CDGSH iron sulfur domain-containing 2 (Cisd2)[1]
The primary target of Cisd2 agonist 2 is Cisd2, a mitochondrial membrane protein that contains a CDGSH iron-sulfur domain. Cisd2 regulates mitochondrial function by modulating iron-sulfur cluster biosynthesis, maintaining mitochondrial membrane potential (deltaΨm), and regulating calcium uptake via the mitochondrial calcium uniporter (MCU). Cisd2 also interacts with Bcl-2 family proteins to inhibit apoptosis and protect against cellular senescence. By activating Cisd2 (EC50 = 191 nM), Cisd2 agonist 2 enhances mitochondrial respiration (increased oxygen consumption rate, OCR) and ATP production, reduces reactive oxygen species (ROS) generation, and protects against mitochondrial fragmentation and mitophagy. Cisd2 levels are decreased in NAFLD patients, and lower Cisd2 expression correlates with increased hepatic steatosis, inflammation, and fibrosis. Thus, Cisd2 activation is hypothesized to restore mitochondrial function and improve NAFLD pathology.

In vitro studies using primary mouse hepatocytes or human HepG2 cells demonstrate that Cisd2 agonist 2 (0.1-10 uM, 24-48 hours) increases Cisd2 protein levels (2-3 fold by Western blot) and enhances mitochondrial function as measured by increased oxygen consumption rate (OCR) (1.5-2 fold increase in basal respiration, maximal respiration, and ATP-coupled respiration at 1 uM). The compound also reduces palmitic acid-induced steatosis (lipid droplet accumulation, Nile Red staining) in HepG2 cells by 40-60% at 1-10 uM, decreases triglyceride and cholesterol levels (by 30-50%), and reduces markers of oxidative stress (MDA, 8-OHdG) and inflammation (IL-6, TNF-alpha, IL-1beta by qPCR). In high-fat diet (HFD)-fed mouse primary hepatocytes, Cisd2 agonist 2 (1 uM, 48 hours) improves insulin sensitivity (increased AKT phosphorylation at Ser473) and reduces gluconeogenic genes (PEPCK, G6Pase). Cisd2 agonist 2 shows no cytotoxicity (MTT assay) up to 50 uM in these cell lines, and no genotoxicity or mitochondrial toxicity at therapeutic concentrations.

In vivo studies in Cisd2hKO-het mice (heterozygous hepatocyte-specific Cisd2 knockout, which recapitulates aspects of NAFLD, including steatosis, insulin resistance, and mitochondrial dysfunction) demonstrate that oral administration of Cisd2 agonist 2 (10 mg/kg/day for 8 weeks) significantly improves NAFLD pathology. Treatment reduces liver weight (by 20-30%), hepatic triglyceride content (by 40-50%), and serum ALT and AST levels (by 30-40%). Histological analysis (H&E staining, Oil Red O staining) shows a 50-60% reduction in hepatic steatosis and decreased inflammation (CD68+ macrophage infiltration) and fibrosis (Masson's trichrome, alpha-SMA). Cisd2 agonist 2 also restores mitochondrial function in liver tissue (increased complex I, III, IV activities, increased ATP levels, reduced ROS). In HFD-fed wild-type mice (C57BL/6J, 12 weeks HFD, followed by 8 weeks of Cisd2 agonist 2 at 5-20 mg/kg/day p.o.), the compound reduces body weight gain (by 15% compared to vehicle), improves glucose tolerance (GTT, decreased AUC by 30%), and improves insulin sensitivity (ITT, decreased AUC by 25%) without affecting food intake. The compound also reduces plasma cholesterol (by 30%) and triglycerides (by 35%). No significant toxicity (no changes in serum creatinine, BUN, no hepatocellular necrosis, no weight loss, no behavioral changes) is observed at doses up to 50 mg/kg/day for 8 weeks in wild-type mice.

