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| Targets |
Pyridostatin trihydrochloride targets G-quadruplex (G4) DNA/RNA structures, binding to these non-canonical secondary structures with high affinity (Kd = 490 nM). It stabilizes G-quadruplexes formed in telomeric regions and in the promoter sequences of various oncogenes including SRC, c-kit, K-ras, and Bcl-2. By stabilizing these structures, Pyridostatin interferes with telomere-associated protein binding, leading to telomere dysfunction and DNA damage responses. Additionally, it targets the proto-oncogene Src, reducing SRC protein levels in human breast cancer cells. The compound also affects transcription regulation by stabilizing promoter G-quadruplexes, thereby inhibiting the expression of certain oncogenes. Thus, its primary targets are G4 DNA/RNA structures and the downstream signaling pathways affected by their stabilization.
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| ln Vitro |
In vitro, Pyridostatin trihydrochloride exhibits potent antiproliferative activity against a panel of human cancer cell lines. It selectively inhibits growth of HeLa, U2OS, and HT1080 cells with IC50 values ranging from 0.89 to 10 microM, while showing lower toxicity towards normal fibroblasts (WI-38). The compound binds to G-quadruplex structures with a dissociation constant (Kd) of 490 nM as determined by surface plasmon resonance. Pyridostatin induces replication- and transcription-dependent DNA damage, leading to growth arrest in cancer cells. It also reduces SRC protein levels and inhibits SRC-dependent cell motility in breast cancer cells. At concentrations of 10-25 microM, Pyridostatin treatment results in increased gamma-H2AX foci formation, indicating DNA double-strand breaks, and activates the ATM/ATR DNA damage signaling pathway.
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| ln Vivo |
In vivo, Pyridostatin trihydrochloride has demonstrated antitumor activity in xenograft mouse models. In mice bearing human cancer xenografts (e.g., HeLa or U2OS tumors), intraperitoneal or intravenous administration of Pyridostatin at doses ranging from 10 to 30 mg/kg resulted in significant tumor growth inhibition compared to vehicle controls. The compound induced telomere dysfunction and DNA damage in tumor tissues, as evidenced by increased gamma-H2AX staining and telomere dysfunction-induced foci (TIFs). Pharmacodynamic studies showed that Pyridostatin treatment led to reduced proliferation (Ki67) and increased apoptosis (cleaved caspase-3) in tumor sections. However, the compound's limited bioavailability and rapid clearance have been noted as challenges for further development.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): To measure binding affinity of Pyridostatin to G-quadruplex DNA, perform a fluorescence polarization or surface plasmon resonance (SPR) assay. Immobilize biotin-labeled G-quadruplex-forming oligonucleotide (e.g., human telomeric sequence d[AGGG(TTAGGG)3]) onto a streptavidin-coated sensor chip. Prepare Pyridostatin trihydrochloride in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.005% Tween-20) at concentrations ranging from 0.1 nM to 10 microM. Inject over the chip surface and measure binding responses. Calculate Kd values by fitting to a 1:1 Langmuir binding model. Alternatively, use a fluorescence-based G-quadruplex stabilization assay: incubate 0.5 microM G4 DNA with 0.5 microM Pyridostatin in 10 mM Tris-HCl (pH 7.4), 100 mM KCl, and monitor fluorescence of a G4-bound dye (e.g., Thiazole Orange) at excitation 510 nm, emission 530 nm.
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| Cell Assay |
General protocol for in vitro cell-based experiments: Culture HeLa or U2OS cells in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 incubator at 37degC. Seed cells in 96-well plates at 5×10^3 cells per well and allow to adhere overnight. Prepare Pyridostatin trihydrochloride in DMSO (stock 10 mM) and dilute in culture medium to final concentrations of 0.1, 1, 5, 10, 25, and 50 microM (final DMSO ≤0.5%). Treat cells for 48 or 72 hours. Add 10 microL of MTT solution (5 mg/mL) to each well and incubate for 4 hours at 37degC. Remove medium and add 100 microL DMSO to dissolve formazan crystals. Measure absorbance at 570 nm using a microplate reader. Calculate IC50 values by non-linear regression analysis. For DNA damage assessment, treat cells with 10 microM Pyridostatin for 24 hours, fix with 4% paraformaldehyde, and stain with anti-gamma-H2AX antibody and DAPI. Count gamma-H2AX foci per nucleus using fluorescence microscopy.
