Small molecule approaches to treat autoimmune and inflammatory diseases (Part II): Nucleic acid sensing antagonists and inhibitors

Xiaoqing Wang, Yafei Liu, Xingchun Han, Ge Zou, Wei Zhu, Hong Shen, HaiXia Liu
Department of Medicinal Chemistry, Roche Innovation Center Shanghai, Roche Pharma Research and Early Development, Shanghai 201203, China

Nucleic acid sensing pathways play an important role in the innate immune system, protecting hosts against infections. However, a large body of evidence supports a close association between aberrant activation of those pathways and autoimmune and inflammatory diseases. Part II of the digest series on small molecule approaches to autoimmune and inflammatory diseases concentrates on recent advances with respect to small molecule an- tagonists or inhibitors of the nucleic acid sensing pathways, including endosomal TLRs, NLRP3 inflammasome and cGAS-STING.

The innate immune system of vertebrate organisms provides the first line of defense against pathogen infections by means of a diverse rangeof germline-encoded immune sensors called pattern recognition re- ceptors (PRRs).1,2 These receptors can recognize pathogen or pathogen-associated molecular patterns (PAMPs) and damage-associated molec- ular patterns (DAMPs). The major classes of PRRs are toll-like receptors (TLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors(NLRs), absent in melanoma 2 (AIM2)-like receptors (ALRs) and C-type lectin-like receptors (CLRs). The nucleic acid sensors3–7 (See Fig. 1) suchas endosomal TLRs and cytosolic RLRs, NLRs and ALRs can recognize DNA, RNA and their derivatives to trigger innate immune responses. In addition to the PRRs, guanosine monophosphate-adenosine mono- phosphate synthase (cGAS) is another cytosolic DNA sensor protein.
The activation of nucleic acid sensing pathways leads to the secretionof inflammatory molecules, particularly type I and type II interferons (IFNs)8,9 which promote a protective response against pathogens by inducing IFN-stimulated genes (ISGs) and by galvanizing a robustadaptive immune response.1 However, over-activation of the pathwaysis closely associated with many autoimmune and inflammatory diseases where a similar ISG signature is identified.10 Given their critical role in the pathogenesis of autoimmune and inflammatory diseases, nucleicacid sensing pathways have become attractive drug targets. Direct blockade of nucleic acid sensors may hold the promise of a comparable or even better efficacy than that of existing therapeutic agents such asJanus kinases (JAK) inhibitors against interferon signaling, and anti- bodies against specific cytokines, owing to a broader coverage of in- flammatory cytokines. Recent advances in understanding the structural basis of various targets in the nucleic acid sensing pathways such as TLR7, TLR8, cGAS and stimulator of interferon genes (STING) have had a profound impact on the discovery of small molecule antagonists or inhibitors in terms of druggability assessment, screening platform development and the following lead identification/optimization. In the sections below, the review focuses on the most investigated targets such as endosomal TLRs, NLRP3 and cGAS-STING, which are amenable totherapeutic targeting by small molecules. It should be noted that given these targets have previously been comprehensively reviewed,11–17 onlythe key advances from 2018 to 2020 are discussed.

Targeting endosomal TLRs
Ten TLRs (TLR1-10) have been identified in humans, among which TLR3, 7, 8 and 9 are mainly localized on the membrane of the endosome to recognize nucleic acids. TLR3 is activated by dsRNA, TLR7 and TLR8 detect ssRNA and TLR9 recognizes DNA containing an unmethylated CpG motif. TLRs comprise an extracellular domain, a transmembrane domain and an intracellular Toll/interleukin-1 receptor (TIR) domain. Upon ligand stimulation, the extracellular domain interacts with the ligand, inducing a conformational change of the TIR domain and initi- ating the recruitment of MyD88 or TRIF to trigger the production ofproinflammatory cytokines (tumor necrosis factor-α (TNF-α), IL-6 etc.) and chemokines.18 TLR7, 8, and 9 signal through a MyD88 cascade; incontrast, TLR3 engages TRIF for downstream signalling (See Fig. 1).
