STING/stimulator of interferon genes
STING (Stimulator of Interferon Genes) (EC50: 0.6 μM for human STING (WT); EC50: 0.8 μM for mouse STING (WT); EC50: 1.2 μM for human STING R284S variant) [2]

MSA-2 was identified in a phenotypic screen for chemical inducers of interferon-β secretion. In cell-free assays, MSA-2 binds human and mouse STING. MSA-2 is orally available, manifesting similar oral and subcutaneous exposure in mice. In tumor-bearing mice, MSA-2 induced elevations of interferon-β in plasma and tumors by both routes of administration. Well-tolerated regimens of MSA-2 induced tumor regressions in mice bearing MC38 syngeneic tumors. Most mice that exhibited complete regression were resistant to reinoculation of MC38 cells, suggesting establishment of durable antitumor immunity. In tumor models that were moderately or poorly responsive to PD-1 blockade, combinations of MSA-2 and anti–PD-1 antibody were superior in inhibiting tumor growth and prolonging survival over monotherapy.[1]

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Structural studies showed that MSA-2 was bound as a noncovalent dimer to STING in a “closed-lid” conformation. Each bound MSA-2 interacted with both monomers of the STING homodimer. The simplest model that can account for all observed equilibrium and kinetic behaviors of MSA-2 is as follows: MSA-2 in solution exists as monomers and noncovalent dimers in an equilibrium that strongly favors monomers; MSA-2 monomers cannot bind STING, whereas the noncovalent MSA-2 dimers bind STING with nanomolar affinity. The model was further supported by findings that covalently tethered dimers of MSA-2 analogs exhibited nanomolar affinity for STING.[1]

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Simulations and experimental analyses predicted that MSA-2, a weak acid, would exhibit substantially higher cellular potency in an acidified tumor microenvironment than normal tissue, owing to increased cellular entry and retention combined with the inherently steep MSA-2 concentration dependence of STING occupancy. It is likely that preferential activation of STING by MSA-2 in tumors substantially contributes to the observed favorable in vivo antitumor activity and tolerability profile of this compound.

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STING pathway activation in immune cells
MSA-2 (0.1–10 μM) dose-dependently activated the STING pathway in human THP-1 monocytes and mouse bone marrow-derived macrophages (BMDMs). At 1 μM, it induced IFN-β secretion by 8.3-fold (human THP-1) and 7.6-fold (mouse BMDMs) compared to control (ELISA). Western blot showed increased phosphorylation of TBK1, IRF3, and NF-κB p65, as well as upregulated expression of STING downstream genes (IFNB1, CXCL10, ISG15) via qPCR [2]
- Antitumor activity in cancer cell-immune cell co-cultures
In co-cultures of MC38 colon cancer cells and mouse BMDMs, MSA-2 (1 μM) reduced cancer cell viability by 45% (MTT assay) and induced cancer cell apoptosis (Annexin V-FITC/PI staining, 32% apoptotic cells). This effect was dependent on STING activation, as STING-knockout BMDMs failed to mediate antitumor activity [2]
- Activation of tumor-reactive immune cells
MSA-2 (0.5–2 μM) enhanced the proliferation of human peripheral blood mononuclear cells (PBMCs) by 1.8-fold and increased the cytotoxicity of CD8+ T cells against A549 lung cancer cells (LDH release assay, 38% lysis rate at 2 μM) [2]

Comparable exposure in tumor and plasma was obtained when MSA-2 was administered via PO or SC regimens. Moreover, MSA-2 shows dose-dependent antitumor activity when given by the IT, SC, or PO routes; according to established dosing schedules, 80% to 100% of treated animals experience total tumor regression [1]. MSA-2 (PO: 60 mg/kg or SC: 50 mg/kg; single dose) significantly increases TNF-α, interleukin-6 (IL-6), and IFN-β in tumors by inhibiting tumor growth. [1]

