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| Targets |
The primary and specific target of DNA2-IN-C5 is the DNA2 nuclease, a multifunctional enzyme that is part of the helicase/endonuclease family. DNA2 plays an essential role in several DNA metabolic pathways, including Okazaki fragment maturation during DNA replication, end resection during homologous recombination repair, and the maintenance of telomere integrity. Its nuclease activity is crucial for processing DNA flaps, while its helicase activity unwinds double-stranded DNA. By specifically inhibiting the nuclease function of DNA2, DNA2-IN-C5 effectively disrupts these critical DNA repair and replication processes. This targeted inhibition makes cancer cells, which often have high levels of replication stress and DNA damage, particularly vulnerable to the compound's effects. The specificity of DNA2-IN-C5 for DNA2 over other related nucleases is a key feature that minimizes off-target effects and makes it a valuable tool for dissecting the precise biological functions of DNA2 within the cell.
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| ln Vitro |
DNA2-IN-C5 demonstrates potent in vitro activity as a competitive and specific inhibitor of the DNA2 nuclease, with a reported half-maximal inhibitory concentration (IC50) of 20 μM. This biochemical activity translates into the inhibition of all associated catalytic functions of the enzyme, including its DNA-dependent ATPase activity, its helicase activity, and its ability to bind to DNA. The compound's mode of action is competitive, meaning it binds to the active site of the enzyme and directly competes with the natural DNA substrate. This leads to a reduction in the enzyme's ability to process DNA. In cellular contexts, this inhibition is expected to induce replication stress and DNA damage accumulation, as the cell's DNA repair mechanisms are compromised. The potency of DNA2-IN-C5, although in the micromolar range, is significant for a research tool compound and allows for its use in various in vitro assays to study DNA2 biology and to validate its role as a target for cancer therapy.
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| ln Vivo |
Currently, there is no publicly available in vivo activity data specifically reported for DNA2-IN-C5. However, based on its well-defined mechanism as a potent DNA2 inhibitor, it is hypothesized that the compound would exhibit significant in vivo activity in preclinical models. By inhibiting DNA2, it is expected to induce replication stress and DNA damage in rapidly dividing cancer cells, potentially leading to tumor growth inhibition, especially when used in combination with DNA-damaging chemotherapeutic agents. The in vivo efficacy of DNA2-IN-C5 would likely depend on its pharmacokinetic properties, including its bioavailability and ability to reach the tumor site. As a lead compound, its in vivo activity is an area of active research interest, and studies are anticipated to evaluate its antitumor potential, alone and in combination with other therapies, in mouse xenograft models of human cancers.
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| Enzyme Assay |
The in vitro enzyme assay protocol for evaluating DNA2-IN-C5 typically employs a fluorescence-based nuclease activity assay using purified recombinant DNA2 protein and a fluorogenic DNA substrate. In a typical setup, varying concentrations of the compound are incubated with a fixed amount of DNA2 enzyme in a reaction buffer optimized for nuclease activity. The reaction is initiated by the addition of a synthetic DNA substrate that contains a fluorophore and a quencher in close proximity. Upon cleavage of the DNA substrate by the active DNA2 nuclease, the fluorophore is released from the quencher, resulting in an increase in fluorescence signal that can be monitored in real-time using a fluorescence plate reader. The initial rate of fluorescence increase, which is proportional to the enzyme's catalytic activity, is measured for each compound concentration. A control reaction without the inhibitor is used to determine the maximum enzyme activity. The data is then plotted as percent inhibition versus compound concentration to generate a dose-response curve, from which the IC50 value of 20 μM is calculated. This protocol is a standard biochemical approach for characterizing nuclease inhibitors.
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| Cell Assay |
In vitro cell-based assays are employed to evaluate the cellular activity of DNA2-IN-C5, typically using cancer cell lines that are known to be sensitive to replication stress. Cells are seeded in multi-well plates and treated with a range of concentrations of DNA2-IN-C5 for a defined period, typically 48 to 72 hours. Following treatment, cell viability is assessed using standard colorimetric or luminescent assays, such as MTT, CCK-8, or CellTiter-Glo, which measure metabolic activity or ATP content as a surrogate for the number of viable cells. To assess the compound's ability to sensitize cells to other agents, combination studies are performed where cells are co-treated with DNA2-IN-C5 and a sub-lethal concentration of a replication stress-inducing drug, such as hydroxyurea or cisplatin. The combination effect is then analyzed using the combination index method to determine if the interaction is synergistic, additive, or antagonistic. These assays are crucial for translating the biochemical inhibition of the enzyme into a functional cellular outcome and for exploring the compound's potential as a chemosensitizer.