Mounting evidence indicates that dysregulation of the endosomal DNA- and RNA-sensing pathways is implicated in the pathogenesis of autoimmune diseases19 such as systemic lupus erythematosus (SLE)20and Sjogren’s syndrome (SS).21 Several review articles11,18,22–25 havesummarized TLR modulators and their therapeutic prospects. The recent discovery of small molecule antagonists targeting TLR7, 8 and 9 led to the identification of diverse chemical series with selective, dual- and pan-TLR antagonism, though it is unclear which TLR selectivity profileof antagonism provides optimal clinical therapeutic efficacy. The emerging co-crystal structures of TLR726 and TLR827 with small mole- cules clearly show that the antagonists’ binding hot spots are located at the interface of the ectodomain of TLR homodimers. TLR7 and TLR8share 40% sequence identity in their ectodomains, and 50% of the res- idues in their binding sites are identical.28 So, it is feasible to identifyselective or dual TLR7/8 antagonists. However, the precise TLR9 antagonist mechanism of action (MoA) remains unclear. Furthermore, the TLR9 ectodomain is related more distantly to TLR8 with 33%sequence identity overall and 25% with respect to residues in their binding sites.28 Therefore, identification of pan-TLR7/8/9 antagonists ismore challenging. Even for those alleged pan-antagonists, TLR9 potency is much weaker than that of TLR7/8. Table 1 summarizes the recently reported endosomal TLR antagonists according to their selectivities. Recent advances with respect to both clinical and preclinical endosomal TLR antagonists are covered in the following section.
At this time, there are at least four small molecules of endosomal TLR antagonists under clinical development (Fig. 2).29 The most advanced molecule, M5049 (1) from Merck KGaA, is undergoing a phase II studyto evaluate its safety and efficacy in COVID-19 pneumonia patients. Meanwhile, a phase I study to evaluate its safety, tolerability and pharmacokinetics in participants with SLE or cutaneous lupus erythe- matosus (CLE) is planned to start in 2021. In addition, BMS-986256 (2), developed by Bristol-Myers Squibb, is in phase I clinical studies to assessits safety and drug levels in subjects with CLE. The other two molecules, E6742 (structure undisclosed) from Eisai and CPG52364 (3) from Pfizer,have completed their phase I studies in healthy volunteers. While M5049,30 BMS-98625631 and E6742 are TLR7/8 dual antagonists, CPG5236432 is a TLR7/8/9 pan-antagonist.
In 2018, Yin’s group identified two distinct chemical scaffolds, pyrazolo[1,5-a]pyrimidine and 4-phenyl-1-(2H)-phthalazinone, as se- lective TLR8 inhibitors through high-throughput screening (HTS).27,47 Medicinal chemistry optimization led to the identification of CU-CPT8m (8), CU-CPT9a-b (9–10) with an IC50 within nanomolar and picomolar range, respectively, in a HEK-Blue TLR8 reporter assay. Both CU-CPT8m and CU-CPT9a doses dependently suppressed TNF-α in peripheral blood mononuclear cells (PBMCs) harvested from rheumatoid arthritis (RA)patients. Importantly, the co-crystal structures of TLR8 with CU-CPT8m and CU-CPT9b indicated that the small-molecule ligands stabilize the pre-formed TLR8 homodimer in its resting state and prevent its activa- tion by binding to a unique site at the dimeric interface. Based on the newly identified unconventional binding site and another HTS hit, they designed a triazole derivative TH1027 (11) as a selective TLR8 antag-onist with an IC50 of 30 2 nM.35 In addition, two more differentiatedchemical scaffolds were discovered with CU-72 (16) as a TLR7/8 dual antagonist and CU-115 (12) and CU-68 (13) as selective TLR8 antago- nists with relatively low potency.36
Based on the work of Shukla et al.,48–50 Karroum et al. discovered a series of compounds with the pyrazolo[1,5-a]quinoXaline scaffold as aselective TLR7 antagonist in 2019.33 The most potent compound (compound 4) showed an IC50 of 8.2 ± 1.6 μM in HEK-blue-hTLR7 cells without any TLR8 activity. According to a modeling study based on Yin’sco-crystal structure of TLR8-CU-CPTs, the difference in amino acids, Thr384 in TLR7 versus Ile403 in TLR8, plays a critical role in the selectivity of compound 4. Mukherjee et al. subsequently reported their discovery of a selective TLR7 antagonist from a well-established purine scaffold TLR7 agonist by a ‘chemical switch’, removing the O-alkylgroup at the C-2 position.34 The leading compound, compound 5, with a low micromolar IC50 and good pharmacokinetic properties was effective in ameliorating cutaneous pathology in a cutaneous autoimmune dis- ease psoriasis mouse model via oral dosing.