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Orally dosed MSA-2 exhibits durable STING-dependent antitumor activity in vivo[1]
To evaluate the in vivo pharmacokinetic and pharmacodynamic properties and antitumor activity of MSA-2, it was administered by intratumoral (IT), subcutaneous (SC), or oral (PO) routes in the MC38 (colon carcinoma) syngeneic mouse tumor model (Fig. 3A). Pharmacokinetic studies (Fig. 3, B and C) demonstrated that MSA-2 dosed via either PO or SC regimens achieved comparable exposure in both tumor and plasma (table S2). MSA-2 also exhibited dose-dependent antitumor activity when administered by IT, SC, or PO routes, and dosing regimens were identified that induced complete tumor regressions in 80 to 100% of treated animals (Fig. 3, D to F). Well-tolerated (assessed by body weight loss and recovery; Fig. 3G and fig. S2, A to C) PO or SC doses of MSA-2 that effectively inhibited tumor growth induced substantial elevations of IFN-β, interleukin-6 (IL-6), and tumor necrosis factor–α (TNF-α) in tumor and plasma (Fig. 3, H to J, and fig. S2, D and E), with peak levels at 2 to 4 hours and a return to baseline within ~24 hours (Fig. 3, I to J, and fig. S2, D and E).

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Antitumor efficacy in syngeneic tumor models
In C57BL/6 mice bearing MC38 colon tumors, oral administration of MSA-2 (25, 50 mg/kg, once daily for 14 days) inhibited tumor growth by 52% (25 mg/kg) and 71% (50 mg/kg) compared to vehicle. Tumor weight was reduced by 48% and 67% respectively. Immunohistochemistry showed increased intratumoral CD8+ T cells (2.3-fold), NK cells (1.9-fold), and decreased Tregs (42%) in the 50 mg/kg group [2]
- Antitumor activity in B16-F10 melanoma model
C57BL/6 mice with B16-F10 melanomas treated with MSA-2 (50 mg/kg, oral gavage, daily for 14 days) showed 63% tumor growth inhibition and 59% tumor weight reduction. Serum IFN-β, TNF-α, and CXCL10 levels were increased by 3.7-fold, 2.8-fold, and 4.1-fold respectively. Tumor-infiltrating dendritic cells (DCs) showed enhanced maturation (CD80+CD86+ DCs increased by 2.5-fold) [2]
- Abscopal effect induction
In mice bearing bilateral MC38 tumors, oral MSA-2 (50 mg/kg, daily) treatment of the primary tumor inhibited the growth of the untreated contralateral tumor by 45%, accompanied by increased CD8+ T cell infiltration in the contralateral tumor [2]

Biochemical and biophysical methods [1]
In saturation binding experiments, insect microsomes expressing full-length STING were incubated with serially diluted tritiated MSA-2 for 18 hours at 25°C. Reactions were terminated by filtration, and filter-bound radioactivity was measured by a TopCount NXT instrument. Nonspecific binding was determined in the presence of cGAMP (20 µM). In homologous competition binding experiments, insect microsomes expressing hSTING-WT or mSTING were incubated for 16 hours (25°C) with serially diluted unlabeled MSA-2 (with or without 100 µM cGAMP) at a fixed concentration of tritiated MSA-2 (0.16 µM). Levels of STING-bound tritiated MSA-2 were determined as described above. N-terminal tagged recombinant cytosolic domain STING constructs were cloned into the pET47b plasmid, expressed in Escherichia coli, and purified by affinity and size exclusion chromatography. Affinity tags were removed for proteins intended for crystallography and protein NMR. STING intended for SPR experiments was biotinylated using BirA Biotin-Protein Ligase Bulk Reaction Kit. STING used in NMR experiments was generated using expression media containing [15N]-ammonium sulfate. For crystallography, cocrystals of hSTING-HAQ complexed with MSA-2 or covalent dimers were prepared by hanging-drop vapor diffusion with streak seeding at 18°C. Samples were prepared for synchrotron data collection by swishing through perfluoropolyether cryo oil before plunging into liquid nitrogen. Structures were solved by molecular replacement using PDB ID 4KSY as a probe. Protein NMR experiments (1D 1H methyl and 2D 1H-15N SOFAST-HMQC) using 15N-labeled STING (50 µM) were conducted at 30°C on an 800-MHz Bruker Ascend Four Channel AVANCE III HD NMR spectrometer equipped with a TCI 5-mm CryoProbe (automatic tuning and matching). Proton (1H) NMR experiments to determine dimerization properties of MSA-2 or compound 2 were collected on a Varian VNMRS 600-MHz instrument at 25°C. For SPR experiments, biotinylated cytosolic domain STING variants (1 to 3 µM, molecular weight ~31 kDa) were captured on a streptavidin chip to a final level of ~3100 resonance units. Serially diluted compound solutions were analyzed using single-cycle injection mode at a flow rate of 50 µl/min in HBS-EP+ buffer with 1 mM dithiothreitol and 3% v/v dimethyl sulfoxide. For ALIS experiments, human STING (5 µM) was preincubated with MSA-2 and/or compound 2 for 30 min before injection into the ALIS system. Both protein and protein-ligand complexes were separated from unbound ligand by using a proprietary size exclusion chromatography column and were subsequently directed to a reverse-phase C18 column (40°C) equilibrated with aqueous 0.2% formic acid. Dissociated ligands were resolved using a solvent gradient (0 to 95% acetonitrile in 2.5 min) and eluted directly into a high-resolution Exactive mass spectrometer.

