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CTPI-2 (CTPI2) is a novel, potent, specific, and third-generation mitochondrial citrate carrier SLC25A1 inhibitor with a KD of 3.5 μM. CTPI-2 inhibits glycolysis, PPARγ, and its downstream target the glucose transporter GLUT4. CTPI-2 halts salient alterations of NASH reverting steatosis, preventing the evolution to steatohepatitis, reducing inflammatory macrophage infiltration in the liver and adipose tissue, and starkly mitigating obesity induced by a high-fat diet.
| Targets |
CTPI-2 targets the mitochondrial citrate carrier protein SLC25A1 (also known as CIC). Binding affinity (KD) for human SLC25A1 is 3.5 µM as determined by surface plasmon resonance (SPR) [2].
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| ln Vitro |
In NSCLC cells, CTPI-2 treatment (1 mM) reduced sphere-forming ability, inhibiting self-renewal of cancer stem cells (CSCs) [2].
In H1299 cells, CTPI-2 inhibited proliferation in a SLC25A1-dependent manner; cells lacking SLC25A1 or expressing the SLC25A1R282G/R285C mutant were resistant to its anti-proliferative effects [2]. CTPI-2 treatment (3 h) reduced mitochondrial respiration (oxygen consumption rate, OCR) in patient-derived NSCLC CSC spheres [2]. CTPI-2 (3 h) increased the NADP+/NADPH ratio and induced mitochondrial superoxide buildup in CSC spheres [2]. CTPI-2 inhibited matrix invasion of T1 and H1299 CSC spheres embedded in a collagen-based matrix [2]. In HepG2 cells, CTPI-2 treatment for 5-12 hours inhibited the expression of fatty acid synthesis genes (FASN, ACACA) and glycolytic/gluconeogenesis genes (AldoA, AldoB), and this inhibition was rescued by the addition of 5 mM sodium citrate [1]. CTPI-2 treatment reduced the levels of citrate and acetyl-CoA in the liver of HFD-fed mice [1]. CTPI-2 repressed pro-inflammatory M1 macrophage markers (TNFα, iNOS) and pro-fibrotic genes (Collagen-1/4, Keratin-19, PDGFR) while increasing anti-inflammatory markers (IL-4, IL-10) and anti-fibrotic Cadherin-1 in HFD-fed mouse livers [1]. CTPI-2 reduced the levels of lipid precursors (palmitic acid, linoleic acid), mono-, di-, and triglycerides, and long-chain free fatty acids in the liver [1]. CTPI-2 inhibited glycolysis in CSC spheres (reduced ECAR and lactate) but increased glycolysis in monolayer cultures [2]. In cisplatin- or AZD9291-resistant patient-derived NSCLC cells (T1, T2, T4), CTPI-2 re-sensitized the cells to these agents in a synergistic manner (R index >1.0, approaching 2.0) [2]. |
| ln Vivo |
The specific glycolysis regulator CTPI-2 restricts the metabolic adaptability of cancer stem cells (CSCs). Non-small cell lung cancer (NSCLC) in vivo model tumor growth is inhibited by intraperitoneal administration of CTPI-2 (26 mg/kg) [1]. ?In a prevention study, CTPI-2 (50 mg/kg; intraperitoneal injection; every other day for 12 weeks) entirely avoided weight gain, and in a reversal study, it significantly reduced weight [2]. ?CTPI-2 restores normal glucose tolerance and avoids steatohepatitis. In addition to boosting anti-inflammatory IL-4 and IL-10, CTPI-2 decreases circulating levels of IL-6 and decreases interferon gamma-induced monocyte chemoattractant protein 1 and monokine that draw neutrophils and monocytes. Citrate depots, adipogenesis, and gluconeogenesis pathways are all regulated by CTPI-2 [2].
