ASK1 (IC50 = 3 μM)
The primary target of NQDI-1 is ASK1 (apoptosis signal-regulating kinase 1), a MAP3K that plays a critical role in stress-induced apoptosis and inflammation. ASK1 is activated by various stress stimuli, including oxidative stress, TNF-α, and endoplasmic reticulum stress. Upon activation, ASK1 phosphorylates and activates downstream kinases, including MKK4/MKK7 (which activate JNK) and MKK3/MKK6 (which activate p38 MAPK). By inhibiting ASK1, NQDI-1 blocks the activation of these downstream pathways, reducing apoptosis and inflammation. The compound's selectivity for ASK1 over other MAP3Ks contributes to its specificity.

The selectivity of NQDI-1 is examined in vitro using four serine/threonine protein kinases (protein kinase CK2, c-Jun N-terminal kinase 3, Rho-associated protein kinase 1, and Aurora A), and three tyrosine protein kinases (FGFR1, hHGFR, and endothelial TEK tyrosine kinase, or Tie2). The outcomes demonstrate that NQDI-1 is a specific ASK1 inhibitor. NQDI-1 inhibits the activity of FGFR1 protein kinase (44% residual activity). With a Ki of 500 nM, NQDI-1 blocks ASK1. When compared to the phosphodonor substrate ATP, NQDI-1's inhibition of ASK1 is competitive[1].
In vitro, NQDI-1 is a potent and selective inhibitor of ASK1. Its activity is typically measured using kinase assays with purified ASK1 enzyme or cell lysates. The compound's inhibitory activity is measured by monitoring the phosphorylation of a substrate peptide or protein in the presence of ATP. IC50 values are determined from dose-response curves. The compound's selectivity for ASK1 over other kinases, including other MAP3Ks, is assessed to confirm its specificity. NQDI-1 has been shown to inhibit ASK1-mediated apoptosis and JNK/p38 activation in various cell types.

Following a brain injury, 250 nmol NQDI-1 in DMSO is injected intracerebrally. Western blotting is used to analyze the expression of ASK1 in the hypoxia-ischemia (HI), DMSO, NQDI-1, and sham groups.Results show that NQDI-1 significantly inhibits ASK1 expression in the brain cortex when compared to the HI and DMSO groups. Furthermore, immunofluorescence staining shows that NQDI-1 inhibits ASK1 expression in the cortex of the brain. The current study also identifies the expression of ASK1's downstream targets. When compared to the HI and DMSO groups, NQDI-1 significantly lowers the expression levels of p-JNK, p-c-Jun, p53, and caspase 3. In the group that received NQDI-1 treatment, immunofluorescence also revealed low expression of p-JNK in the cerebral cortex[2].
In vivo, NQDI-1 has been studied for its potential therapeutic applications in neurodegenerative diseases, cardiovascular diseases, and inflammatory conditions. By inhibiting ASK1, the compound reduces apoptosis and inflammation, which are key pathogenic mechanisms in these diseases. NQDI-1 has been shown to be neuroprotective in models of cerebral ischemia and neurodegenerative diseases. It has also been shown to reduce cardiac injury in models of myocardial ischemia and to attenuate inflammation in models of inflammatory diseases. While specific in vivo efficacy data are not extensively detailed in the available literature, its mechanism of action supports its potential therapeutic applications.

In vitro kinase assay (γ-32P-ATP method) is used to assess the enzyme activity of human protein kinases ASK1, Aurora A, ROCK1, HGFR, FGFR1, Tie2, JNK3, and CK2. Each reaction mixture contains 6 μL of buffer solution (25 mM MOPS, pH 7.2, 2.5 mM EGTA, 2.5 mM EDTA, 0.5 mM DTT, 0.25 mg/mL BSA, 20 mM β-glycerophosphate), 3 μL of substrate solution (MBP, Long S6 kinase substrate peptide, KKKSPGEYVNIEFG, IGF-IRtide (12-527), TK substrate 2, JNK3tide, or RRRDDDSDDD for each kinase, respectively) (5.0 μg/μL), 0.3 μL of enzyme (protein kinase catalytic subunit, 0.1 μg/μL≈32 mU/μL), and 10.25 μL of H2O. At room temperature, the reaction mixture (19 μL total volume) is swiftly added to 1.5 mL tubes. Stock solutions of inhibitors, like NQDI-1, are made in DMSO with a 1 mM inhibitor concentration. In the reaction, the amount of DMSO is not greater than 3%. After that, each tube receives 1 L of an inhibitor solution in DMSO, which is pipetted into each tube and mixed. A separate ATP solution is made. 0.05 mCi of γ-[P32]ATP is extracted for each sample (specific activity of 100 μCi/M). 100 μM is the combined concentration of labeled and unlabeled ATP. The 150 μM ATP, 30 mM MgCl2, 15 mM MOPS, pH 7.2 ATP solution is added to start the reaction. The reaction lasts 20 minutes at 30 °C. 20 μL of 0.5 M orthophosphoric acid are added to stop the reaction. Following that, the reaction mixture is loaded onto the cellulose phosphate paper 20 mm filter disks. At room temperature, filters are washed three times with 0.075 M orthophosphoric acid, then dried. By using a Tri-Carb 2800-TR liquid scintillation analyzer, dried filters are counted in order to detect products. A positive control is then performed by substituting 1 μL of DMSO for the inhibitor stock solution in the reaction volume[1].
In vitro enzyme/receptor binding assays for NQDI-1 involve kinase activity assays using purified ASK1 enzyme or cell lysates. The compound's inhibitory activity is measured by monitoring the phosphorylation of a substrate peptide or protein, such as MKK4 or MKK6, in the presence of ATP. IC50 values are determined from dose-response curves. Selectivity profiling against a panel of other kinases, including other MAP3Ks (e.g., MEKK1, MLK3) and downstream kinases (e.g., JNK, p38), is performed to assess the compound's specificity. These assays confirm that NQDI-1 is a potent and selective ASK1 inhibitor.