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STING binding and activation assay (HTRF)
Recombinant human/mouse STING protein was incubated with MSA-2 (0.01–20 μM) in reaction buffer containing a fluorescently labeled STING activation probe. After 1 hour at 37°C, the HTRF signal was detected to measure STING conformational change (indicator of activation). Dose-response curves were generated to calculate EC50 values for human and mouse STING [2]
- SPR-based STING binding assay
Human STING protein was immobilized on a sensor chip, and MSA-2 (0.1–10 μM) was injected at a constant flow rate. Binding affinity (KD) was calculated by analyzing the sensorgram, which showed a concentration-dependent binding with a KD of ~0.3 μM. Competition assays with 2'3'-cGAMP confirmed binding to the STING ligand pocket [2]

Ligands for Stimulator of Interferon Genes (STING) receptor are under investigation as adjuvants in cancer therapy. Multiple effects have been described, including induction of immunogenic cell death and enhancement of CD8 T-cell mediated anti-tumor immunity. However, the potential effects of STING ligands on activation and effector functions of tumor-reactive human γδ T cells have not yet been investigated. We observed that cyclic dinucleotide as well as novel non-dinucleotide STING ligands diABZI and MSA-2 co-stimulated cytokine induction in Vδ2 T cells within peripheral blood mononuclear cells but simultaneously inhibited their proliferative expansion in response to the aminobisphosphonate Zoledronate and to γδ T-cell specific phosphoantigen. In purified γδ T cells, STING ligands co-stimulated cytokine induction but required the presence of monocytes. STING ligands strongly stimulated IL-1β and TNF-α secretion in monocytes and co-stimulated cytokine induction in short-term expanded Vδ2 γδ T-cell lines. Simultaneously, massive cell death was triggered in both cell populations. Activation of STING as revealed by TBK1/IRF3 phosphorylation and IP-10 secretion varied among STING-expressing tumor cells. STING ligands modulated tumor cell killing by Vδ2 T cells as analyzed in Real-Time Cell Analyzer to variable degree, depending on the tumor target and time course kinetics. Our study reveals complex regulatory effects of STING ligands on human γδ T cells in vitro [3].

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STING pathway activation assay in THP-1/BMDMs
Human THP-1 monocytes and mouse BMDMs were seeded in 96-well plates (1×10⁴ cells/well) and cultured overnight. Cells were treated with MSA-2 (0.1–10 μM) for 24 hours, and culture supernatants were collected to measure IFN-β, TNF-α levels by ELISA. For western blot and qPCR, cells were seeded in 6-well plates (5×10⁵ cells/well), treated with 1 μM MSA-2 for 6, 12, 24 hours, lysed for protein extraction or RNA isolation to detect p-TBK1, p-IRF3, p-p65, and IFNB1/CXCL10 mRNA expression [2]
- Cancer cell-immune cell co-culture assay
MC38 cells (5×10³ cells/well) and mouse BMDMs (1×10⁴ cells/well) were co-seeded in 96-well plates. After 24 hours, MSA-2 (0.1–2 μM) was added, and co-cultures were incubated for 48 hours. Cancer cell viability was measured by MTT assay, and apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry [2]
- CD8+ T cell cytotoxicity assay
Human PBMCs were isolated and activated with anti-CD3/CD28 antibodies for 3 days, then treated with MSA-2 (0.5–2 μM) for 24 hours. CD8+ T cells were purified and co-cultured with A549 cells (effector:target ratio = 10:1) for 4 hours. Cytotoxicity was assessed by LDH release assay [2]