In 2020, Tojo et al. reported their discovery of potent and selective TLR7 antagonists.26 They successfully identified compounds 6 and 7with TLR7 IC50s of 15 and 25 nM, respectively, and high selectivity over TLR8 and TLR9, through modification of 8-oXoadenine derivatives of TLR7 agonists. More importantly, using crystallography and cryo- electron microscopy, they identified that both TLR7 agonists and an- tagonists bind to the interface of the TLR7 homodimer. The agonists induced a closed-form conformation, whereas antagonist 6 induced an open-form conformation. Interestingly, the less potent compound 7 could induce both conformations, which were in dynamic equilibrium. Moreover, compound 6 was shown to ameliorate autoimmunity in lupus-prone NZB/W F1 mice through decreasing proteinuria, increasing survival rate and reducing glomerular lesion scores in a dose-dependent manner via oral dosing.
In 2018 Ganguly’s group developed a series of potent and selectiveTLR9 antagonists.37 By modulating the substituents with varying ba- sicity, lipophilicity and flexibility at the quinazoline core, they suc-cessfully determined compound 14 to be the most selective TLR9 antagonist over TLR7 (>600-fold). By introducing lipophilic basic pro- pylpyrrolidine moiety, the compound could easily penetrate the cells and be trapped inside the acidic (pH 6.0–6.5) endosomal compartment, which increased the ligand concentration around the target. Using the same strategy, they also developed another purine series (e.g. compound15) with even more potent TLR9 antagonistic activity.38
In 2020, Novartis reported their discovery of TLR7/8 dual antagonist 17, discovered by means of a high throughput TLR8 antagonist competition assay to specifically identify binders of the ectodomain.39 Compound 17 inhibited IFN-α release in a dose-dependent manner in a ssRNA challenged mouse model through oral dosing, correlating withthe compound’s good in vitro efficacy and pharmacokinetic properties. In addition, Novartis reported two other distinct scaffolds, 1840 and 22,43 as TLR7/8 dual antagonist and TLR7/8/9 pan-antagonist,respectively, with very good in vitro activity.
In addition to the clinical TLR7/8 dual antagonist, BMS-986256, Bristol-Myers Squibb reported the discovery of compound 21 as a TLR7/8/9 pan-antagonist in 2020.28 However, the TLR9 activity wasrelatively weak in comparison to that of TLR7/8 activity. In mouse pharmacodynamics study, oral dosing of compound 21 could dose- dependently decrease agonist-induced IL-6 production. Compound 21 also dose-dependently alleviated the severity of lupus and psoriasis manifestation in MRL/lpr mice and imiquimod-induced psoriasis mice.
Recently, Roche has published several patentapplications41,42,44,45,51–55 which have disclosed several chemical scaf- folds as TLR7/8/9 pan-antagonists or TLR7/8 dual-antagonists. Some compounds such as compound 23 and compound 24 showed excellent TLR7/8/9 pan-antagonistic activity with IC50s below the assay detection limits, while other molecules such as compounds 19 and 20 exihibitedgood TLR7/8 dual-antagonistic activity with a >1000-fold selectivity over TLR9.
In 2020, Choi’s group reported the small molecule pan-TLR3/7/8/9 antagonist TAC5-a (25) that could inhibit all four of the nucleic acid sensing TLRs.46 It dose-dependently inhibited the secretion of proin- flammatory cytokines (TNF-α and IL-6) mediated by TLR3/7/8/9 in the dose range of 1–50 μM in cell-based assays without affecting other TLRs.
Although the in vitro potency was relatively weak, TAC5-a was confirmed to be effective in alleviating psoriasis and lupus symptoms and reducing disease markers in mouse models.