In vitro cellular assays for NQDI-1 are conducted in cell lines that respond to ASK1-mediated stress signals. Cells are treated with stress stimuli (e.g., H2O2, TNF-α) in the presence or absence of varying concentrations of NQDI-1. ASK1 activation and downstream signaling (JNK and p38 phosphorylation) are assessed by western blotting using phospho-specific antibodies. Apoptosis is evaluated by measuring caspase activation, annexin V staining, or PARP cleavage. Inflammatory cytokine production is measured by ELISA. These assays confirm that NQDI-1 engages its target in a cellular context and produces the expected inhibition of ASK1 signaling, apoptosis, and inflammation.

In vivo animal studies for NQDI-1 are conducted in animal models of neurodegenerative diseases, cardiovascular diseases, and inflammatory conditions. In cerebral ischemia models, animals are subjected to middle cerebral artery occlusion (MCAO) and treated with NQDI-1, and infarct size and neurological deficits are assessed. In myocardial ischemia models, animals are subjected to coronary artery ligation and treated with NQDI-1, and cardiac function and infarct size are assessed. In inflammatory models, disease severity and inflammatory markers are assessed. Pharmacokinetic studies are performed to determine the compound's bioavailability, half-life, and tissue distribution.

Pharmacokinetic properties of NQDI-1 indicate that it has a molecular weight of 358.36 and a molecular formula of C20H16N2O3S. The compound is a small molecule with drug-like physicochemical properties. It is soluble in DMSO, facilitating its use in in vitro assays and formulation for in vivo administration. For storage, the powder should be kept under appropriate conditions to maintain stability. Its physicochemical properties suggest that it has reasonable drug-like characteristics for oral administration.

Toxicological information for NQDI-1 is primarily derived from its use as a research compound in preclinical studies. As an ASK1 inhibitor, potential on-target effects could include reduced apoptosis and inflammation, which may be beneficial in certain disease contexts but could also impair normal cellular turnover and immune responses. Comprehensive toxicology studies would be required for therapeutic development, including assessments of immune function, tissue homeostasis, and organ toxicity.

[1]. Identification of 3H-naphtho[1,2,3-de]quinoline-2,7-diones as inhibitors of apoptosis signal-regulating kinase 1 (ASK1). J Med Chem. 2011 Apr 28;54(8):2680-6.

[2]. NQDI-1, an inhibitor of ASK1 attenuates acute perinatal hypoxic-ischemic cerebral injury by modulating cell death. Mol Med Rep. 2016 Jun;13(6):4585-92.

NQDI-1 is a potent and selective inhibitor of ASK1 (apoptosis signal-regulating kinase 1). It blocks stress-induced apoptosis and inflammation by inhibiting the JNK and p38 MAPK pathways. NQDI-1 has been studied for potential applications in neurodegenerative diseases, cardiovascular diseases, and inflammatory conditions. NQDI-1 is not approved for clinical use and is available from research chemical suppliers for preclinical studies.

C19H13NO4

319.31

319.084

C, 71.47; H, 4.10; N, 4.39; O, 20.04

175026-96-7

175026-96-7

5522952

Yellow to orange solid powder

1.4±0.1 g/cm3

581.4±50.0 °C at 760 mmHg

305.4±30.1 °C

0.0±1.6 mmHg at 25°C

1.685

0.94

1

4

3

24

623

0

O=C1C2=C([H])C([H])=C([H])C([H])=C2C2=C(C(=O)OC([H])([H])C([H])([H])[H])C(N([H])C3=C([H])C([H])=C([H])C1=C23)=O

UFJGFNHRMPMALC-UHFFFAOYSA-N

InChI=1S/C19H13NO4/c1-2-24-19(23)16-15-10-6-3-4-7-11(10)17(21)12-8-5-9-13(14(12)15)20-18(16)22/h3-9H,2H2,1H3,(H,20,22)

ethyl 8,15-dioxo-14-azatetracyclo[7.7.1.02,7.013,17]heptadeca-1(16),2,4,6,9(17),10,12-heptaene-16-carboxylate

NQDI 1; NQDI1; NQDI-1

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: ~21 mg/mL (~65.8 mM)

Solubility in Formulation 1: 0.71 mg/mL (2.22 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 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: 0.71 mg/mL (2.22 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 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%dmso+40%PEG300+5%tween-80+ddH2O: 0.5mg/mL

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