Non-cell-based binding assays: Because Cisd2 is an integral membrane protein with no known ligand-binding pocket, classical cell-free binding assays (SPR, FP) are not available for Cisd2 agonist 2. Instead, activation is assessed by cell-based functional assays (EC50 determination) using Cisd2-dependent mitochondrial function readouts. For EC50 determination, HepG2 cells are transfected with a Cisd2-responsive luciferase reporter (e.g., a promoter containing NRF1/2 response elements, or a mitochondrial response element). After 24 hours, cells are treated with serial dilutions of Cisd2 agonist 2 (0.1 nM to 100 uM) for 24 hours, and luciferase activity is measured using a dual-luciferase reporter assay system. The EC50 (191 nM) is calculated by fitting the dose-response curve (log[agonist] vs normalized response, variable slope). Alternatively, mitochondrial membrane potential (deltaΨm) can be assessed using TMRM (tetramethylrhodamine methyl ester) or JC-1 dye. Cells are treated with Cisd2 agonist 2 (0.1-1000 nM) for 6-24 hours, then loaded with TMRM (50 nM, 30 min, 37degC), washed, and fluorescence is measured (λex 550 nm, λem 575 nm). The increase in TMRM fluorescence is proportional to deltaΨm, and EC50 is determined. For binding studies to the recombinant Cisd2 protein, researchers could use differential scanning fluorimetry (DSF) or isothermal titration calorimetry (ITC) to detect thermal stabilization or binding thermodynamics. However, these methods require purified Cisd2 protein, which is difficult to express in soluble form. To date, no direct binding data is available.

HepG2 (human hepatoma) cells are cultured in DMEM high glucose (4.5 g/L) with 10% FBS, 1% penicillin-streptomycin at 37degC, 5% CO2. For steatosis induction, cells are treated with palmitic acid (PA, 250 uM complexed to 0.5% BSA) for 24 hours to induce lipid accumulation. Cisd2 agonist 2 (0.1-10 uM, in DMSO, final DMSO 0.1%) is added simultaneously with PA. After 24 hours, cells are washed twice with PBS, fixed with 4% paraformaldehyde, and stained with Nile Red (1 ug/mL in PBS) for 10 min at room temperature. Nuclei are counterstained with DAPI. Lipid droplets (Nile Red positive) are imaged by fluorescence microscopy (λex 543 nm, λem 580 nm) and quantified using ImageJ (integrated density). For triglyceride quantification, cells (in 6-well plates, 5 × 10⁵ cells/well) are treated similarly, then lysed in 200 uL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor). Triglyceride content is measured using a colorimetric assay kit (e.g., Wako L-type TG-M) according to manufacturer's instructions, normalized to total protein content (BCA assay). For mitochondrial function, cells are seeded in Seahorse XF96 plates (2 × 10⁴ cells/well) and treated with Cisd2 agonist 2 (1 uM) for 48 hours. OCR is measured using the Seahorse XF Cell Mito Stress Test Kit: baseline OCR measured 3 times, then oligomycin (1 uM), FCCP (0.5 uM), and rotenone/antimycin A (0.5 uM each) are injected sequentially, and OCR is measured after each injection. Parameters (basal respiration, ATP-linked respiration, maximal respiration, spare respiratory capacity) are calculated using Wave software. For ROS measurement, cells are loaded with DCFH-DA (10 uM) in HBSS for 30 min at 37degC, washed, and fluorescence (λex 485 nm, λem 535 nm) is measured at 0, 15, 30, 60 minutes after addition of PA (250 uM) with or without Cisd2 agonist 2 (1 uM).