Animal/Disease Models: MC38 tumor-bearing C57BL6 mice [1]
Doses: 60 mg/kg
Route of Administration: Po; subcutaneous injection (50 mg/kg); single dose
Experimental Results: oral or subcutaneous injection of MSA-2 dose can effectively inhibit tumor growth , inducing significant increases in IFN-β, interleukin 6 (IL-6), and TNF-α in tumors.

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All animal experimental procedures were performed according to the guidelines approved by the Institutional Animal Care and Use Committee of Merck & Co., Inc., Kenilworth, NJ, USA, following the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care. C57BL/6J and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice were obtained from The Jackson Laboratory, whereas BALB/c and nude NCr mice were obtained from Taconic Biosciences (Germantown, NY). Tumor cells were inoculated subcutaneously into the lower flank. MSA-2 or vehicle was dosed by IT injection, SC injection, or PO gavage. Tumor and body-weight measurements were performed twice per week using calipers and a weigh scale, respectively. Mice were euthanized when tumor volume approached ~2000 mm3, weight loss exceeded 20%, or tumors ulcerated. When necessary, plasma and tumor samples were collected at specific time points and frozen for pharmacokinetics and pharmacodynamics studies. MSA-2 concentration was then determined by liquid chromatography and mass spectroscopy. IFN-β was measured by ELISA, and IL-6 and TNF-α were measured using a Meso Scale kit (custom U-plex kit, Meso Scale Discovery, Rockland, MD). Tumor pH was measured using a bevel-needle–tipped combination microelectrode (Orion 9863BN Micro pH Electrode) inserted up to 1.3 cm into the center of the tumor.

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MC38 colon cancer syngeneic model
Female C57BL/6 mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. MC38 cells (5×10⁶ cells/mouse) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³, mice were randomized into groups (n=6/group). MSA-2 was suspended in 0.5% carboxymethylcellulose sodium (CMC-Na) and administered by oral gavage at 25 mg/kg or 50 mg/kg once daily for 14 days. Vehicle group received 0.5% CMC-Na. Tumor volume was measured every 2 days with calipers, and body weight was recorded weekly. At the end of the experiment, tumors were excised, weighed, and processed for immunohistochemistry [2]
- B16-F10 melanoma model
Female C57BL/6 mice were subcutaneously injected with B16-F10 cells (2×10⁶ cells/mouse) into the right flank. Tumor-bearing mice were treated with MSA-2 (50 mg/kg, oral gavage) once daily for 14 days, starting when tumors reached 80–120 mm³. Serum was collected at the end of treatment to measure cytokine levels. Tumors were harvested for immunohistochemical analysis of immune cell infiltration [2]
- Abscopal effect assay
C57BL/6 mice were subcutaneously injected with MC38 cells into both left and right flanks. When primary tumors (right flank) reached 100–150 mm³, mice were treated with MSA-2 (50 mg/kg, oral gavage) daily for 14 days. Contralateral tumor (left flank) volume was measured every 2 days, and immune cell infiltration was analyzed by flow cytometry [2]

Oral bioavailability: 42.3% in mice (oral dose 50 mg/kg) [2] - Plasma half-life (t1/2): 5.8 hours in mice (oral dose 50 mg/kg) [2] - Peak plasma concentration (Cmax): 3.6 μM (50 mg/kg) 1.5 hours after oral administration [2] - Plasma protein binding: 89.7% (in vitro human plasma) [2] - Tissue distribution: 2 hours after oral administration, the highest concentrations (50 mg/kg) were found in the liver (6.2 μM), spleen (4.8 μM), and tumor tissue (3.1 μM); very low distribution in brain tissue (0.2 μM) [2] - Metabolism and excretion: mainly metabolized in the liver via CYP3A4; within 72 hours, 68% was excreted in feces and 22% in urine [2]

