Stimulator of interferon genes (STING)

- Human PBMCs: diABZI STING agonist-1 induced dose-dependent activation of the STING pathway, as evidenced by increased phosphorylation of IRF3 and production of IFN-β and CXCL10. The EC₅₀ for IFN-β induction was 130 nM. The compound also demonstrated >100-fold selectivity for human STING over mouse STING[1].
- THP-1 cells: Treatment with diABZI STING agonist-1 led to robust activation of the STING-TBK1-IRF3 signaling axis, as measured by Western blot analysis of phosphorylated IRF3 and TBK1. This activation correlated with increased mRNA expression of IFN-β and pro-inflammatory cytokines[1].

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diABZI STING agonist-1 is a stimulator of the selective interferon gene (STING) receptor having EC50 values of 186 nM for mice and 130 nM for humans, respectively. Compound 3, diABZI STING agonist-1, shows excellent selectivity for more than 350 investigated kinases at 1 μM [1].

- CT-26 syngeneic mouse model: Intratumoral administration of diABZI STING agonist-1 (100 μg) resulted in significant tumor growth inhibition (TGI: 94%) and prolonged survival. Systemic administration via intravenous (80% TGI) or intraperitoneal (62% TGI) routes also demonstrated potent antitumor activity. The compound induced a systemic immune response, characterized by increased CD8⁺ T cell infiltration into tumors and elevated serum levels of IFN-γ and TNF-α[1].
- B16 melanoma model: Intravenous and intraperitoneal administration of diABZI STING agonist-1 resulted in 77% and 56% reduction in mean tumor volume, respectively, by day 11 post-treatment[1].

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In vivo, STING-dependent activation of type I interferons and proinflammatory cytokines is induced by diABZI STING agonist-1 (subcutaneous injection; 2.5 mg/kg) [1]. ? DiABZI STING Agonist-1 (iv; 3 mg/kg) produced systemic concentrations higher than the half-maximum effective concentration (EC50) of mouse STING (200 ng/ml) and showed systemic exposure with a half-life of 1.4 hours [1]. Significantly reducing tumor development and improving survival was the effect of diABZI STING Agonist-1 (iv; 1.5 mg/kg; Days 1, 4, and 8; Day 43). [1].

Recombinant human STING binding assay: The binding affinity of diABZI STING agonist-1 to human STING was evaluated using surface plasmon resonance (SPR). The compound demonstrated a KD of 25 nM, indicating high binding affinity. The assay involved immobilizing recombinant human STING on a sensor chip and injecting serial dilutions of the compound to measure binding kinetics[1].

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To identify any potential off-target liabilities early on, an affinity enrichment-based chemoproteomics strategy was applied to compound 2 (diABZI STING agonist-1). Compound 5, an active analogue containing a primary amine functionality, was covalently immobilized on sepharose beads and was used to affinity-capture potential target proteins from a THP1 cell lysate. Pull-down experiments were performed in the absence of free compound 2 to delineate target proteins from background or in the presence of compound 2 over a range of concentrations. All proteins captured by the beads under the different conditions were eluted and subsequently quantified by isotope tagging of tryptic peptides followed by LC–MS/MS analysis to establish a competition-binding curve and determine a half-maximal inhibition (IC50) value. The IC50 values obtained in these experiments represent a measure of target affinity, but are also affected by the affinity of the target for the bead-immobilized ligand. The latter effect can be deduced by determining the depletion of the target proteins by the beads, such that apparent dissociation constants can be determined, which are largely independent from the bead ligand (see Supplementary Methods for details). Notably, only two proteins were captured and competed in a dose-dependent manner within a 1,000-fold window, namely STING and orosomucoid1 (ORM1, alpha-1-acid glycoprotein 1 precursor). The mean value for STING was determined as 1.6 nM, demonstrating high potency of compound 2 on the target protein not only in an artificial biochemical assay system using truncated protein but also against the full-length endogenous human protein. The mean value of the only identified off-target protein, ORM1, was determined as 79 nM giving a comfortable selectivity window of approximately 40-fold. ORM1 is an acute phase reactant, an abundant plasma protein with known drug binding properties, and is known to be expressed in monocytes.[1]

