1 exploit the active site cysteine residue found in this kinase, but more recently both type I ATP hinge-binding inhibitors and type II DFG-out inhibitors have been described. This article will review the emerging role of TAK1 kinase in inflammation, the current state of the art for small molecule inhibitor development and opportunities for chemical biology approaches.

Medicinal chemistry techniques, including structure-based molecular design, fragment replacement and synthetic library enablement, were used to create potent inhibitors of bromodomain and extraterminal domain (BET) and CREB bindingprotein bromodomains. One inhibitor featured the homoaromatic tropolone methyl ether motif as a mimic of acetyl lysine, as confirmed by X-ray crystallography. The intrinsic photoreactivity of the tropone fragment was harnessed successfully to directly photolabel recombinant bromodomains which inspired further development of a clickable probe to assess BRD4 target engagement by (+)-JQ1 as a representative BET inhibitor in a complex proteome. The antimitoticnatural productcolchicine and related natural products that contain the tropolone ring system were also found to be bromodomain inhibitors, and surprisingly, our probe was shown to photolabel tubulin. These results highlight the caution that should be exercised when considering the selectivity of pharmacological agents, and photoreactive chemical probes should be assessed for their intrinsic ability to directly label their biological targets with a view to creating useful chemoproteomic tools.

Drug attrition in clinical trials due to inadequate preclinical target validation is a huge problem facing drug discovery. The issue is compounded by concerns over the reproducibility of published ...

The reactivity of amino acid residues in proteins is context-dependent and difficult to predict. Chemical biology can be used to understand the chemical modifications of proteins to help elucidate the nature of the reactive proteome. The resulting insights can be applied to pharmacoproteomics, target identification and molecular pathology.

Professor Eranthie Weerapana and Professor Lyn Jones introduce this Protein Labelling themed issue.

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.