Targeting the NLRP3 inflammasome
Inflammasomes are multiprotein complexes consisting of NLR family members NLRP1, NLRP3 and NLRC4, and non-NLR receptors such as AIM2 and IFI16.56 The NLRP3 inflammasome is the most widelyinvestigated NLR given its important role in host defense and innateimmunity. The NLRP3 inflammasome is formed by three components: NLRP3 protein, procaspase-1 and ASC. NLRP3 protein contains PYD at the N-terminus, an array of 12 LRR at the C-terminus and the NACHT.57
PYDs interact with ASC, which contains a CARD. LRR senses microbial ligands and endogenous alarmins. NACHT contains ATPase activity and requires ATP binding for NLRP3 activation. Under normal conditions, the connection between NACHT and LRRs prevents the formation of inflammasome via inhibiting the interaction between NLRP3 protein and ASC.
NLRP3 responds to various stimuli, including viral RNA and extra- cellular ATP.58,59 The activation of NLRP3 inflammasome depends on two successive signals.58 In the initial priming step, internal and external DAMPs and PAMPs are recognized by TLRs to activate the nuclear NF-κB mediated signal, which results in the upregulation of inactive NLRP3 protein, proIL-1β and proIL-18. The subsequent activation step is trig- gered by further stimuli. Firstly, the conformation of NLRP3 protein ischanged to facilitate the interaction between PYDs in NLRP3 protein and ASC. Secondly, the procaspase-1 binds to ASC via CARD, leading to the formation of NLRP3 inflammasome. Thirdly, self-cleavage of procaspase-1 generates active caspase-1, leading to maturation of proIL- 1β and proIL-18 to IL-1β and IL-18. Then, inflammatory and immune responses are then induced (See Fig. 1). In addition, the active caspase-1 also cleaves and activates Gasdermin-D, which causes pyroptosis to formpore in the cell wall and the release of cytokines and cellular contents into extracellular space.60
There is a lot of evidence to support the hypothesis that the inap- propriate activation of NLRP3 inflammasome correlates with multiple diseases, such as autoimmune diseases61 (e.g. systemic sclerosis,IBD,61,62 RA and SLE), inflammatory diseases58 and neurodegenerativediseases.63–65 Therefore, the NLRP3 inflammasome might be a potential target to treat these diseases.
Until now, at least five NLRP3 inflammasome inhibitors have been tested in human clinical trials.66 OLT1177 (34 in Fig. 3) is an orally available and specific NLRP3 inflammasome inhibitor by directlybinding to NLRP3.67 In phase I study, OLT1177 was determined to be safe and well-tolerated in healthy volunteers receiving oral doses up to 1000 mg/day for 8 days. OLT1177 also showed a meaningful exposurewith a long half-life. Currently, it is undergoing a phase II clinical study for the treatment of acute gouty arthritis.68 NT-0167 (structure undis- closed) is under phase I clinical study to evaluate safety, tolerability,pharmacokinetics and pharmacodynamics responses. IFM-2427 wasdeveloped by IFM Tre and later acquired by Novartis. As an inhibitor of NLRP3 by directly binding to NLRP3 protein,69 IFM-2427 is now un- dergoing a phase I clinical study for autoimmune diseases. Recently,
Novartis has advanced IFM-2427 (renamed as DFV890) into phase IIor therapeutic effects on several disease models such as gouty arthritis, cryopyrin-associated autoinflammatory syndromes, and T2D.