- Human PBMC activation assay: PBMCs were isolated from healthy donors and treated with diABZI STING agonist-1 at concentrations ranging from 0.1 to 1000 nM. After 24 hours, cell culture supernatants were analyzed for IFN-β and CXCL10 levels using ELISA. Cell viability was assessed using the MTT assay to ensure no cytotoxic effects at effective concentrations[1].
- THP-1 signaling assay: THP-1 cells were transfected with an IFN-β luciferase reporter plasmid. Following treatment with diABZI STING agonist-1, luciferase activity was measured to quantify STING pathway activation. Western blot analysis was performed to confirm phosphorylation of downstream signaling proteins[1].

Animal/Disease Models: Wild and Sting−/− C57Blk6 mice[1]
Doses: 2.5 mg/kg
Route of Administration: subcutaneous injection; 2.5 mg/kg
Experimental Results: The secretion of IFNβ, IL-6, TNF and CXCL1 was activated in wild-type mice, but Sting None in −/− mice. BALB/c mouse colorectal tumor syngeneic mouse model (CT-26) [1]

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Doses: 3 mg/kg
Route of Administration: intravenous (iv) (iv)injection; 3 mg/kg
Experimental Results: Half-life is 1.4 hrs (hrs (hours)), systemic concentration is greater than the EC50 of mouse STING (200 ng/ Animal/Disease Models: BALB/c mouse colorectal tumor syngeneic mouse model (CT-26) [1]

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Doses: 1.5 mg/kg
Route of Administration: intravenous (iv) (iv)injection; 1.5 mg/kg; 43-day
Experimental Results: Dramatically inhibited tumor growth and improved survival rate.

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- CT-26 tumor model: Female C57BL/6 mice were implanted subcutaneously with CT-26 cells. When tumors reached ~100 mm³, mice were randomized into treatment groups. diABZI STING agonist-1 was formulated in 10% DMSO/90% PBS and administered via intratumoral, intravenous, or intraperitoneal routes at indicated doses. Tumor volumes were measured twice weekly, and survival was monitored daily[1]. - B16 melanoma model: Mice were inoculated with B16 cells and treated with diABZI STING agonist-1 via intravenous or intraperitoneal routes. Tumor growth was assessed by caliper measurements, and immune cell infiltrates were analyzed by flow cytometry[1].

- Plasma pharmacokinetics: Following intravenous administration in mice, diABZI STING agonist-1 exhibited a half-life (t₁/₂) of 2.1 hours and a volume of distribution (Vd) of 0.8 L/kg. The compound demonstrated moderate plasma protein binding (~75%)[1].
- Tissue distribution: Biodistribution studies revealed significant accumulation of diABZI STING agonist-1 in tumors, spleen, and liver. The compound was rapidly cleared from the bloodstream, with >80% eliminated within 24 hours[1].

- Acute toxicity: Single-dose intravenous administration of diABZI STING agonist-1 in mice up to 100 mg/kg did not result in mortality or significant adverse effects. Clinical signs, body weight, and organ weights were monitored for 14 days post-treatment[1].
- Chronic toxicity: Repeat-dose toxicity studies in rats (daily intraperitoneal injections for 28 days) showed no dose-limiting toxicities. Hematology, clinical chemistry, and histopathology analyses revealed no significant abnormalities[1].

[1]. Design of amidobenzimidazole STING receptor agonists with systemic activity. Nature. 2018 Dec;564(7736):439-443.

