STING
SP-23 targets STING (stimulator of interferon genes), a key adaptor protein in the innate immune response to cytosolic DNA. STING activation triggers the production of type I interferons and pro-inflammatory cytokines. By targeting STING for degradation via the PROTAC mechanism (proteolysis-targeting chimera), SP-23 reduces STING protein levels and attenuates STING-mediated inflammatory signaling. The compound has a DC50 of 3.2 μM for STING degradation.

SP23 demonstrated the highest STING-degrading effect with a DC50 of 3.2 μM. Western blot analysis confirmed that SP23 induced the degradation of STING via the PROTAC mechanism, which is the proteasome-dependent pathway.In addition, the STING/TBK1/NF-κB signaling pathway was downregulated in THP-1 cells.[1]
In vitro, SP-23 degrades STING with a DC50 of 3.2 μM. It is a novel, first-in-class STING-targeting PROTAC degrader based on C-170 (STING inhibitor). The compound's ability to degrade STING rather than merely inhibit it makes it a valuable tool for studying STING biology and inflammation. It shows anti-inflammatory activity in cell-based assays.

SP23 exhibited high in vivo anti-inflammatory efficacy in a cisplatin- AKI mouse model and effectively protected mouse kidneys from cisplatininduced injury in vivo. Mechanistically, SP23 inhibited the downstream signals of the STING signaling pathway in vivo, which is consistent with the results from in vitro experiments.SP23 also demonstrated benign toxicity profiles in the AKI mouse model.[1]
In vivo, SP-23 exerts high anti-inflammatory efficacy in a cisplatin-induced acute kidney injury mouse model by modulating the STING signaling pathway. By degrading STING, the compound reduces STING-mediated inflammatory responses and protects against kidney injury. However, specific dosing and efficacy details have not been extensively detailed in the available literature. Further preclinical studies would be required.

Enzyme-linked Immunosorbent Assay. Culture medium supernatant and serum from mouse eyeball blood samples were collected. Levels of mouse and human IFN-β, CXCL10 and IL-6 were determined by enzyme-linked immunosorbent assay kits (B Neobioscience) according to the product manual.[1]
For in vitro degradation assays, cells are treated with SP-23 at concentrations ranging from 0.1-100 µM for 4-24 hours. STING protein levels are assessed by Western blotting. DC50 values (concentration for 50% degradation) are calculated from dose-response curves.

Cytotoxicity (MTT) Assay. The newly synthesized compounds SP21-23 were assessed for their cytotoxicity against various normal cell lines, including human normal hepatocyte (LO2), human embryonic kidney cells (HEK293A) and mouse neural stem cells (C17.2), using the MTT assay. Cells were seeded into 96-well plates at a density of 5000 cells/well. After treatment with compounds SP21-23 at the indicated concentration, MTT (5 mg/mL in PBS) was added and the cells were incubated for another 4 h. Cell viability was detected with a microplate reader at a wavelength of 570 nm.[1]
For cell-based assays, immune cells or other relevant cell types are treated with SP-23 at concentrations ranging from 0.1-100 µM. STING degradation is confirmed by Western blotting. Inflammatory cytokine production (such as IFN-β, IL-6, TNF-α) is measured by ELISA. Cell viability is assessed to confirm that effects are not due to cytotoxicity.