Recently, Xu and co-workers reported a novel tetrahydroquinoline, compound 37, as a specific NLRP3 inflammasome inhibitor without affecting NLRC4 or AIM2 inflammasomes.97 The results from a biolayer interferometry (BLI) assay indicated that compound 37 directly boundto the NACHT domain of NLRP3 and inhibited NLRP3 ATPase activity. It showed an IC50 of 7.8 μM on IL-1β release in LPS/ATP-stimulated THP-M cells. Compound 37 alleviated the shortening of colon length and dose- dependently decreased the secretion of IL-1β in a dextran sulfate sodium (DSS)-induced colitis mouse for COVID-19-related pneumonia.70 SomaliX and Inzomelid
Bharate and co-workers reported their discovery of a computation-(structures undisclosed) were developed by Inflazome, but were recentlyacquired by Roche.71–76 As a potent, selective, orally available and peripherally-restricted NLRP3 inflammasome inhibitor for inflamma-tory diseases, SomaliX showed excellent safety, tolerability and phar- macokinetics in healthy subjects.77 This compound is ready to enter intophase II study. Inzomelid, an orally available, brain-penetrant inhibitor used in the treatment of debilitating inflammatory diseases of the brain, showed excellent safety, tolerability and pharmacokinetic properties in healthy subjects in the completed phase I study, and also deliveredpreliminary positive results in a patient with cryopyrin-associated pe- riodic syndrome (CAPS).78,79
Structure-diverse NLRP3 inflammasome inhibitors have been re- ported (Fig. 3), including Glyburide (26),80 JC-121 (27),81 JC-171(28),82 MCC950 (29),83–85 MNS (30),86 NBC-6 (31),87 BOT-4-one(32)88 and Oridonin (33)89. Recent review articles17,66,90–94 have nicely summarized theNLRP3 activation mechanism, inhibitors and related indications. The following section focuses on non-natural se- lective NLRP3 inflammasome inhibitors that have been reported since 2018 and which have MoA of inhibiting ATPase activity, suppressing caspase-1 activation and blocking recruitment of ASC (Table 2, Fig. 4). Discovered by Deng group through modification of a HTS hit, the 4-oXo-2-thioXo-thiazolidinone derivative CY-09 (35) is a potent and se- lective inhibitor of NLRP3.95 It showed inhibitory activity with an IC50 of 2.40 0.10 μM on IL-1β release in LPS-primed bone marrow derived macrophages (BMDMs) under the stimulation of nigericin. It also dose- dependently inhibited IL-1β secretion in freshly isolated synovial fluid cells (SFCs) from patients with gout.104 CY-09 showed specific inhibitionon NLRP3 by directly binding to NACHT and inhibiting ATPase activity without affecting NLRC4 and AIM2 inflammasomes.
Tranilast (36), a previously approved anti-allergic drug, was found tospecifically suppress NLRP3 inflammasome activation without affecting NLRC4 or AIM2 inflammasomes.96 Mechanistic study indicated that Tranilast directly binds to the NACHT domain and blocks NLRP3 olig-omerization. In a dose-dependent manner, Tranilast inhibited caspase-1 activation and IL-1β production at 50 and 100 μM, respectively, in SFCs from patients with gout. Importantly, it showed remarkable preventiveally designed compound 38 by using a NLRP3 homology model.98 The inhibition IC50 of compound 38 was 5 μM on IL-1β release in ATP- stimulated J774A.1 cells. Molecular modeling indicated that com- pound 38 occupies the ATP-binding site of NLRP3 protein, the same site as MCC950 with additional cation-π interaction with Leu 171 contrib- uted by the nitro-group.
Sharma and co-workers discovered compound 39 to be a highly potent, orally bioavailable and selective NLRP3 inflammasome inhibitor by optimizing the sulfonylurea moiety of MCC950 (clogP = 3.27).99
Compound 39 has an IC50 of 6.8 ± 0.5 nM on IL-1β release in THP1 cells.
In comparison to MCC950 (IC50 of 8 0.7 nM), compound 39 has equivalent potency and lower lipophilicity (clogP 2.45). It specifically blocked recruitment of ASC during NLRP3 activation without affecting the AIM2 inflammasome. In addition, it decreased NLRP3 dependent IL- 1β release by 44% at 10 mg/kg oral dosing in LPS/ATP challenged mice.
The Zhang group reported the identification of JC-124 (40) as a se- lective NLRP3 inhibitor by inhibiting the activation of caspase-1.100 Thein vivo efficacy was demonstrated in three models: a transgenic mouse AD model by reducing AD pathology at 50 mg/kg/day;81 an AMI model by limiting the infarct size and protecting cardiac functions at 30 mg/kg;100 and a traumatic brain injury (TBI) model by decreasing inflam-matory response in an injured brain and cortical lesion volume after TBI in adult male Sprague-Dawley rats via multiple i.p. dosing.105 Encour- aged by the promising pharmacodynamics effect of JC-124, Zhang’s group further optimized the benzenesulfoamide scaffold and identified a series of JCP-124 close analogues, among which, YQ128 (41) showed an
IC50 of 0.30 ± 0.01 μM on IL-1β release in J774A.1. In addition, YQ-128 dose-dependently suppressed IL-1β release in peritoneal macrophages upon LPS/ATP challenge with an IC50 of 1.59 0.60 μM. YQ128 spe-cifically inhibited NLRP3 inflammasome without affecting NLRC4 or AIM2 inflammasome. However, pharmacokinetic studies in rats showed an extensive clearance and tissue distribution, and low oral bioavail- ability (%F 10), likely due to poor absorption and high first-pass metabolism.