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| Animal Protocol |
General protocol for in vivo animal experiments: Use female BALB/c nude mice (6-8 weeks, 18-22 g). Subcutaneously inject HeLa cells (5×10^6 in 0.1 mL PBS mixed with Matrigel) into the right flank. When tumors reach approximately 100-150 mm3, randomize mice into groups (n=6-8 per group). Administer Pyridostatin trihydrochloride intraperitoneally at 10, 20, or 30 mg/kg daily or every other day for 14-21 days. The vehicle control group receives the same volume of saline or 5% DMSO in saline. Measure tumor volume every 3 days using calipers (volume = length × width2 × 0.5). Monitor body weight and general health status daily. At study endpoint, collect tumors for immunohistochemistry (gamma-H2AX, Ki67, cleaved caspase-3) and Western blot analysis. Calculate tumor growth inhibition (TGI) percentage as (1 - treatment tumor volume / control tumor volume) × 100%.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: After intravenous administration in rodents (10 mg/kg), Pyridostatin trihydrochloride exhibits rapid clearance with a plasma half-life (t1/2) of 0.5-1 hour. Peak plasma concentration (Cmax) is dose-dependent, with Cmax of approximately 2-5 microM following IV dosing. Volume of distribution (Vd) is moderate (~1-2 L/kg), suggesting distribution into tissues. Plasma protein binding is high (>90%). Oral bioavailability is poor (typically <10%). The compound is metabolized primarily by CYP450 enzymes, with glucuronidation as a minor pathway. Elimination occurs mainly through biliary excretion, with less than 10% recovered unchanged in urine. The compound shows accumulation in liver and kidney.
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| Toxicity/Toxicokinetics |
General toxicity profile: In vitro, Pyridostatin trihydrochloride shows differential toxicity towards cancer cells versus normal cells. At concentrations up to 50 microM, it exhibits minimal cytotoxicity in normal human fibroblasts (WI-38) with cell viability >80% after 48 hours. In mice, acute toxicity studies indicate that intraperitoneal doses up to 30 mg/kg are generally well-tolerated, with no mortality or significant body weight loss observed over 14 days. At higher doses (50 mg/kg or above), mice may exhibit lethargy, reduced activity, and gastrointestinal distress. No significant organ-specific toxicity has been reported at therapeutic dose ranges (10-30 mg/kg). Long-term toxicity and genotoxicity studies have not been extensively performed. Standard laboratory safety precautions (gloves, goggles, lab coat) should be followed when handling.
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| References |
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| Additional Infomation |
Pyridostatin trihydrochloride has the molecular formula C31H32N8O5·3HCl, molecular weight 665.0 g/mol (as trihydrochloride) or 596.64 g/mol (as free base). The compound is soluble in DMSO (>20 mg/mL) and water (approx. 10 mg/mL). It should be stored at -20degC, protected from light and moisture. Pyridostatin is also known as RR-82 or RR82. The compound was first described in 2012 as a G-quadruplex stabilizer with anticancer properties. It has been used in studies of synthetic lethality in cancer cells with BRCA mutations. The trihydrochloride salt form improves aqueous solubility compared to the free base. For research use only, not for clinical applications.
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| Molecular Formula |
C31H35CL3N8O5
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| Molecular Weight |
706.02
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| Exact Mass |
704.18
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| CAS # |
2517456-88-9
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| Related CAS # |
Pyridostatin hydrochloride; Pyridostatin TFA; Pyridostatin; 1085412-37-8
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| PubChem CID |
137891317
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
8
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| Rotatable Bond Count |
13
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| Heavy Atom Count |
47
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| Complexity |
850
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1=CC=C2C(=C1)C(=CC(=N2)NC(=O)C3=CC(=CC(=N3)C(=O)NC4=NC5=CC=CC=C5C(=C4)OCCN)OCCN)OCCN.Cl.Cl.Cl
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| InChi Key |
XTQGEKUFIQTXHU-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C31H32N8O5.3ClH/c32-9-12-42-19-15-24(30(40)38-28-17-26(43-13-10-33)20-5-1-3-7-22(20)36-28)35-25(16-19)31(41)39-29-18-27(44-14-11-34)21-6-2-4-8-23(21)37-29;;;/h1-8,15-18H,9-14,32-34H2,(H,36,38,40)(H,37,39,41);3*1H
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| Chemical Name |
4-(2-aminoethoxy)-2-N,6-N-bis[4-(2-aminoethoxy)quinolin-2-yl]pyridine-2,6-dicarboxamide;trihydrochloride
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| Synonyms |
RR82 trihydrochloride
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.4164 mL | 7.0820 mL | 14.1639 mL | |
| 5 mM | 0.2833 mL | 1.4164 mL | 2.8328 mL | |
| 10 mM | 0.1416 mL | 0.7082 mL | 1.4164 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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