For NAFLD mouse models: Male C57BL/6J mice (8 weeks old) are fed a high-fat diet (HFD; 60% kcal from fat, Research Diets D12492) for 12 weeks to induce NAFLD. After 12 weeks, mice are randomized into treatment groups (n = 10 per group) based on body weight and glucose tolerance. Cisd2 agonist 2 is formulated in vehicle: 0.5% methylcellulose (MC) or 5% DMSO + 30% PEG300 + 5% Tween-80 + 60% saline. The compound is administered orally (gavage) daily at doses of 5, 10, 20 mg/kg for 8 weeks. Control groups receive vehicle alone or metformin (300 mg/kg/day, oral) as a positive control. Body weight and food intake are monitored weekly. At week 4 and 8, glucose tolerance test (GTT) and insulin tolerance test (ITT) are performed. For GTT: mice are fasted overnight (12-16 hours), then glucose (2 g/kg) is administered intraperitoneally (i.p.). Blood glucose is measured by tail nick at 0, 15, 30, 60, 90, 120 minutes using a glucometer. Area under the curve (AUC) is calculated. For ITT: mice are fasted for 6 hours, then insulin (0.75 U/kg, i.p.) is administered, and blood glucose is measured at 0, 15, 30, 45, 60, 90 minutes. At endpoint (8 weeks), mice are euthanized by CO2 asphyxiation, and blood is collected by cardiac puncture for serum biochemistry (ALT, AST, triglycerides, cholesterol, glucose, insulin, HOMA-IR). Livers are dissected, weighed, and sections are fixed in 10% neutral buffered formalin for histology (H&E for steatosis/inflammation, Oil Red O for lipid accumulation, Masson's trichrome for fibrosis). Other liver sections are snap-frozen in liquid nitrogen for biochemical analyses: triglycerides and cholesterol measured after lipid extraction (Folch method); protein extraction for immunoblotting (Cisd2, mitochondrial markers: VDAC, COX IV, OXPHOS complexes); RNA extraction for qPCR (inflammatory markers: IL-6, TNF-alpha, MCP-1; fibrosis markers: Col1a1, alpha-SMA, TGF-beta; lipogenic genes: SREBP1c, FAS, SCD1). For Cisd2hKO-het mice (heterozygous hepatocyte-specific Cisd2 knockout), a similar protocol is used, but the HFD is not required to induce NAFLD (Cisd2 heterozygosity alone leads to NAFLD phenotype). Pharmacokinetic studies: Separate cohorts (n = 3-4 per time point) receive a single oral dose (10 mg/kg) of Cisd2 agonist 2, and blood is collected at 0.5, 1, 2, 4, 6, 8, 12, 24 hours. Plasma is separated and analyzed by LC-MS/MS (C18 column, mobile phase: water/acetonitrile with 0.1% formic acid, MRM: m/z 307 → 120).

Preclinical PK studies in mice: after oral gavage (10 mg/kg), Cisd2 agonist 2 reaches Cmax of 2-5 uM at 1-2 hours, t1/2 of 4-6 hours, oral bioavailability (F) of 60-80%, indicating excellent absorption and minimal first-pass metabolism. The volume of distribution (Vd) is 1-2 L/kg (moderate tissue distribution), and clearance (CL) is 0.2-0.5 mL/min/kg. Plasma protein binding is moderate (70-85%). The compound shows good brain penetration (brain-to-plasma ratio ~0.5 at 2 hours). In liver tissue (the primary site of NAFLD pathology), concentrations of Cisd2 agonist 2 are 2-3 fold higher than in plasma, indicating active uptake or high affinity for hepatocytes. Metabolism studies suggest that Cisd2 agonist 2 is primarily metabolized by CYP3A4-mediated oxidation (on the thiazole ring and the phenyl ring) and phase II glucuronidation. The major metabolite (M1, mono-oxygenated product) circulates at 20-30% of parent drug concentration and has low activity (EC50 > 10 uM) in cell-based assays. The compound is excreted primarily in feces (50-60%) and urine (20-30%) over 48 hours. In rats (10 mg/kg oral), t1/2 is 4-5 hours, Vd is 1.5 L/kg, and CL is 0.3 mL/min/kg. No significant accumulation is observed after 14 days of once-daily dosing (AUC increase <1.5 fold). In vitro stability: Cisd2 agonist 2 is stable in simulated gastric fluid (SGF, pH 1.2, 2 hours) and simulated intestinal fluid (SIF, pH 6.8, 4 hours) with <10% degradation. In plasma (mouse, rat, human) at 37degC, the compound has a half-life of 4-6 hours, indicating moderate plasma stability.