The natural product parthenolide (PTL) exhibits anti-inflammatory activity, and has been reported to target the NLRP3 inflammasome;106

DNA-cGAS interaction modulators
The anti-malarial drugs hydroXychloroquine (HCQ) and quinacrine have been identified as inhibiting cGAS activity and IFN-β secretion through blocking the formation of DNA-cGAS complex.129,143 Elkon’s group further reported a series of quinacrine derivatives,144 amongwhich X6 (45) showed modest potency with an IC50 of 14 μM in THP-1cells. In Trex1—/— mice, X6 reduced cGAMP production in the heart and attenuated the expression of ISGs in the spleen and heart. In addition, X6reduced ISG expression in the PBMCs of SLE patients. Suramin (46), a drug used to treat river blindness and African sleeping sickness, was recently identified as a cGAS inhibitor through the screening of a focused library. Mechanistically, suramin displaced the bound DNA from cGAS. It dose-dependently inhibited cGAMP production and fully reduced IFN-β expression in THP-1 cells at 5 μM. Li and coworkers un- covered that acetylation at one of the three lysine residues of cGAS,K384, K394 and K414, impaired cGAS’s binding to dsDNA, therebysuppressing its enzymatic function.145 Importantly, they identified thatcatalytic domain.The binding of the initial virtual screening hits wasthe widely used drug, aspirin (47), directly acetylated those lysine res- idues and efficiently inhibited cGAS-mediated immune responses. More interestingly, they further demonstrated that aspirin significantlyreduced the ISGs in the PBMC of AGS patients and attenuated self-DNA- induced autoimmunity in the Trex1—/— mice (See Fig. 5).

cGAS dimerization modulator
Yin’s lab recently reported a new class of h-cGAS inhibitors that target the dimeric interface, presumably, to disrupt cGAS dimeriza- tion.146 A virtual screening was conducted to target the key residues Lys335 (human Lys347) and Lys382 (human Lys394) crucial for cGASoligomerization139 based on the reported mouse cGAS structure. Z918(48) was identified as a weakly active h-cGAS inhibitor. Subsequent medicinal chemistry optimization produced CU-76 (49), which inhibi- ted cGAS activity in both ATP consumption assay with an IC50 of 0.24 µM and in THP-1 cells (See Fig. 6).

cGAS active site inhibitor
PF-06928215142 and RU.521147 are the first reported cGAS in- hibitors targeting h-cGAS and mouse cGAS (m-cGAS) active sites, respectively. They usually occupy the cGAMP pocket, and picked up key stacking interactions with Agr376 and Try436. The Tuschl grouprecently reported the discovery of h-cGAS inhibitors by using a high throughput ATP-coupled luminescence-based assay.148 Medicinalchemistry optimization of two initial hits led to the identification of more potent compounds such as J014 (50) and G150 (51) with IC50 values of 0.100 µM and 0.0102 µM, respectively. J014 was active in THP-1 cells with an IC50 value of 2.63 µM, but was deprioritized due to cytotoXicity and poor selectivity. G150 showed a higher activity in THP- 1 cells with an IC50 value of 1.96 µM and good activity in primary human macrophages (IC50 less than 1 µM). The crystal structure of G150 bound to the h-cGAS apo catalytic domain demonstrated its localization in the cGAMP pocket. Zhao and coworkers reported the discovery of novel cGAS active site inhibitors by utilizing a virtual screening approachconfirmed by thermal shift assay (TSA), and the enzymatic activity was evaluated in a PPiase-coupled assay, identifying compound 52 as a moderately effective cGAS inhibitor with an IC50 of 29.88 3.20 µM. Subsequent similarity research into compound 52, followed by a second virtual screening, led to the identification of more potent compounds S2(53) and S3 (54) with IC50 values of 13.1 0.09 µM and 4.9 0.26 µM, respectively. The crystal structure of compound S2 bound to the h-cGAS catalytic domain was resolved (PDB code: 6LRL). A Bellbrook Labspatent disclosed a series of tricyclic benzofuropyrimidine analogues as cGAS inhibitors.150 The co-structure of compound 55 indicated that the tricyclic core sat in the middle of Arg376 and Try436. Compound 56 showed the best cellular potency with an IC50 of 13 µM in an IFN-β ELISA assay using THP-1 cells (See Fig. 7).