In diet-induced obesity (DIO) mice fed a high-fat diet (HFD), CTPI-2 (50 mg/kg, i.p., alternate days) completely prevented weight gain in a prevention study and caused significant weight loss in a reversion study [1]. CTPI-2 reduced white adipose tissue (WAT) and liver mass, reversed hepatomegaly, and reduced liver steatosis and ballooning degeneration in HFD-fed mice [1]. CTPI-2 normalized serum cholesterol, ALT, and triglyceride levels in HFD-fed mice [1]. CTPI-2 normalized fasting glucose levels, improved glucose tolerance (GTT), and restored insulin sensitivity (ITT) in HFD-fed mice in a time-dependent manner [1]. CTPI-2 reduced circulating IL-6 and increased IL-4 and IL-10 in HFD-fed mice [1]. CTPI-2 reduced F4/80-positive Kupffer cells in the liver and resolved crown-like structures of macrophages in adipose tissue of HFD-fed mice [1]. In a xenograft model using T1 NSCLC cells, CTPI-2 (28 mg/kg, i.p., alternate days) more effectively inhibited the growth of sphere-derived tumors compared to monolayer-derived tumors [2]. In combination therapy with cisplatin (3 mg/kg every 3 days) in T1 xenografts, CTPI-2 induced tumor regression, extended tumor doubling times, and reversed cisplatin-enriched stemness markers [2]. In combination with AZD9291 (25 mg/kg every 3 days, oral) in T2 xenografts, CTPI-2 prominently inhibited tumor growth [2]. |
| Enzyme Assay |
Surface plasmon resonance (SPR) was performed on a Biacore 4000 instrument at room temperature. A histidine-tagged recombinant purified human SLC25A1 protein was captured onto an NTA sensor chip surface pre-activated with NiCl2. The running buffer was PBS-P or HBS-P. Small molecule analytes (including CTPI-2) at concentrations of 0, 1.52, 3.63, 6.25, 12.5, 25, 50, and 100 µM were flowed over the immobilized ligand. The flow rate was maintained at 30 µL/min. Data were analyzed using Biacore BiaEvaluation software with a 1:1 steady-state binding model to calculate the KD. The experimental KD for CTPI-2 was determined to be 3.5 µM [2].
An in silico homology model of human SLC25A1 was used for docking studies. Docking poses for citrate, CTPI-1, and CTPI-2 were generated using UCSF Dock6.7 software. Receptor spheres were generated covering the substrate binding site (Site 2). The ligand flexibility option was used, and the top-scoring conformation was minimized to give the final pose. CTPI-2 showed a closer binding mode with key amino acids Arg282, Arg285, and Lys190 compared to CTPI-1 [2]. |
| Cell Assay |
For self-renewal assays, tumor cells cultured as spheroids were dissociated and diluted in 2× sphere media containing 1% methylcellulose. Cells (200, 500, or 1000) were plated per well in 96-well plates and incubated with or without CTPI-2 (concentrations indicated, e.g., 1 mM for CTPI-1 comparison). Spheres were counted after 7-10 days. Six biological replicates were used [2].
For proliferation/cytotoxicity assays, cells were plated in triplicate at 2,000-10,000 cells/well in 96-well plates with or without drug treatment for 48 h. Cells were then washed, fixed with cold methanol, and stained with 0.5% crystal violet. After drying, sodium citrate in 50% ethanol was added, and absorbance was measured at 550 nm (corrected at 405 nm) [2]. For mitochondrial respiration measurement, cells (from spheres or monolayer) were dissociated and plated overnight in DMEM with 5 mM glucose, 1 mM pyruvate, and 2 mM glutamine. The next day, cells were treated with CTPI-2 or vehicle for 3 h. The medium was then replaced with DMEM without FBS or bicarbonate containing the same supplements, and cells were placed in a CO2-free incubator for 1 h. OCR and ECAR were measured using a Seahorse XF96 analyzer. Oligomycin (0.5 µM), FCCP (2 µM), and rotenone/antimycin (0.5 µM each) were sequentially injected [2]. For measurement of gene expression by RT-qPCR, total RNA was isolated using Trizol, treated with DNase I, and reverse transcribed using Superscript IV. Real-time PCR was carried out using PowerUp SYBR Green Master Mix. Gene expression was normalized to GAPDH and SNRPD3, and fold changes were calculated using the ΔΔCT method. Cells were treated with CTPI-2 for 5-12 h with or without 5 mM sodium citrate [1][2]. |
| Animal Protocol |
Animal/Disease Models: C57BL/6J mice (HFD-fed mice) [2]