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| Animal Protocol |
Specific in vivo animal study protocols for DNA2-IN-C5 are not widely documented in the public domain. However, based on the standard practices for evaluating similar DNA repair inhibitors with anticancer potential, a typical study would involve the use of immunocompromised mice bearing subcutaneous human tumor xenografts. Once the tumors have reached a specific volume, the animals would be randomized into treatment groups, including a vehicle control group and groups receiving the compound at various doses. DNA2-IN-C5 would likely be administered via oral gavage or intraperitoneal injection, as its formulation permits. The treatment regimen would involve daily or intermittent dosing over several weeks, during which tumor volumes and body weights would be monitored regularly to assess efficacy and tolerability. At the end of the study, tumors would be harvested for further analysis, including histology, immunohistochemistry (e.g., for markers of DNA damage like γH2AX), and measurement of drug concentrations to correlate pharmacokinetics with pharmacodynamics. This type of study design is standard for evaluating the in vivo antitumor activity of novel compounds.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic (PK) data for DNA2-IN-C5 is not specifically reported in the available literature. However, based on its physicochemical properties, some general PK predictions can be made. The compound has a molecular weight of 234.17 and a calculated LogP of 2.07, indicating moderate lipophilicity. This suggests that the compound is likely to have reasonable cell permeability and, potentially, moderate oral bioavailability. It is soluble in DMSO at concentrations up to ~31.25 mg/mL, and formulation studies have shown it can be dissolved in a vehicle consisting of 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. Such a formulation is typically used for in vivo administration. Comprehensive PK parameters such as half-life (t½), maximum concentration (Cmax), area under the curve (AUC), and volume of distribution would need to be determined in future studies to fully understand the compound's absorption, distribution, metabolism, and excretion (ADME) profile.
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| Toxicity/Toxicokinetics |
Specific toxicity data for DNA2-IN-C5 is not available in the public domain, as it is currently a research compound and has not undergone formal preclinical toxicology evaluation. As an inhibitor of DNA2, which is a critical enzyme for DNA replication and repair, the compound is expected to exhibit a degree of cytotoxicity, particularly in rapidly dividing cells. This intrinsic on-target effect could limit its therapeutic window if not carefully managed. The potential for off-target toxicity would also need to be assessed in standard safety pharmacology studies. The compound is currently classified as a research tool and is not intended for human use; therefore, a comprehensive toxicological profile has not been established. Any future development of the compound would require extensive in vitro and in vivo toxicology studies to determine its safety margin, identify target organs of toxicity, and assess its genotoxic and carcinogenic potential.
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| References | |
| Additional Infomation |
DNA2-IN-C5 is a valuable research tool for investigating the biology of the DNA2 helicase/nuclease and its role in maintaining genomic stability. Its mechanism of action involves the competitive inhibition of DNA2, which induces replication stress, making it a promising candidate as a sensitizer for cancer therapies that target DNA replication. The compound has not advanced to clinical trials and does not have FDA approval for any indication; it remains strictly in the preclinical research stage. Its primary application is in laboratory settings for studying the DNA damage response and exploring the potential of targeting DNA repair pathways for therapeutic benefit. In the broader context, inhibitors like DNA2-IN-C5 are crucial for validating novel drug targets and for understanding the complex interplay between DNA replication, repair, and cell survival, which is fundamental to the development of next-generation cancer therapeutics.
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| Molecular Formula |
C10H6N2O5
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|---|---|
| Molecular Weight |
234.17
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| Exact Mass |
234.028
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| CAS # |
35973-25-2
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| PubChem CID |
225906
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.624g/cm3
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| Boiling Point |
437.2ºC at 760mmHg
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| Flash Point |
218.2ºC
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| Index of Refraction |
1.674
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| LogP |
2.07
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
17
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| Complexity |
411
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BMIZBCVEHSUUNO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C10H6N2O5/c13-9-5-2-1-3-7(12(16)17)8(5)11-4-6(9)10(14)15/h1-4H,(H,11,13)(H,14,15)
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| Chemical Name |
8-nitro-4-oxo-1H-quinoline-3-carboxylic acid
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| Synonyms |
DNA2INC5; DNA2 IN C5; DNA2-IN-C5
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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 : ~31.25 mg/mL (~133.45 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (8.88 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 (8.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 4.2704 mL | 21.3520 mL | 42.7040 mL | |
| 5 mM | 0.8541 mL | 4.2704 mL | 8.5408 mL | |
| 10 mM | 0.4270 mL | 2.1352 mL | 4.2704 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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