cGAS inhibitor with undisclosed mechanism
Aduro Biotech recently disclosed multiple series of cGAS inhib- itors.151–153 C1089 (57) with a pyrazolopyrimidinone core was active ina cGAS biochemical assay and in THP-1 cells with an IC50 value less than 20 µM.151 It also showed inhibitory activity against cytokines such as RANTES and monocyte chemoattractant protein-1 (MCP-1) secretion inTrex1—/— BMDM with IC50s of 1.251 µM and 6.973 µM, respectively. Triazine compounds were disclosed in another patent application.
Compound TA1065 (58) was the most potent one in THP-1 cells with an IC50 value between 20 and 100 µM.152 In addition, new compounds with an imidazopyridazinone core have been recently disclosed.153 Com- pound 59 is a representative showing an IC50 of less than 1 µM in the cGAS biochemcial assay and an IC50 of less than 10 µM in THP-1 cells. Bellbrook Labs disclosed another patent application on their cGAS in- hibitors with an undisclosed binding MoA.154 Compound 60 inhibited cGAS enzymatic activity with an IC50 of 0.391 µM, and was weakly active in THP-1 cells (See Fig. 8).

Targeting the STING
STING is an endoplasmic-reticulum protein111 consisting of an N-terminal domain (~140 aa) that contains four transmembrane helices, and a cytoplasmic C-terminal domain that includes a globular ligand binding domain (aa 153 – 340) and a C-terminal tail (aa 342 – 379). The cytoplasmic parts of STING form a butterfly-shaped dimer with the ligand binding pocket located at the dimer interface. Crystal structures of the cytosolic domain of human STING in both apo and in complexwith natural and synthetic ligands have been determined to be highly structured dimers.155–162
All the reported STING antagonists are at the discovery stage. In terms of the binding MoA, two types of STING antagonists have beenreported, including competitive binding (Fig. 9) and allosteric binding (Fig. 10). The STING competitive pocket is large and polar with highaffinity endogenous ligand cGAMP (Kd 4.6 nM),163 and as such, it would be challenging to develop a potent and orally available small molecule antagonist.164 Alternatively, inhibition of the agonist-induced,post-translational palmitoylation by covalently modifying Cys88/91 at an allosteric pocket could inhibit STING signaling,165 presumably, by blocking STING polymerization. This new MoA provides a novelapproach to identify STING signaling inhibitors.166
Merck scientists have reported the discovery of orally availableSTING antagonists that bind to the STING competitive binding site.164 To leverage the intrinsic symmetric nature of STING, the team focused on identifying small molecules that can bind to the STING homodimer ina 2:1 ratio while maintaining physicochemical properties suitable for oral drugs. Compound 61 was firstly identified as a weak hit (IC50 = 7.3 µM in HAQ STING cGAMP displacement assay), and its crystal structure indicated a 2:1 binding mode with intermolecular interaction of the twoligands. Further medicinal chemistry optimization led to the discovery of compound 62 with improved binding affinity (IC50 of 0.068 µM). Compound 62 inhibited the IFN-β production with an IC50 of 11 µM in cGAMP stimulated THP-1 cells. In addition, compound 62 demonstrated good oral bioavailability (%F 60) in a rat pharmacokinetics study.
Astin C (63), a cyclopeptide isolated from the medicinal plant Astertataricus, was identified as a STING antagonist by Wang lab through a screening of a Compositae-type cyclopeptide library.167 It was found tospecifically bind to the STING C-terminal domain by utilizing a biotin pull-down assay. Further in silico virtual simulation analysis suggested that astin C may block the cGAMP binding pocket. The binding affinity of astin C (Kd 53 nM) is comparable to that of cGAMP in an isothermal titration calorimetry (ITC) assay. In addition, astin C showed good ac- tivity in inhibiting IFN-β production in human fetal lung fibroblasts withan IC50 of 10.83 1.88 µM. Moreover, astin C significantly attenuated the IFN-β and a set of pro-inflammatory cytokines in Trex1—/— BMDMcells and mice.
