STING/stimulator of interferon genes

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.

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).

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.

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].

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.

[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.

Pharmacological activation of the STING (stimulator of interferon genes)-controlled innate immune pathway is a promising therapeutic strategy for cancer. Here we report the identification of MSA-2, an orally available non-nucleotide human STING agonist. In syngeneic mouse tumor models, subcutaneous and oral MSA-2 regimens were well tolerated and stimulated interferon-β secretion in tumors, induced tumor regression with durable antitumor immunity, and synergized with anti-PD-1 therapy. Experimental and theoretical analyses showed that MSA-2 exists as interconverting monomers and dimers in solution, but only dimers bind and activate STING. This model was validated by using synthetic covalent MSA-2 dimers, which were potent agonists. Cellular potency of MSA-2 increased upon extracellular acidification, which mimics the tumor microenvironment. These properties appear to underpin the favorable activity and tolerability profiles of effective systemic administration of MSA-2.[1]

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In summary, the study by Pan et al. identified an orally available STING agonist, MSA-2, through high-throughput screening. As a single medication, MSA-2 suppressed tumor with both innate and adaptive immune responses and was well tolerated. Toward immunologically “cold” tumors, the combination of MSA-2 and anti-PD-1 therapy outperformed monotherapy. Owing to the unique mechanism, MSA-2 exhibited a higher potency in the acidic tumor microenvironment, where the small molecule underwent noncovalent dimerization to form a bioactive ligand. It is believed that the powerful MSA-2 will encourage researchers to discover other human STING agonists.[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.)