Stimulator of interferon genes (STING) is a receptor in the endoplasmic reticulum that propagates innate immune sensing of cytosolic pathogen-derived and self DNA. The development of compounds that modulate STING has recently been the focus of intense research for the treatment of cancer and infectious diseases and as vaccine adjuvants. To our knowledge, current efforts are focused on the development of modified cyclic dinucleotides that mimic the endogenous STING ligand cGAMP; these have progressed into clinical trials in patients with solid accessible tumours amenable to intratumoral delivery3. Here we report the discovery of a small molecule STING agonist that is not a cyclic dinucleotide and is systemically efficacious for treating tumours in mice. We developed a linking strategy to synergize the effect of two symmetry-related amidobenzimidazole (ABZI)-based compounds to create linked ABZIs (diABZIs) with enhanced binding to STING and cellular function. Intravenous administration of a diABZI STING agonist to immunocompetent mice with established syngeneic colon tumours elicited strong anti-tumour activity, with complete and lasting regression of tumours. Our findings represent a milestone in the rapidly growing field of immune-modifying cancer therapies.[1]

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- Mechanism of action: diABZI STING agonist-1 is a small-molecule STING agonist that binds to the ligand-binding domain of STING, triggering conformational changes that activate the TBK1-IRF3 signaling pathway, leading to the production of type I interferons and pro-inflammatory cytokines[1].
- Development rationale: The compound was designed to overcome limitations of cyclic dinucleotide (CDN) STING agonists, such as poor stability and systemic delivery challenges. Its amidobenzimidazole scaffold provides enhanced potency, selectivity, and oral bioavailability[1].
- Preclinical efficacy: diABZI STING agonist-1 demonstrated robust antitumor activity in multiple syngeneic mouse models, both as monotherapy and in combination with immune checkpoint inhibitors[1].

C42H51N13O7

849.937247514725

849.403

C, 59.35; H, 6.05; N, 21.42; O, 13.18

2138299-33-7

diABZI STING agonist-1 trihydrochloride;2138299-34-8;diABZI STING agonist-1 (Tautomerism);2138498-18-5

131986624

Typically exists as White to off-white solids

1.9

4

12

18

62

1570

0

CCN1C(=CC(=N1)C)C(=O)NC2=NC3=C(N2C/C=C/CN4C5=C(C=C(C=C5OCCCN6CCOCC6)C(=O)N)N=C4NC(=O)C7=CC(=NN7CC)C)C(=CC(=C3)C(=O)N)OC

JGLMVXWAHNTPRF-CMDGGOBGSA-N

InChI=1S/C42H51N13O7/c1-6-54-31(19-25(3)49-54)39(58)47-41-45-29-21-27(37(43)56)23-33(60-5)35(29)52(41)12-8-9-13-53-36-30(46-42(53)48-40(59)32-20-26(4)50-55(32)7-2)22-28(38(44)57)24-34(36)62-16-10-11-51-14-17-61-18-15-51/h8-9,19-24H,6-7,10-18H2,1-5H3,(H2,43,56)(H2,44,57)(H,45,47,58)(H,46,48,59)/b9-8+

1-[(E)-4-[5-carbamoyl-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]-7-(3-morpholin-4-ylpropoxy)benzimidazol-1-yl]but-2-enyl]-2-[(2-ethyl-5-methylpyrazole-3-carbonyl)amino]-7-methoxybenzimidazole-5-carboxamide

diABZI STING agonist-1; 2138498-18-5; diABZI STING agonist-3; 2138299-33-7; STING agonist 3; diABZI STING agonist-1; 2138498-18-5; diABZI STING agonist-3; 2138299-33-7; Tautomerism; STING agonist 3; diABZI STING agonist-1 (Tautomerism); L7DUG75C36; diABZI STING agonist-1 (Tautomerism); diABZI STING agonist-1 Tautomerism; L7DUG75C36;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~100 mg/mL (~117.66 mM)

Solubility in Formulation 1: ≥ 3.5 mg/mL (4.12 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 35.0 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: ≥ 3.5 mg/mL (4.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 35.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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