GlaxoSmithKline disclosed a series of N-methyl amidobenzimida- zoles STING antagonists in a recent patent application,168 derived from a previously reported agonist scaffold that occupies the STING competi-tive pocket at the dimeric interface.160 Some antagonists exhibitedexcellent potency in both biochemical and cellular assays. For example, compound 64 showed excellent binding affinity with a pIC50 value of >9.9 in a displacement assay, and strongly inhibited IFN-β secretion inboth THP-1 cells and human PBMCs with pIC50 values of 8.9 and 7.1, respectively.
Ablasser’s group reported the identification of covalent STING an-tagonists from a cell-based chemical screening.166 The identified nitro- furan analogues, as exemplified by C-178 (65), covalently modified the transmembrane Cys91 through a nucleophilic addition of the thiol to the 4-position of the furan ring followed by an intramolecular rearrange- ment. C-178 potently and selectively inhibited STING activation inmouse BMDMs. A more soluble close analogue, C-176 (66), suppressedIFN-β production in agonist challenged mice and in Trex1—/— mice. In addition, C-176 attenuated various markers of systemic inflammation inTrex1—/— mice by means of chronic treatment. However, both C-176 and C-178 are mouse specific STING antagonists, with a following additionalchemical screen revealing a human STING antagonist H-151 (67), which similarly inhibited human STING at submicromolar level through co- valent modification of Cys91. H-151 doesn’t contain a typical cysteine reactive scaffold, and covalent product was observed in a mass spec- trometry study; however, the reaction mechanism of covalent bond formation has not been explained. H-151 exhibited notable efficacy in agonist-challenged mice and in the bioluminescent IFN-β reporterTrex1—/— mice. Notably, another two research groups have discovered that endogenous ligands such as nitro-fatty acid (68)169 and 4-HNE (69)170 inhibit STING activation in a similar fashion by forming a co-valent bond with Cys91/Cys88 and Cys88 respectively through Michael addition.
In 2020, IFM Therapeutics released a series of patent applications to protect their STING antagonists,171–178 with the clinical trial of the lead molecule expected to be conducted in 2021.179 Their antagonists seemto have been derived from the pharmacophore of H-151, containing a(n) (un)substituted (aza)indole connecting with a hydrophobic scaffold through a linker such as urea, amide, aminosulfoXimine or 3,4-diamino- cyclobut-3-ene-1,2-dione, the exact MoA of which has not been dis-closed. The compounds showed very good potency in THP-1 cells as exemplified by compounds 70,172 71,173 72,174 and 73175 with IC50s lessthan 1.0 μM (See Fig. 11).

Concluding remarks and future prospects
Unlike the various kinase inhibitors, as outlined in the first part of the review,180 which treat autoimmune and inflammatory diseases fromdownstream of either innate or adaptive immune signaling, the nucleic acid sensing antagonists/inhibitors address the diseases from the initi- ation of the pathogenic signaling, which might bring differentiated therapeutic benefit. Meanwhile, the discovery and development of nucleic acid sensing antagonists face several challenges. Firstly, auto- immune and inflammatory diseases, in particular SLE, are very hetero- geneous diseases, thus selecting the right patients is critical for clinical proof-of-concept. Secondly, the pathogenic nucleic acids may activate multiple pathways; so, to achieve satisfactory efficacy may require blocking several nucleic acid sensors simultaneously, which may not be technically achievable by a single molecule. In addition, the technical challenge of obtaining orally available small molecule antagonists/in-hibitors for certain targets is high. For example, the cGAS-STING pathway has fostered enthusiasm since the identification of cGAS141; however, progress has been relatively slow, which might be related tothe poor druggability of the protein pocket. Despite these obstacles, several small molecule antagonists/inhibitors of nucleic acid sensors such as TLR78 and NLRP3 have been advanced into clinical trials as noted beforehand. The cGAS-STING antagonists are approaching the entry into clinical development. In addition, a growing understanding of the structural basis of nucleic acid sensing will continue to accelerate the discovery process. Moreover, new technologies such as targeted protein degradation and RNA-targeted small molecules provide alternative ap- proaches to inhibiting nucleic acid sensing. Richer small molecule nucleic acid sensing antagonist/inhibitor pipelines are expected to be seen in both preclinical and clinical stages in the coming years. More importantly, the clinical outcomes of the pilot molecules will further guide preclinical compound optimization and design of clinical studies.

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