In Vivo Anti-inflammatory Efficacy Study.
Eight-week-old C57BL/6J male mice were purchased from Guangzhou Southern Medical University Experimental Animal Technology Development Co, certificate number: 110324201104674226. All experimental procedures were approved by Ethical Committee at SMU (Southern Medical University) and implemented accordingly. After 24 h of diurnal circulation and free diet and water, mice were randomly assigned to the following four groups: control group and cisplatin induction model group (25 mg/kg), low-dose treatment group (SP23, 30 mg/kg), and high-dose treatment group (SP23, 60 mg/kg). Mice were pretreated with SP23 administered intraperitoneally 1 h prior to cisplatin injection and administered continuously at the same time each day. Seventy-two hours after cisplatin treatment, all mice were euthanized, blood samples and heart, liver, and kidney tissues were collected. One sample from each tissue in each group was randomly selected and fixed in 4% paraformaldehyde for histological examination, and the remaining tissues were immediately frozen in liquid nitrogen and stored at -80°C. Urea nitrogen (BUN) and creatinine concentrations were determined at the Guangzhou Huayin Medical Laboratory Center using a serum biochemical auto-analyzer. Renal imaging was monitored by the Small Animal Magnetic Resonance Imaging System (PharmaScan70/16, USA) at the Southern Medical University Experimental Center.[1]
Pharmacokinetic Study in Male SD Rats.
Male SD rats (300-360 g) were purchased from Liaoning Changsheng Biotechnology Co., Ltd. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.5 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.0833, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24, 36, and 48 h after oral (20 mg/kg) or intravenous (2 mg/kg) administration of compound SP23. Waters Acquity UPLC (Waters Corp., Milford, MA, USA) was used for the separation of SP23 and internal standard compound. The effective separation of SP23 and internal standard compound was achieved on Acquity UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm, Waters Corporation, Milford, MA, USA) used at a column temperature of 40 °C. The mobile phase consisted of a mixture of solvent A (acetonitrile) and solvent B (formic acid/ ultrapure water, 1:1000, v/v). The linear gradient program for the mobile phase was set as follows: 0 min 10% A; 0.5 min 30% A; 1 min 95% A; 2 min 95% A; 2.3 min 10% A. The flow rate of the mobile phase was 0.4 mL/min and the injection volume was 2 μL. The XEVO TQD triple quadrupole mass spectrometer was equipped with an electrospray ionization (ESI) source; multiple reaction monitoring (MRM) mode was selected for quantitation. The Mass Lynx 4.1 software (Waters Corp.) was used for data acquisition. Mass spectral data were obtained in positive electrospray mode (ESI+) in MRM mode.
For in vivo efficacy studies, the cisplatin-induced acute kidney injury mouse model is used. Mice are treated with SP-23 via appropriate routes at doses determined from pharmacokinetic studies. Kidney injury markers (such as BUN and creatinine), inflammatory cytokine levels, and STING protein levels in kidney tissues are assessed.

SP-23 is soluble in DMSO (25 mg/ml). Storage is recommended at -20°C for long-term stability. Further detailed PK parameters including half-life, oral bioavailability, and tissue distribution would be required for in vivo studies. The compound is for research use only and is not intended for human or veterinary use.

Toxicological data for SP-23 have not been extensively reported. As a research compound, it is intended for laboratory use only and is not for human or veterinary use. Standard safety precautions should be followed when handling this compound. Comprehensive toxicology studies would be required before any clinical development.

[1]. Novel CRBN-Recruiting Proteolysis-Targeting Chimeras as Degraders of Stimulator of Interferon Genes with In Vivo Anti-Inflammatory Efficacy. J Med Chem. 2022 May 12;65(9):6593-6611.

SP-23 (SP23) is a novel, first-in-class STING-targeting PROTAC degrader with a DC50 of 3.2 μM. It exerts high anti-inflammatory efficacy in a cisplatin-induced acute kidney injury mouse model. SP-23 is a research tool for studying STING biology and inflammation and is not approved for clinical use.

C34H33N7O10

699.666727781296

699.23

C, 58.37; H, 4.75; N, 14.01; O, 22.87

2762552-74-7

164886634

Light yellow solid

2.9

5

11

14

51

1390

0

C1CC(=O)NC(=O)C1N2C(=O)C3=C(C2=O)C(=CC=C3)NCCCCCCNC(=O)/C=C/C(=O)NC4=CC=C(C=C4)NC(=O)C5=CC=C(O5)[N+](=O)[O-]

TXNQXRGOKABDOB-FOCLMDBBSA-N

InChI=1S/C34H33N7O10/c42-26(15-16-27(43)37-20-8-10-21(11-9-20)38-32(46)25-13-17-29(51-25)41(49)50)36-19-4-2-1-3-18-35-23-7-5-6-22-30(23)34(48)40(33(22)47)24-12-14-28(44)39-31(24)45/h5-11,13,15-17,24,35H,1-4,12,14,18-19H2,(H,36,42)(H,37,43)(H,38,46)(H,39,44,45)/b16-15+

(E)-N-[6-[[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino]hexyl]-N'-[4-[(5-nitrofuran-2-carbonyl)amino]phenyl]but-2-enediamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~142.92 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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