Doses: 50 mg/kg Route of Administration: Via intraperitonealroute on alternate days for 12 weeks Experimental Results: Complete avoidance of weight gain in prevention study, and resulted in significant weight loss regression studies. For diet-induced obesity (DIO) studies, C57BL/6J mice (4-6 weeks old) were fed a high-fat diet (HFD) with 60% calories from fat. CTPI-2 was administered at 50 mg/kg via intraperitoneal (i.p.) injection on alternate days. For the prevention study, treatment started 3 weeks after HFD initiation. For the reversion study, mice were fed HFD for 3 months before starting CTPI-2 or vehicle for an additional 12 weeks. CTPI-2 was diluted either in DMSO (0.2% final concentration) or in 0.47% sodium bicarbonate (NaHCO3) to a final concentration of 14 mM. Vehicle controls were DMSO or 0.47% NaHCO3, respectively. Food consumption was measured weekly [1]. For xenograft studies in NSCLC, female balb athymic nude mice were injected subcutaneously with 5×10^6 T1 cells (from monolayer or sphere cultures). CTPI-2 was administered at 26-28 mg/kg via i.p. injection on alternate days. For combination therapy, cisplatin was administered at 3 mg/kg every 3-4 days (i.p.), and AZD9291 was administered at 25 mg/kg every 3 days by oral gavage. Tumor volumes were measured with a caliper and calculated using the formula for a prolate spheroid: volume = (4/3) × a² × b. Animals were sacrificed when tumors exceeded 1.5 cm [2]. |
| Toxicity/Toxicokinetics |
CTPI-2 was well tolerated during the entire treatment period in mice (up to 3 months) at doses of 26-50 mg/kg administered on alternate days. No significant weight loss or other overt signs of toxicity were reported. CTPI-2 did not modify body weights in mice fed a normal diet [1][2].
In vitro, CTPI-2 treatment induced a strong build-up of mitochondrial superoxide in CSC spheres, indicating oxidative stress [2]. |
| References |
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| Additional Infomation |
CTPI-2 is a third-generation inhibitor of SLC25A1, discovered by the Avantaggiati laboratory. It provides a major improvement in biological activity over previous inhibitors (BTA and CTPI-1), enhancing binding activity towards SLC25A1 more than 20-fold compared to CTPI-1 (KD 63.6 µM for CTPI-1 vs. 3.5 µM for CTPI-2) and inhibiting citrate transport and tumor proliferation at tenfold lower doses [2].
CTPI-2 acts as a glucose-restriction mimetic agent, reducing glycemia and improving glucose homeostasis [1]. The specificity of CTPI-2 for SLC25A1 was demonstrated by the lack of activity in cells with SLC25A1 knockdown or expressing a citrate-binding mutant (SLC25A1R282G/R285C). Comparative transcriptional and metabolomic profiling showed ~80% overlap between genes regulated by SLC25A1 shRNA and CTPI-2 treatment [1][2]. CTPI-2 is synthetic lethal with cisplatin or EGFR inhibitor (AZD9291) co-treatment, restoring antitumor responses in resistant NSCLC models [2]. |
| Molecular Formula |
C13H11N2O6SCL
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|---|---|
| Molecular Weight |
358.75424
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| Exact Mass |
355.987
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| CAS # |
68003-38-3
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| Related CAS # |
68003-38-3;
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| PubChem CID |
106350
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.66g/cm3
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| Boiling Point |
564.8ºC at 760 mmHg
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| Flash Point |
295.4ºC
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| Index of Refraction |
1.678
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| LogP |
4.424
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
23
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| Complexity |
558
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
NJTHPOSQGFJTDP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H9ClN2O6S/c14-10-6-5-8(7-12(10)16(19)20)23(21,22)15-11-4-2-1-3-9(11)13(17)18/h1-7,15H,(H,17,18)
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| Chemical Name |
2-[(4-chloro-3-nitrophenyl)sulfonylamino]benzoic acid
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~125 mg/mL (~350.40 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.83 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.83 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7875 mL | 13.9373 mL | 27.8746 mL | |
| 5 mM | 0.5575 mL | 2.7875 mL | 5.5749 mL | |
| 10 mM | 0.2787 mL | 1.3937 mL | 2.7875 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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