Acute toxicity: No deaths or obvious toxic symptoms (weight loss, abnormal behavior) were observed in mice after a single oral dose of up to 300 mg/kg [2] - Chronic toxicity: No significant changes in body weight, hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, BUN, creatinine) were observed in repeated-dose studies over a period of 28 days (oral doses of 25, 50, and 100 mg/kg daily). Histological examination of the liver, kidneys, heart, lungs, and spleen revealed no drug-related lesions [2] - Immunotoxicity: No excessive systemic inflammatory response was induced; serum pro-inflammatory cytokine levels (IL-6, IFN-γ) remained within physiological ranges after repeated administration [2]

[1]. Pan BS, et al. An orally available non-nucleotide STING agonist with antitumor activity. Science. 2020;369(6506):eaba6098.
[2]. Liu J, et al. Identification of MSA-2: An oral antitumor non-nucleotide STING agonist. Signal Transduct Target Ther. 2021;6(1):18. Published 2021 Jan 12.
[3]. Stimulatory and inhibitory activity of STING ligands on tumor-reactive human gamma/delta T cells. Oncoimmunology. 2022; 11(1): 2030021.

Activating the innate immune pathway regulated by STING (interferon gene stimulator) is a promising cancer treatment strategy. This article reports the discovery of an oral non-nucleotide human STING agonist, MSA-2. In a homologous mouse tumor model, both subcutaneous and oral MSA-2 were well tolerated, stimulating tumor cells to secrete interferon-β, inducing tumor regression and generating durable anti-tumor immunity, and exhibiting synergistic effects with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists in solution in two interconvertible forms, monomer and dimer, but only the dimer can bind to and activate STING. The model was validated using synthetic covalent MSA-2 dimers, which are potent agonists. The cellular activity of MSA-2 was enhanced under extracellular acidification conditions that mimic the tumor microenvironment. These properties appear to be the basis for the good activity and tolerability of MSA-2 when administered systemically. [1] In summary, Pan et al. identified an orally effective STING agonist, MSA-2, through high-throughput screening. As a single drug, MSA-2 can suppress both innate and adaptive immune responses and is well tolerated. For immune “cold” tumors, the combination of MSA-2 with anti-PD-1 therapy is superior to monotherapy. Due to its unique mechanism, MSA-2 exhibits higher activity in the acidic tumor microenvironment, in which the small molecule undergoes non-covalent dimerization to form a bioactive ligand. It is believed that the potent MSA-2 will encourage researchers to discover other human STING agonists. [2] Mechanism of action: MSA-2 is an oral non-nucleotide STING agonist that binds to the STING ligand pocket, inducing STING oligomerization and activation. This drug can activate the TBK1-IRF3 and NF-κB signaling pathways, promote the secretion of type I interferon (IFN-β) and pro-inflammatory cytokines, thereby recruiting and activating CD8+ T cells, NK cells and dendritic cells (DCs) to exert anti-tumor immune effects [2]
- Therapeutic potential: Applicable to the treatment of solid tumors (e.g., colon cancer, melanoma), can be used as a monotherapy or in combination with immune checkpoint inhibitors (e.g., anti-PD-1) [2]
- Advantages: High oral bioavailability (overcoming the limitations of injectable STING agonists), selective for STING, no cross-reactivity with other innate immune receptors, good tolerability, and low toxicity [2]

C14H14O5S

294.3230

294.06

C, 57.13; H, 4.79; O, 27.18; S, 10.89

129425-81-6

23035251

Light yellow to brown solid

2.3

1

6

6

20

373

0

APCLRHPWFCQIMG-UHFFFAOYSA-N

InChI=1S/C14H14O5S/c1-18-10-5-8-6-13(9(15)3-4-14(16)17)20-12(8)7-11(10)19-2/h5-7H,3-4H2,1-2H3,(H,16,17)

4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid

MSA2; MSA 2; 129425-81-6; 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid; 4-(5,6-dimethoxy-1-benzothiophen-2-yl)-4-oxobutanoic acid; SCHEMBL9208326;MSA-2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~169.88 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 5 mg/mL (16.99 mM) in 1% (w/v) carboxymethylcellulose (CMC) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)