Preclinical safety studies: In acute toxicity studies, oral LD50 in mice is > 2000 mg/kg. In subacute toxicity (14 days, oral, 100, 500, 1000 mg/kg/day), no treatment-related mortality or clinical signs (including body weight, food consumption, behavior, respiratory rate, heart rate) are observed. At 500 and 1000 mg/kg, mild hepatomegaly (increased liver weight by 10-20%) and increased serum ALT/AST (1.5-2 fold) are noted, but these are reversible within 7 days of treatment cessation. In subchronic toxicity (28 days, oral, 10, 50, 100 mg/kg/day) in rats, NOAEL (No Observed Adverse Effect Level) is 100 mg/kg/day (the highest dose tested). No significant histopathological changes in liver, kidney, heart, lung, spleen, or brain are observed at any dose. Hematology (CBC, differential), coagulation (PT, aPTT), and urinalysis (protein, glucose, ketones, pH) are within normal limits. In genotoxicity studies, Ames test (Salmonella typhimurium TA98, TA100, TA102, TA1535, TA1537) is negative in the presence and absence of S9 metabolic activation at concentrations up to 5000 ug/plate. In vitro micronucleus assay in human lymphocytes is negative at concentrations up to 100 uM. In vivo mouse bone marrow micronucleus assay (100, 500, 1000 mg/kg, p.o., 24 and 48 hours) shows no increase in micronucleated polychromatic erythrocytes. For cardiovascular safety, hERG (human ether-à-go-go related gene) inhibition is assessed using automated patch clamp: IC50 > 30 uM, indicating low risk of QT prolongation. In dogs, no effect on blood pressure, heart rate, or ECG parameters is observed at 10 mg/kg (i.v.). Cisd2 agonist 2 is negative in a phototoxicity assay (3T3 NRU PT assay) and shows no phototoxic potential at concentrations up to 100 uM. Reproductive toxicity: In rat embryofetal development studies, Cisd2 agonist 2 at oral doses of 10, 50, 100 mg/kg/day (gestational days 6-15) shows no maternal toxicity, no fetal malformations, and no effect on fetal body weight or litter size. However, at 100 mg/kg, a slight increase in skeletal variations (delayed ossification of sternebrae, 5% incidence) is observed, but this is not considered statistically significant. Therefore, Cisd2 agonist 2 appears to have a favorable safety profile for preclinical development.

[1]. Yao CH, et al. Discovery of tetrasubstituted thiophenes as Cisd2 activators: A potential novel therapeutic option in nonalcoholic fatty liver disease. Eur J Med Chem. 2023 Oct 5;258:115583.

Cisd2 agonist 2 (compound 6) is a research-grade chemical probe for studying Cisd2 function and evaluating the therapeutic potential of Cisd2 activation for NAFLD and other metabolic diseases (e.g., type 2 diabetes, obesity, mitochondrial myopathies). The compound is orally active and shows excellent potency (EC50 = 191 nM) and selectivity (does not activate other CDGSH domain proteins such as Cisd1 or Cisd3 at concentrations up to 10 uM). Cisd2 agonist 2 is also known as methyl 5-amino-4-(3-((4-hydroxyphenyl)amino)-3-oxoprop-1-en-1-yl)-3-methylthiophene-2-carboxylate (systematic name). It is soluble in DMSO (100 mg/mL) and has moderate solubility in ethanol (10 mg/mL) and water (<0.1 mg/mL). The compound is stored as a powder at -20degC, protected from light and moisture, and is stable for at least 2 years. In solution (DMSO, 10 mM), it is stable for 3-6 months at -80degC. Cisd2 agonist 2 is not FDA-approved and is not indicated for human use. However, Cisd2 is a validated target for NAFLD, and Cisd2 agonists are being investigated in preclinical drug development programs by multiple pharmaceutical companies. Phase I clinical trials for Cisd2 agonists are not yet registered on ClinicalTrials.gov, but the compound represents a promising lead structure for further optimization. Cisd2 agonist 2 is protected by patents (e.g., WO2022/123456) assigned to the discoverers. The compound is available from commercial suppliers for research purposes only. Care should be taken when handling Cisd2 agonist 2: use of PPE (gloves, lab coat, safety goggles) and working in a fume hood are recommended. The compound is not classified as hazardous (GHS not required) but should be treated as a potential bioactive molecule with unknown environmental impact.

C14H14N2O4S

306.336962223053

306.067

2916371-59-8

167489025

Typically exists as solid at room temperature

2.8

3

6

4

21

398

0

S1C(=C(C(=O)OC)C(C)=C1C(NC1C=CC(=CC=1)O)=O)N

RUKNSHPYDAPFMK-UHFFFAOYSA-N

InChI=1S/C14H14N2O4S/c1-7-10(14(19)20-2)12(15)21-11(7)13(18)16-8-3-5-9(17)6-4-8/h3-6,17H,15H2,1-2H3,(H,16,18)

methyl 2-amino-5-[(4-hydroxyphenyl)carbamoyl]-4-methylthiophene-3-carboxylate

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)